Editors
Dr. Sangeeta Desai, MD, DTM
Professor, Pathology
Dr. Tanuja Shet, MD, DNB, DPB, DTM
Professor, Pathology
Dr. Preetha Rajasekharan, MD
Associate Professor, Pathology
Dr. Epari Sridhar, MD
Assistant Professor, Pathology

Published by
Tata Memorial Hospital
Mumbai

1. Inherited Cancer Predisposition and Issues in Genetic Testing

2. HPV testing for cervical Cancer screening

3. Translocation based diagnosis of bone and soft tissue sarcoma

4. Molecular classification of breast cancer: Ready for clinical application?

5. Circulating tumour cells as predictive and prognostic marker of breast cancer

6. Evaluation of Prognostic and Predictive Role of HER 2 in Breast Cancer

7. Prognostic and Predictive Markers of Gliomas

8. Evidence for Use of Molecular Methods in Lymphomas

9. An Algorithm for molecular monitoring of Chronic Myeloproliferative Disorders

10. Role of molecular methods in prognostication of neuroblastoma

11. Molecular Genetics of Colorectal Carcinoma

12. Molecular markers and selection of Targeted therapy

Preface

Medicine has long been practised by data, often of unproven validity and insufficient to answer clinically relevant questions pertaining to individual patients for a long time now. Recent times however has seen the emergence of Evidence based medicine (EBM); defined as the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients” in the practice of oncology. It integrates the recent medical research evidence with clinical expertise to improve patient care.

This is the seventh volume on Evidence Based Management Guidelines brought out by the Tata Memorial Centre, which represents the commitment of the Centre to improvement of cancer care in India. The current volume focuses on Central Nervous System Tumours, role of Molecular Diagnostics and Interventional Radiology in oncology practice.

With improvement in survival in most cancers, the emphasis has now shifted to individualization of treatment based on predictive and prognostic factors, leading to a paradigm shift in the management of cancers. In this regard, there is an urgent need to review and redefine the practice guidelines based on compelling evidence.

The growing momentum of molecular analysis has enormous promise for Molecular diagnostics and numerous research applications.  The vast arsenal of newer modalities in the field of molecular pathology available today is bewildering and ever expanding. They provide opportunities to study etiopathogenesis, to aid diagnosis, to prognosticate and to devise targeted therapy in cancer. The challenge for molecular diagnostics is to provide valuable molecular information for tumors using reproducible, cost-effective, and time-efficient assays that can be integrated into the current framework of specimen processing in anatomic pathology. The theme of the conference and this volume is the integration of diagnostic skills and best available, appropriate “molecular” evidence, which would help in decision-making and clinical management especially in pediatric round cell tumors, soft tissue sarcomas, breast cancers amongst other tumors.
We look forward to your feedback and inputs that will prove invaluable in improving the quality and applicability of these guidelines in our country.

Dr. (Ms.) K.A. Dinshaw,
DMRT (Lond), FRCR (Lond)
Director, Tata Memorial Centre

Contributors

Inherited Cancer Predisposition and Issues in Genetic Testing

Rajiv Sarin, Pradnya Kowtal

While all cancers may be considered a genetic disease as they arise from somatic mutations, 2-5% of cancers arise due to a germ line mutation in high penetrance genes causing inherited predisposition. Most common hereditary cancer syndromes such as the breast ovarian cancer syndrome, hereditary CRC or retinoblastoma (RB) follow a Mendelian autosomal dominant pattern of inheritance but a few like Xeroderma Pigmentosum have recessive inheritance. Coordinated epidemiological and molecular studies in cohorts of families predisposed to specific cancers have led to the discovery of several cancer associated genes in the past 2 decades. Tumour suppressor genes such as BRCA1, BRCA2, TP53, RB, MMR and proto-oncogenes such as RET have been identified and shown that germline mutation in these genes are responsible for some common inherited cancer syndromes. Within a decade of identifying these genes, translational research has enabled the use of information on the specific mutations in these genes and molecular signature of hereditary cancers for clinical management of cancer affected persons and for assessing cancer risk and guiding preventive approaches for high risk healthy members of such families. The most recent progress specific therapeutic approaches for some hereditary cancers such as BRCA associated breast or ovarian cancer.

When to suspect inherited predisposition to cancers
There are several indicators of possible inherited predisposition to cancer in an individual and within a family. The most important pointer is the occurrence of similar or related cancers in first or second degree relatives, often with a Mendelian pattern of inheritance. Except for de novo germline mutations, where the index case or proband may develop a hereditary cancer without any family history of cancer, most hereditary cancers will affect closely related individuals in successive generations. In addition to the family history, germline mutations in the genes responsible for inherited predisposition may also be responsible for bilateral, multifocal or multiple primary cancers in an individual, cancers at unusually young age, other syndromic features and sometimes characteristic histological or biological features. Probability of identifying a germline mutation in BRCA1 or BRCA2 in hereditary breast ovarian cancer, in MMR genes in HNPCC and in TP53 mutation in Li Fraumeni syndrome (LFS) is well predicted by various definitions of these syndromes and by computer models that take into account personal and family history of cancer, age at cancer diagnosis, bilaterality, histological subtypes etc.

Need for Mutation Analysis and Molecular Diagnostics in Hereditary Cancers
Molecular or biological features such as the triple negative cancer or specific gene expression profile in breast cancer or MSI in HNPCC indicate the presence of specific germline mutation. Confirmation of a deleterious or pathogenic germline mutation in the predisposing gene by sequencing is important to confirm the diagnosis of hereditary cancer or a particular hereditary cancer syndrome and this may have implications for cancer prognosis, for advising optimal local and systemic treatment, and for genotype– phenotype correlation. In families where the cancer associated germline mutation is known or in ethnic groups such as the Ashkenazi Jews where founder germline mutations are prevalent in the population, screening for these specific mutations in the healthy but at risk individuals is useful for risk estimation and advising appropriate screening strategies and sometimes prophylactic medical or surgical treatments as discussed later. For many well defined hereditary cancer syndromes such as the hereditary breast ovarian cancer syndrome or Li-Fraumeni Like (LFL) Syndrome, germline mutation is not identified in 20 - 50% of the families fulfilling the criteria.

Ethical, legal and social issues in genetic testing
There are various ethical, legal and social issues related to genetic testing. While the basic issue centers on disclosure of results and possibility of genetic discrimination, specific issues may vary according to the existing protection laws and their enforcement in different countries and the health care set up. Despite the existence of relevant laws in several countries, serious concerns still exist about discrimination by the employer and insurance companies based on the genetic risk for a serious illness like cancer. Purely on ethical grounds, the matter is further complicated with the emerging possibilities of pre-implantation or pre-natal genetic testing for genes which may have variable penetrance for developing cancer, cancers may be diagnosed after several decades and be highly curable when diagnosed. While there would always be several emerging issues pertaining to the ethical and social aspects of genetic testing, the current balance is in favor of genetic testing in well defined hereditary cancer syndromes after pre-test counseling and informed consent regarding the advantages and limitation of genetic testing (1).

While the Health Insurance Portability and Accountability Act of 1996 of USA prohibits disclosure of health information unless there is a serious and imminent threat to the health or safety of a person or the public, physicians have been sued in USA for failing to warn of genetic risk from an inherited cancer predisposition as highlighted by Harris et al (1). In the Pate versus Threkel case, the court judged that a physician should have warned his patient with medullary thyroid cancer of the same risk to her children, since this risk could be tested and the cancer could have been prevented by prophylactic measures. Other examples include the case of Safer versus Estate of Pack which highlighted failure to warn a relative of the risk of FAP; a New Jersey court ruled that a doctor’s duty was to take reasonable steps to warn directly immediate family members at risk of harm from an inherited disorder. The ethics pertaining to patenting genetic testing and unfair monopolistic practices has been highlighted by the challenge of the Myriad Genetics patent for BRCA1 and BRCA2 in the European Patent Office. The prohibitive cost of over Rs. 100,000 for mutation analysis of BRCA1 and BRCA2 at the Myriad Genetics precludes the use of this test for families who may benefit from this genetic testing. This has prompted several research laboratories to offer genetic testing services. However, since the results of genetic testing could have far reaching consequences on the individual and the family, it is important to employ highly sensitive and specific mutation analysis techniques and ensure quality control.

Issues in clinical management of patients with hereditary cancers
As opposed to the sporadic cancers, individuals with hereditary cancer could pose special clinical management problems due to a variety of reasons. This includes younger age at diagnosis, multifocal or bilateral involvement and a higher risk of second primary cancer. Clinical management of these patients is further complicated by a higher psychosocial burden arising from past experiences in the family. Confirmation of a germline mutation in a cancer predisposing gene is sometimes useful in the management algorithm. Knowledge of mutation in BRCA1 or BRCA2 is used to counsel the patients about the long term risks and possible therapeutic options, e.g. in familial breast cancer the risk of ipsilateral breast cancer recurrence, risk after breast conservation and the contralateral breast cancer risk and also the type of chemotherapy- platinum agents or the investigational PARP inhibitors could be discussed; like wise the higher risk of metachronous lesions in individuals with colon cancer and mutations in MMR genes influence the extent of resection with the recommended surgical procedure being the total colectomy with ileorectal or ileosigmoid anastomosis.

Counseling, Screening and prophylactic treatments for high risk individuals
Based on a detailed pedigree analysis of the family tree (genogram) regarding the number of cancer affected cases, site and histology of cancers, age at diagnosis and the relationship of the individual with the proband, members of cancer prone families can be counseled for their cancer risk and need for genetic test. Such specific genetic and psychosocial evaluation and counseling play a major role in the decision making process to opt for genetic testing. Healthy individuals from families where cancer is being inherited in a Mendelian autosomal dominant or recessive pattern are at substantial risk of developing cancer. In a cancer prone family where the cancer causing germline mutation has been identified in a cancer affected case, mutation analysis in the unaffected high risk relatives after a pre-test counseling could be very useful for determining their risk for a particular cancer and the type and frequency of screening required. Prior to the identification of BRCA1 and BRCA2, prophylactic mastectomy and oopherectomy has been done for high risk healthy women based on their family history and this was shown to protect them from cancer. However, with the availability of genetic testing, it would now be considered unethical to subject a healthy woman to prophylactic mastectomy on the basis of family history alone, especially if a deleterious BRCA1 or BRCA2 mutation has previously been identified in her family.

It is important to note that germline mutation in the commonly tested high penetrance genes may not be identified in many families that show classical Mendelian autosomal dominant pattern of inheritance. These families have to be evaluated in specialised genetics clinic and counselled accordingly, but with less precise estimates of future cancer risk in the unaffected relatives. The situation is further complicated by variable penetrance.

Cancer genetics services in India
There is very scanty data on the hereditary aspects of cancers outside the European and North American Caucasian population. The cancer genetics clinic initiated at TMH in 2003 is currently one of only two places in the country where specialized cancer genetic services are routinely offered within and outside research setting. To date the clinic has evaluated and registered over 400 families. The Cancer Genetics Lab at ACTREC became functional in 2007 and provides genetic analysis to aid counseling and medical management for high risk families; maintains a DNA bank and is evaluating genotype – phenotype correlation for high penetrance cancer predisposition genes such as BRCA1; BRCA2, TP53, Chk2 in hereditary breast and ovarian cancers and RET proto oncogene in medullary thyroid cancers. Similar studies will be shortly undertaken for Xeroderma Pigmentosa, HNPCC and RB. Here we outline our approach to three autosomal dominant hereditary cancer syndromes where the clinic is offering full range of services from clinical evaluation, pedigree analysis, risk assessment, pre-test counseling, mutation analysis and post test counseling and appropriate clinical management.

Hereditary breast and ovarian cancer and BRCA1 and BRCA2 genes
The BRCA1 gene is located on chromosome 17q21 and has 24 exons of which 22 exons are transcribed and translated into a protein. The BRCA2 gene is located on chromosome 13q12-13. It comprises of 26 exons, of which exon 1, is a part of the 5’ untranslated region. The BRCA2 protein is made up of 3418 amino acids. Both BRCA1, BRCA2 have a large exon 11. Several copies of BRC repeats are found in this gene. Both these proteins are considered to function as tumor suppressor genes and play a role in DNA repair. Mutations involving an insertion or deletion of nucleotides in the BRCA1 / 2 genes, can lead to an altered reading of codons, in turn giving rise to the “frame-shift’ mutations. These constitute nearly 50% and 30% of all the mutations reported in BRCA1 and BRCA2, respectively (2). On the other hand, single nucleotide changes lead to ‘point mutations’ causing substitution of a functionally and / or structurally different amino acid. These ‘mis-sense’ mutations can affect the function of the protein depending on the site as well as the nature of the amino acid introduced. Single nucleotide base pair change that result in the generation of a ‘stop codon’ (amino acid chain termination signals), termed as ‘nonsense mutations’, lead to the production of a truncated protein. Missense and nonsense mutations constitute nearly 25% and 10% of reported mutations in BRCA1, respectively (2). Missense mutations have been found to be more common in BRCA2 (~50%).

DNA sequencing of BRCA 1/ 2 genes aids in the detection of mutations which can be interpreted as positive for pathogenic / deleterious mutations, negative for pathogenic mutations and genetic variants. The genetic variants can be divided into three classes: a) favor polymorphism b) suspected deleterious and c) uncertain clinical significance. In various literature reports, BRCA1 and BRCA2 have been screened by various methodologies like dHPLC, CSGE, SSCP, and PTT, and / or followed by DNA sequencing. Considering the implications of a false negative genetic test, it is important to use highly sensitive methodologies like dHPLC or CSGE for screening of possible mutation in BRCA1 and BRCA2.

Specific mutations found in multiple unrelated families of a given population are designated as founder mutations. Three founder mutations (185delAG and 5382insC in BRCA1 and 6174delT in BRCA2) have been observed in 2.5% of the Ashkenazi Jewish population and account for majority of breast cancers in this community. In India the mutation 185delAG, detected in different ethnic groups is supposed to have arisen independently of any Ashkenazi Jewish origin (3) and has been identified in 3 families seen at our Genetics Clinic.

The National Institute for Health and Clinical Excellence (NICE), UK (4) and several other expert recommendations are available for risk assessment and breast screening of women from high-risk families. The suggested practice is monthly breast self examination and a six-monthly clinical breast examination by trained oncologist, surgeon or gynecologist. An annual radiological screening using a mammography, MRI or ultrasonography, as appropriate for woman’s age and breast density is recommended. While in 2004 NICE did not recommend breast MRI (4), in its July 2006 update [www.nice.org.uk/CG041], NICE has recommended bilateral breast MRI ensuring high temporal and spatial resolution and dynamic sequences with post contrast. They should be double-read where possible. Screening guidelines for women at high risk of ovarian cancer are not very clear. The recommendations, to date, include transvaginal ultrasound and CA-125 measurements once or twice a year, starting from the age of 25 to 30 years with consideration of prophylactic 35 years (5). The impact of such screening is uncertain with the available evidence showing no definite advantage (6).

The preventive strategies in high risk population vary from non-invasive chemoprevention with anti-oestrogens to invasive prophylactic oophorectomy. Chemoprevention using selective estrogen receptor modulators has shown to decrease the incidence of estrogen-receptor positive tumors to a great extent. The fact that most breast cancer associated with BRCA1 mutation are often estrogen receptor negative has also to be kept in mind. An alternative, yet a drastic method used to prevent breast cancer development is prophylactic bilateral mastectomy with immediate reconstruction which is shown to reduce the risk for breast cancer by 85-100% as per four observational studies and a meta-analysis (7-10). Prophylactic oopherectomy reduces risk of ovarian cancer by 85-100% and that of breast cancer by 53-68%. The risk reduction is greater in BRCA1 carriers (11). It is important to discuss these aspects of clinical management with the high risk patients who can then make an informed decision.

The safety of breast conservative therapy in women with BRCA associated breast cancer was an issue after some earlier reports suggesting very high rates of ipsilateral breast recurrence up to 40% in these women. However, international collaborative data (12) from 160 women with a deleterious germline BRCA1/2 mutation and 445 controls showed no significant excess ipsilateral breast recurrence rates in mutation carriers with 10 and 15-year estimates being 12% (95% CI 9–15%) and 24% (95% CI 17–33%) for carriers and 9% (95% CI 7–10%) and 17% (95% CI 12–21%) for controls, respectively (hazard ratio [HR] 1.37, P = 0.19). On multivariate analysis, excluding carriers who had undergone oophorectomy, BRCA1/2 mutation status was an independent predictor of ipsilateral breast recurrence (HR 1.9; P = 0.04). No significant difference was und between carriers who had undergone oophorectomy and sporadic controls for incidence of IBTR (P = 0.37). In mutation carriers who had not undergone oophorectomy, there were no local failures following tamoxifen treatment, in comparison with rates of 8%, 17% and 31% at 5, 10 and 15 years, respectively, without tamoxifen treatment. Tamoxifen use also reduced risk of CBCs in mutation carriers (HR 0.31; P = 0.05).

Multiple endocrine neoplasia (MEN) and RET proto Oncogene
MEN is characterized by occurrence of tumors that involve two or more endocrine glands in a single patient or in close relatives. There are two types of MEN syndromes, MEN type 1 sometimes called Wermer syndrome, and MEN type 2, also referred to as Sipple syndrome. MEN 2 is divided into three subtypes depending on the organs involved. The predisposing gene for MEN 2 is RET. The RET proto oncogene has been localized to chromosome 10q11.2, comprises of 21 codons. The RET (REarranged during Transfection) proto oncogene codes for a receptor tyrosine kinase (RTK) protein. RTKS transduce the extracellular signals for processes as diverse as cell growth, differentiation, survival and programmed cell death. The signals are transduced by dimerization of RTK and autophosphorylation through effectors that recognize and interact with the phosphorylated protein. Though a number of downstream processes are triggered, the interaction between effector ligand and RTK is very specific (13). Germline mutations in RET that produce constitutively activated receptors cause MEN type 2 and several endocrine and neural-crest-derived tumors, whereas mutations resulting in nonfunctional RET or lower expression of RET are found in individuals affected with Hirschsprung disease (14). Genetic testing of RET proto oncogene comprises of mutation analysis of the highly conserved, cysteine-rich, extracellular domain (exons 10 and 11), intracellular tyrosine kinase domains (exons 13, 14, and 15), and an intracellular catalytic core (exon 16). The specific mutations influence the type of cancers and to some extent their prognosis. By using a worst-case scenario, grouping RET genotypes into highest risk category (codon 918 mutation), high-risk category (codon 609, 611, 618, 620, 630, and 634 mutations), and least-high risk category (codon 768, 790, 791, 804, and 891 mutations) is useful for planning the timing of prophylactic surgery in RET carriers. Since it is not well understood how polymorphisms modify the cancer risk, the magnitude of benefit by mutation based prophylactic thyroidectomy is not well known (15). Calcitonin stimulation test compliments the RET mutation analysis in such situations.

Li-Fraumeni syndrome (LFS) and TP53 mutation
This rare autosomal dominant cancer syndrome arises from germline mutation in the tumor suppressor gene TP53. LFS was initially defined using stringent criteria of 1) Proband with a sarcoma diagnosed before the age of 45; 2) 1st degree relative with cancer before the age of 45; and 3) Another 1st or 2nd degree relative with either a sarcoma diagnosed at any age or any cancer diagnosed under the age of 45. Li – Fraumeni Like (LFL- Birch) syndrome was defined as a proband with any childhood tumour or sarcoma, brain tumour, or adrenocortical tumour under 45 years, plus a first or second degree relative with a typical LFS tumour at any age and another first or second degree relative with any cancer under the age of 60.  A more relaxed criterion was proposed for LFL-Eles with a clustering of two typical LFS tumours in subjects who are first or second degree relatives at any age. Germline TP53 mutation has been reported in 77% families with LFS and 40% families with LFL syndrome (16). The IARC maintains a database of all somatic and germline mutations in p53 [http://www-p53.iarc.fr/]. The mutation detection in p53 gene for LFS is carried out by amplification of various exons of the gene using

HPV testing for cervical Cancer screening

R. Sankaranarayanan

1. Introduction

Cervical cancer is a major public health problem globally, accounting for an estimated 493,000 incident cases, 1.4 million prevalent cases and 273,000 deaths in the world around 2002 (1). It is a major cause of mortality and premature death among women in their most productive years in low- and medium-resourced countries in Asia, Africa and Latin America, which accounts for four-fifths of the global burden, reflecting the grim reality of the lack of effective control measures in many high-risk countries. If effective preventive interventions are not currently implemented, over 1 million new cervical cancer cases will be diagnosed annually by the year 2030.

Early detection of asymptomatic cervical precancerous lesions by Pap smear screening has been an established and effective preventive measure for several decades, and has led to the successful prevention of invasive cervical cancer and premature death from it in developed countries. The recognition that cervical cancer is caused by persistent infection by one or more of the 15 oncogenic high-risk human papillomavirus (HPV) types have resulted in the development and evaluation of a number of HPV detection tests for cervical cancer screening (2). HPV testing has three main applications as a screening test: first as a primary screening test to detect cervical intraepithelial neoplasia (CIN); secondly as a triaging investigation to select women with minor cytological abnormalities for colposcopy and treatment; thirdly as a follow-up test for women treated for high-grade CIN (CIN 2 and 3) to rapidly identify treatment failures / successes and for continuing management of women referred for colposcopy in whom no lesion could be visualized or histology turns out to be negative (3, 4). Published experience from randomized controlled trials (level 1 evidence), cohort and cross- sectional studies regarding the utility of HPV testing in cervical cancer screening is reviewed and discussed in this chapter. The end points discussed are accuracy (sensitivity and specificity) to detect high-grade precursor lesions and cancer, detection rates of CIN 2 or worse lesions as compared to cytology, incidence of CIN 2 and 3 lesions and incidence and mortality from cervical cancer.

2. HPV detection tests
HPV testing relies exclusively on molecular biology techniques that detect HPV DNA in cervical cell samples using nucleic acid probes. Since there are so many HPV types with differing carcinogenic potential, HPV tests are designed to determine whether one or more of high-risk types are in the specimen. The two broadly used techniques for HPV testing include signal-amplified nucleic acid assay and target amplified techniques such as PCR.

Hybrid capture II (HC II) test is a commercially available and FDA approved HPV test that uses signal amplification to detect HPV DNA. HC II test involves a laboratory process that produces light signals proportional to the amount of HPV DNA present in the specimen. Cervical samples are classified as HPV positive if the relative light unit (RLU) reading obtained from the luminometer of the HC II assay equipment is equal to or greater than the mean of the positive control (PC) values supplied by the HC II kit. A positive result is recorded for specimens with RLU/PC ratio of 1 or more, corresponding to a viral load of 1 pg/ ml or 5000 or more viral copies. The process requires technologically advanced equipment and the test, as of now, is expensive. These requirements currently make the use of HC II too expensive and difficult to implement in many low-resource settings. For all practical purposes, it has the same sensitivity as that of PCR for detecting HPV DNA. HC II detects 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), is standardized and highly reproducible.

Target amplified HPV assays produce highly concentrated samples of a specific DNA genetic sequence which are probed to identify specific HPV genotypes. PCR is the most common target-amplified technique and is capable of detecting small amounts of HPV DNA. General or consensus primer-mediated PCR assays have enabled screening for broad spectrum of HPV types in clinical specimens using a single PCR reaction. Following amplification using consensus primers, individual HPV types are identified using a variety of methods. Using consensus primers in a test format known as real-time QPCR, it is possible to generate viral load data. The considerable skills, advanced equipment and costs involved make PCR inappropriate for large screening programs in low-resource settings.

Recognizing the importance of developing rapid, simple, accurate and affordable HPV test formats suitable for use in low-resource settings, there is an on-going effort (START project: Screening Technologies to Advance Rapid Testing) conceived and coordinated by Program for Appropriate Technology in Health (PATH) in collaboration with the Cancer Institute of the Chinese Academy of Medical Sciences (CICAMS), Beijing, the Tata Memorial Centre (TMC), Mumbai, Nargis Dutt Memorial Cancer Hospital (NDMCH), Barshi, the IARC, Lyon, and industrial partners, funded through the Bill & Melinda Gates Foundation to develop such tests. A rapid batch test format, based on HC technology, targets oncogenic HPV types and another assay targets the detection of the E6 protein of oncogenic HPV types in a lateral flow strip test format. The commercial availability of these tests is eagerly awaited.

Of the oncogenic HPV types, HPV-16 and to a lesser extent HPV 18/45 may carry a greater risk than the others, as evidenced by sustained increase in risk of developing CIN3 or cancer for up to 10 years after an initial positive HPV-16 result as compared to other oncogenic types (5). For this reason, it may be efficient to genotype women testing positive by pooled assays, allowing the intensity of follow-up to be tailored.

New technologies are being developed to identify persistent infections, thereby avoiding repeated HPV DNA tests, and to improve the specificity without substantially reducing the sensitivity of HPV testing. Persistent expression of the viral oncogenes E6 and E7 is a necessary step for HPV induced carcinogenesis (6). A high detection rate of E6 and E7 transcripts has been found in cervical cancer tissues (7) and a relationship with histological severity has been observed in cervical biopsies (8). Since HPV infection most often does not lead to cervical neoplasia, and since E6/ E7 appear to be involved in the progression of neoplastic changes following infection, tests that detect the expression of these viral proteins have the potential to improve specificity of HPV testing. Different mRNA detection tests are currently being developed and evaluated.

3. HPV testing as primary cervical screening test

3.1 Absolute accuracy

The accuracy of HPV testing in detecting high-grade CIN and cancer has been investigated in several cross-sectional studies in which women were concurrently screened with cytology and HPV testing in the context of primary screening and the results have from these studies have been summarized in recent meta-analyses and reviews (3, 4, 9). In the most recent meta-analysis, 26 such cross-sectional studies were reviewed (4). In 10 studies, women were referred for confirmation of disease status only when at least one screening test was positive; in 8 studies a random sample of screen- negatives received reference standard investigations allowing adjustment for verification bias, and in 6 studies all women had colposcopy with biopsy if colposcopically suspicious.

The pooled results are given in Table 1 (4). Overall, the pooled sensitivity of HC II based on 18 studies at test cut off value of 1 pg/ml in detecting high-grade CIN and cancer was 90.7% (95% CI: 87.5-93.8) and the specificity was 88.1% (95% CI: 86.1-90.1). The range in sensitivity and specificity among the included studies were 50-100% and 61-95%, respectively. The observed sensitivity of HC II for CIN 2 or worse lesions was lower in the 3 Indian cross-sectional studies than other reported studies in the West: 50%, 70% and 80%, respectively and was also lower than average in other developing countries (77% in Peru, 81% in Zimbabwe, 83% in Brazil and 88% in South Africa) (10, 11, 12, 13). The sensitivity of HC II for CIN 2 or worse lesions was consistently high in 8 studies conducted in Europe and North America, yielding pooled estimate of 98.1% (95% CI: 96.8-99.4%); the pooled specificity was 91.3%.

The pooled sensitivity of PCR, based on 7 studies, was 84.4% (95% CI: 77.4-91.5) and the specificity was 95.1% (95% CI: 93.5-96.7) (4). The range in sensitivity and specificity among the included studies were 64-95% and 79-99%, respectively. Thus, its pooled sensitivity was lower, but the pooled specificity was higher as compared to the HC II test. However, these studies used different primers and detection of amplified sequences, and significant heterogeneity was seen. For example, the sensitivity was 95% in a study where GP5+/GP6+ primers were used followed by hybridization with a cocktail of oligonucleotides of 14 high-risk HPV types as compared to a sensitivity of 64% in another study where the PCR/Sharpassay (MY09/MY 11 primers, hybridization with 10 high-risk types) (14, 15). The variability is much less when good quality control procedures are used for currently used consensus PCR assays. Guidelines for quality assurance are currently being developed.

It is pertinent to point out that the accuracy of HPV testing in detecting CIN 2 or worse disease showed substantial heterogeneity and the main factor that explains this variation is the geographical location of the studies. Studies conducted in Europe or North America, where HC II test was used, showed better performance as compared to studies in developing countries.

3.2 Relative accuracy compared to cytology

Comparison of the accuracy of HPV testing with that of Pap smear at atypical squamous cells of undetermined significance (ASCUS) or worse, or low-grade squamous intraepithelial neoplasia (LSIL) or worse, threshold for referral, based on 18 studies, indicate that the pooled sensitivity of the of HC II was 23% (95% CI: 13-25%) higher and its pooled specificity was 6% lower than cytology (ratio: 0.94; 95% CI: 0.92-0.96; range 0.67-1.09) (4). In one randomized trial in India, the detection rate of CIN 2 or worse disease was lower in the HPV arm compared to the cytology group (16). In all other studies, the sensitivity of HC II was higher, ranging from 1 to 115%.

3.3. Combination of HPV testing and cytology
The combination of Pap smear with HC II resulted in a 45% (95% CI: 1.31-1.60) and 39% (95% CI: 11-73%) higher detection of CIN2 or worse or CIN3 or worse disease respectively than cytology alone (at ASCUS or worse threshold for referral), whereas the specificity was 7% lower (95% CI: 6-8%). Adding a Pap smear (ASCUS cut off for positive test) to the HC II test increased the sensitivity of HC II for CIN2 or worse or CIN3 or worse by 7% and 4% respectively, but resulted in a loss in specificity of 5% (95% CI: 4-6%) and 7% (95% CI: 5-9%). In general, combining cytology with HPV testing adds little to overall sensitivity, but does reduce specificity. It would appear that the most useful role for cytology is to triage women whose primary HPV screening test is positive to avoid referral and over-treatment of women with minimal and no detectable cytologic abnormality and who are likely to have transient infections.

3.4 Evidence from randomized trials
Results from 6 randomized trials comparing HPV testing with cytology have been recently published (16- 22). In four of the six studies, HC II test was used (16-18, 21). In one study GP5+/GP+ PCR followed with reversed line blot assay was used (22) and in another GP5+/GP6+ PCR followed by confirmation with a cocktail detecting 14 high- risk types was used (20). In two studies HPV testing alone was used in the experimental arm (16, 17), but in four cytology was used in addition to HPV testing in the experimental arm (18- 22). In all trials cytology at ASCUS/atypical glandular cells of unspecified significance (AGUS) threshold was used in the control arm.

The baseline relative sensitivity for detection of CIN 2 or worse lesions of HPV testing compared to cytology varied between 1.46 (17) and 1.74 (21) and was statistically significantly different from unity in the trials conducted in Europe or Canada (pooled ratio: 1.55; 95% CI: 1.32-1.83) (17, 18, 20-22), but was not statistically significantly different from unity in the Indian trial (relative sensitivity of 0.88; 95% CI: 0.76 to 1.03) (16). The relative sensitivity for CIN3 or worse disease pooled from four studies, reporting this outcome and excluding the Indian trial, yielded an estimate of 1.31 (95% CI: 1.06 to 1.62). The pooled relative sensitivity of combined HPV and cytology screening compared to HPV testing alone, was 1.04 (95% CI: 0.87 to 1.25) (18, 20, 21).

In a randomized trial in South Africa, cryotherapy for HPV test-positive women triaged by visual screening with acetic acid (VIA) resulted in 77% and 74% decline in the prevalence of CIN 2-3 lesions at 6 and 12 months respectively (23). In the Swedish trial, at subsequent screening examinations over 4 years, the proportion of women in the intervention group who were found to have CIN 2 or 3 lesions or cancer was 42% less and the proportion with CIN 3 lesions or cancer was 47% less than the proportions of control women (22). The number of CIN 3 or worse lesions detected in the subsequent round in the Dutch randomized trial was lower in the intervention group than in the control group (24/8413 vs. 54/8456, 55% decrease, 95% CI 28-72; p=0.001) (20). These results indicate that a higher detection rate of high grade precursor lesions, when HPV testing was used as part of the initial screening process, led to lower rates of disease at the subsequent screening.

No studies have yet reported the impact of HPV testing on cervical cancer incidence and mortality and results from an Indian randomized trial in Osmanabad district are expected in 2008 (16).

3.5 Duration of protection

It has been shown in studies that HPV negativity alone, or in combination with negative cytology, gives a longer duration of protection against CIN2 or worse disease than being cytology negative alone.

In a 10-year follow-up of 21,000 women new lesions developed much more rapidly in those who were HPV positive compared to women who were HPV negative (24). In a Danish study, HPV testing at 5-yearly intervals provided similar protection as cytology testing at 3-yearly intervals (25). In a French study, 5 of 4401 women with negative cytology and HPV tests followed-up for a median of 34 months developed high-grade lesions, compared to 29 of 501 women who were initially cytology-negative, but HPV positive (26). In a 5-year follow-up of 2810 cytology-negative women, 4/62 HPV-positive women developed CIN3 or worse lesions compared to 1/2175 HPV-negative women (27). In a Brazilian cohort of 2404 women, cytologic lesions persisted longer, and progressed more rapidly, in HPV-positive women (28). In a Swedish study, HPV-negative women had a relative risk of 0.53 (95% CI: 0.29- 0.92) compared to cytology-negative women (22).

4. HPV testing in the triage of ASCUS and LSIL
In a meta-analys is of 22 studies, where the accuracy of HC II for triage of women with ASCUS could be assessed, 8.7% underlying CIN2 or worse lesions were found. Overall, HC II had a sensitivity of 93.1% (95% CI: 91.1-95.1%) for detecting CIN2 or worse lesions and the pooled specificity was 62.3% (95% CI: 57.6-67.1%) (4). In 7 studies, where a repeat Pap smear was also taken, the sensitivity of HC II was, on average, 14% higher than repeat cytology, considering ASCUS or worse as a positive result, for detection of CIN2 or worse lesions (ratio: 1.14; 95% CI: 1.08-1.20) and HC II testing and cytology triage showed a similar specificity (ratio: 0.99; 95% CI: 0.88-1.10).

The pooled sensitivity of HC II triage of women with an index smear showing LSIL in 12 studies was very high: 97.2% (95% CI: 95.6-98.8%) for the outcome of CIN2 or worse lesions; its specificity was very low: 31.2% (95% CI: 23.6-38.8%) (Table 1) (3). The very large majority of women with LSIL had a positive HC II result (pooled estimate of 76.6%). However, for women aged 35 or more, the HPV-positivity rate was much lower than for younger women. The potential value of HPV testing as an adjunct to cytology in the 35 years and above age group was substantially better than for younger women (29, 30). HPV-16 infected women with an initially equivocal or mildly abnormal cytology have a 50% chance of biopsy confirmed CIN2 or worse lesion within 2 years, indicating that HPV-16 detection may be useful for the triage of LSIL (27, 31).

5. Post-colposcopy management of negative findings
For women referred for colposcopy because of abnormal smears, but who do not have any visible lesion on colposcopy or for whom a biopsy yields negative histology, a negative HPV test at or near the time of colposcopy provides additional reassurance that there is unlikely to be any detectable disease, while being HPV-positive (especially for types 16 and 18), indicates a continuing risk (24, 31, 32) and a need for short-term repeat testing. Especially for type 18 the possibility of glandular lesions should be excluded by careful examination of the endocervical canal.

6. Surveillance after treatment of CIN
In a meta-analysis of 16 studies with variation in design, timing of visits, choice of HPV testing methods and the assessment of disease status at entry and end of follow-up, treatment failure, expressed in terms of residual or recurrent CIN, occurred in 10.2% of treated cases. The sensitivity of HPV testing in predicting treatment failure ranged from 67% to 100% and was, on average, 94.4% (95% CI: 90.9-97.9%) (Table 1). The specificity of HPV testing for predicting treatment success varied between 44% and 100%.

Similarly, HPV testing after treatment predicted residual or recurrent CIN with significantly higher sensitivity (ratio: 1.16; 95% CI: 1.02-1.33) and lower specificity (ratio: 0.96; 95% CI: 0.91-1.01) than follow-up cytology. When CIN lesions were treated by excision, HPV testing predicted treatment outcome with higher sensitivity than histological assessment of the section margins (relative sensitivity: 1.31 [95% CI: 1.11-1.55]; relative specificity: 1.05 [95% CI: 0.96-1.15]).

7. Self-sampling for HPV testing
An overview of several studies that have evaluated the diagnostic accuracy of self-collected vaginal specimens using swabs, tampons, or brushes for HPV compared to clinician collected samples indicate an overall relative sensitivity of 74% and a specificity of 84% for the self-collected sample (33). Although clearly not as good as a clinician taken sample, this sensitivity compares favorably to cytology whose sensitivity for CIN2 or worse lesions is less than 70%. It has been found that women prefer self-sampling to a clinician taken sample (34-36). However, with currently existing technologies, a clinician taken sample is to be preferred when this is possible.

8. Age group for HPV testing
The peak incidence of HPV infection occurs around age 20, the peak incidence of CIN 2-3 occurs around age 30, and invasive cancers occur around age 40-50 years. About 20-40% of sexually active women in the 20-30 year group may test positive HPV at any given time and most of these infections are transient and will clear by themselves. Thus, HPV testing

younger women (<30 years of age) will result in detection of transient HPV infections and thus will result in decreased specificity, increased investigation/treatment rates and costs. It is recommended that HPV testing be resorted to in women aged 30 years and above (37).

9. Psycho-social aspects of HPV testing
Public understanding of HPV infection and its consequences is very limited and it is important that women understand the role of HPV infection when HPV testing is used for screening. Currently, women are largely unaware of the role of HPV infection in cervical cancer and the role of sexual behavior in acquiring HPV infection. Awareness that a sexually-transmitted HPV infection (STI) is the causal agent in cervical cancer could affect attitudes towards the disease, and may cause confusion and anxiety on issues of fidelity and trust in relationships. Thus, care is needed to minimize any reduction in screening coverage that could come from the association with STIs. In populations with high prevalence of HPV infection, large numbers of women will have positive HPV results and will, therefore, need advice and support. Promoting understanding of HPV without creating anxiety is critically important. In many cultures, explaining that HPV is an infection of the surface epithelium in the reproductive tract rather than explicit reference to a STI, may be a preferable explanation to prevent women shying away from testing.

10. Conclusions
HPV testing is substantially more sensitive and somewhat less specific (due to the detection of transient infections) than cytology in detecting high-grade CIN. HPV testing provides an objective, highly reproducible, automated and sensitive test for CIN detection and allows better quality assurance. A possible new algorithm for screening is applying the more sensitive HPV test first and using a more specific test such as cytology for the 5-15% HPV-positive women to decide on management. For HPV-positive and cytology-negative women, both tests can be repeated after 1 year and positives could be referred for colposcopy. In low-resource settings where cytology is not feasible, VIA can be used to provide immediate treatment for HPV positive women in a single visit strategy, which has been shown to be safe and effective in reducing the prevalence of high-grade CIN in a randomized trial in South Africa (23). HPV testing is very effective in the triage of ASCUS cytology. HPV testing accurately identifies women with incomplete excision of CIN2 or 3 lesions. The continued detection of the same type of HPV for 18 months or more after treatment indicates a high possibility of residual disease. Self-sampling for HPV could be a valuable screening method for women who refuse to attend clinician-based screening and an important way of improving population coverage of screening. An international working party of the IARC concluded based on surrogate end points that HPV testing is expected to be at least as effective as cytology screening in reducing cervical cancer burden (37).

However, the currently available HPV tests are too sophisticated, cumbersome and costly to be feasible to implement in public health programs in low- and medium-resourced countries. Current efforts to develop simple, inexpensive / affordable, rapid and accurate HPV tests with simple supporting equipment for use in developing countries through the START project is a promising initiative to make HPV testing a feasible screening method in low-resource settings. Effective education and counseling messages need to be developed for HPV-positive women.

References

1. Ferlay J, Bray F, Pisani P, Parkin DM (2004) GLOBOCAN 2002. Cancer Incidence, Mortality and Prevalence Worldwide. IARC Cancer Base No. 5 version 2.0. IARC Press: Lyon

2. MuHoz N, Bosch FX, de SS, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348: 518-27.

3. Arbyn M, Sasieni P, Meijer CJ, et al. Chapter 9: Clinical applications of HPV testing: A summary of meta-analyses. Vaccine 2006; 24 Suppl 3: S78-S89

4. Cuzick J, Arbyn M, Sankaranarayanan R, et al. Chapter 2: Overview of the evidence from cervical screening studies. Vaccine 2008. In Press

5. Khan MJ, Castle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 2005; 97: 1072-9.

6. zur Hausen H, de Villiers EM. Human papillomaviruses. Annu Rev Microbiol 1994; 48: 427-47.

7. Nakagawa S, Yoshikawa H, Yasugi T, et al. Ubiquitous presence of E6 and E7 transcripts in human papillomavirus-positive cervical carcinomas regardless of its type. J Med Virol 2000; 62: 251-8.

8. Kraus I, Molden T, Erno LE, et al. Human papillomavirus oncogenic expression in the dysplastic portio; an investigation of biopsies from 190 cervical cones. Br J Cancer 2004; 90: 1407-13.

9. Koliopoulos G, Arbyn M, Martin-Hirsch P, et al. Diagnostic accuracy of human papillomavirus testing in primary cervical screening: a systematic review and meta-analysis of non-randomized studies. Gynecol Oncol 2007; 104: 232-46.

10. Sankaranarayanan R, Chatterji R, Shastri S, et al. Accuracy of human papillomavirus testing in primary screening of cervical neoplasia: results from a multicenter study in India. Int J Cancer 2004; 112:
341-7.

11. Blumenthal PD, Gaffikin L, Chirenje ZM, et al. Adjunctive testing for cervical cancer in low resource settings with visual inspection, HPV, and the Pap smear. Int J Gynaecol Obstet 2001; 72: 47-53.

12. Kuhn L, Denny L, Pollack A, et al. Human papillomavirus DNA testing for cervical cancer screening in low-resource settings. J Natl Cancer Inst 2000; 92: 818-25.

13. Sarian LO, Derchain SF, Naud P, et al. Evaluation of visual inspection with acetic acid (VIA), Lugol’s iodine (VILI), cervical cytology and HPV testing as cervical screening tools in Latin America. This report refers to partial results from the LAMS (Latin AMerican Screening) study. J Med Screen 2005; 12: 142-9.

14. Schneider A, Hoyer H, Lotz B, et al. Screening for high-grade cervical intra-epithelial neoplasia and cancer by testing for high-risk HPV, routine cytology or colposcopy. Int J Cancer 2000; 89: 529-34.

15. Cuzick J, Beverley E, Ho L, et al. HPV testing in primary screening of older women. Br J Cancer 1999; 81:
554-8.

16. Sankaranarayanan R, Nene BM, Dinshaw KA, et al. A cluster randomized controlled trial of visual, cytology and human papillomavirus screening for cancer of the cervix in rural India. Int J Cancer 2005; 116: 617-23.

17. Kotaniemi-Talonen L, Nieminen P, Anttila A, Hakama M. Routine cervical screening with primary HPV testing and cytology triage protocol in a randomised setting. Br J Cancer 2005; 93: 862-7.

18. Ronco G, Segnan N, Giorgi-Rossi P, et al. Human papillomavirus testing and liquid-based cytology: results at recruitment from the new technologies for cervical cancer randomized controlled trial. J Natl Cancer Inst 2006a; 98: 765-74.

19. Ronco G, Giorgi-Rossi P, Carozzi F, et al. Human papillomavirus testing and liquid-based cytology in primary screening of women younger than 35 years: results at recruitment for a randomised controlled trial. Lancet Oncol 2006; 7: 547-55.

20. Bulkmans NW, Berkhof J, Rozendaal L, et al. Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial. Lancet 2007; 370: 1764-72.

21. Mayrand MH, Duarte-Franco E, Rodrigues I, et al. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med 2007; 357: 1579-88.

22. Naucler P, Ryd W, Tornberg S, et al. Human papillo-mavirus and Papanicolaou tests to screen for cervical cancer. N Engl J Med 2007; 357: 1589-97.

23. Denny L, Kuhn L, De SM, et al. Screen-and-treat approaches for cervical cancer prevention in low-resource settings: a randomized controlled trial. JAMA 2005; 294: 2173-81.

24. Sherman ME, Lorincz AT, Scott DR, et al. Baseline cytology, human papillomavirus testing, and risk for cervical neoplasia: a 10-year cohort analysis. J Natl Cancer Inst 2003; 95: 46-52.

25. Kjaer SK, van den Brule AJ, Paull G, et al. Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study. BMJ 2002; 325: 572.

26. Clavel C, Cucherousset J, Lorenzato M, et al. Negative human papillomavirus testing in normal smears selects a population at low risk for developing high-grade cervical lesions. Br J Cancer 2004; 90: 1803-8.

27. Bulkmans NW, Berkhof J, Bulk S, et al. High-risk HPV type-specific clearance rates in cervical screening. Br J Cancer 2007; 96: 1419-24.

28. Schlecht NF, Platt RW, Duarte-Franco E, et al. Human papillomavirus infection and time to progression and regression of cervical intraepithelial neoplasia. J Natl Cancer Inst 2003; 95: 1336-43.

29. Ronco G, Cuzick J, Segnan N, et al. HPV triage for low grade (L-SIL) cytology is appropriate for women over 35 in mass cervical cancer screening using liquid based cytology. Eur J Cancer 2007; 43: 476-80.

30. Cuzick J, Szarewski A, Cubie H, et al. Management of women who test positive for high-risk types of human papillomavirus: the HART study. Lancet 2003; 362: 1871-6.

31. Castle PE, Solomon D, Schiffman M, Wheeler CM. Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities. J Natl Cancer Inst 2005; 97: 1066-71.

32. Khan MJ, Castle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 2005; 97: 1072-9.

33. Ogilvie GS, Patrick DM, Schulzer M, et al. Diagnostic accuracy of self collected vaginal specimens for human papillomavirus compared to clinician collected human papillomavirus specimens: a meta-analysis. Sex Transm Infect 2005; 81: 207-12.

34. Nobbenhuis MA, Helmerhorst TJ, van den Brule AJ, et al. Primary screening for high risk HPV by home obtained cervicovaginal lavage is an alternative screening tool for unscreened women. J Clin Pathol 2002; 55: 435-9.

35. Brink AA, Meijer CJ, Wiegerinck MA, et al. High concordance of results of testing for human papillomavirus in cervicovaginal samples collected by two methods, with comparison of a novel self-sampling device to a conventional endocervical brush. J Clin Microbiol 2006; 44: 2518-23.

36. Szarewski A, Cadman L, Mallett S, et al. Human papillomavirus testing by self-sampling: assessment of accuracy in an unsupervised clinical setting. J Med Screen 2007; 14: 34-42.

37. IARC (2004) IARC Handbooks on Cancer Prevention. Volume 10. Cervix Cancer Screening. IARC Press: Lyon.

Translocation based diagnosis of bone and soft tissue sarcoma

Selected Abstracts
1. Diagnostic gold standard for soft tissue tumours: morphology or molecular genetics?

Pfeifer JD, Hill DA, O’Sullivan MJ, Dehner LP. Histopathology 2000; 37: 485-500.
The recognition of recurrent genetic alterations in specific tumour types has provided the basis for the reclassification of certain soft tissue neoplasms, and molecular analysis of patient material has the potential to provide both diagnostic and prognostic information. In this review, we evaluate the role of molecular genetic testing as the prospective ‘gold standard’ for sarcoma diagnosis. Molecular genetic testing, as with every new method, promises to improve accuracy and to be more sensitive and less subjective, claims that have been made previously by histochemistry, electron microscopy and immunohistochemistry. Technical limitations in molecular assays, as well as more general specificity issues, decrease the clinical usefulness of molecular pathological testing significantly and suggest that, at present, molecular evaluation is best considered an ancillary technique that neither supersedes other ancillary techniques nor eclipses traditional pathological examination.

2. Practical application of molecular genetic testing as an aid to the surgical pathologic diagnosis of sarcomas: a prospective study.

Hill DA, O’Sullivan MJ, Zhu X, et al. Am J Surg Pathol 2002; 26: 965-77.
The strong correlation of specific reciprocal translocations with individual tumor types and the demonstration that polymerase chain reaction (PCR)-based methods can detect translocations in tissue samples have stimulated interest in the role of molecular genetic testing in diagnostic surgical pathology. To evaluate the clinical utility of PCR-based molecular analysis of soft tissue neoplasms in routine surgical pathology, 131 consecutive soft tissue tumors submitted for molecular genetic testing at a tertiary care teaching hospital were prospectively analyzed over a 36-month period. RT-PCR was used to test tumor RNA for fusion transcripts characteristic of malignant round cell tumors (including Ewing sarcoma/primitive neuroectodermal tumor, desmoplastic small round cell tumor, and alveolar rhabdomyosarcoma), spindle cell tumors (including synovial sarcoma and congenital fibrosarcoma), and fatty tumors (myxoid liposarcoma). DNA sequence analysis was used to confirm the identity of all PCR products, and the PCR results were compared with the histopathologic diagnosis. We found that sufficient RNA for RT-PCR-based testing was recovered from 96% of the 131 cases and the percentage of tumors that tested positive for the associated characteristic fusion transcript was in general agreement with those reported in the literature. DNA sequence analysis of PCR products identified both variant transcripts and spurious PCR products, underscoring the value of product confirmation steps when testing formalin-fixed, paraffin-embedded tissue. Only in rare cases did testing yield a genetic result that was discordant with the histopathologic diagnosis. We conclude that PCR-based testing is a useful adjunct for the diagnosis of malignant small round cell tumors, spindle cell tumors, and other miscellaneous neoplasms in routine surgical pathology practice.

3. Use of reverse transcriptase polymerase chain reaction for diagnosis and staging of alveolar rhabdomyosarcoma, Ewing sarcoma family of tumors, and desmoplastic small round cell tumor.

Athale UH, Shurtleff SA, Jenkins JJ, et al. J Pediatr Hematol Oncol 2001; 23: 99-104.

PURPOSE: To compare the use of reverse transcriptase polymerase chain reaction (RT-PCR) with that of morphology-based methods for diagnosis, staging, and detection of metastatic disease in pediatric alveolar rhabdomyosarcoma (ARMS), Ewing sarcoma family of tumors (ESFT), and desmoplastic small round cell tumors (DSRCT).

MATERIALS AND METHODS: RT-PCR assays for the EWS-FLII, EWS-ERG, PAX3-FKHR, PAX7-FKHR, and EWS-WTI fusion transcripts were performed on RNA extracted from the primary tumor tissue, bone marrow, and body fluids obtained at initial presentation and relapse. Molecular findings were compared with original histologic diagnoses and results of staging procedures.

RESULTS: Eighty-eight samples from 47 patients with ARMS (n = 13), ESFT (n = 31), or DSRCT (n = 3) were analyzed. The detection rate of metastatic disease was significantly higher with RT-PCR (95%) as compared with the morphologic methods (70%) for the three pediatric sarcomas studied. In primary tumors with characteristic fusion transcript, RT-PCR was positive in all cases with morphologic evidence of metastatic disease. Moreover, in six patients (3 with ARMS, 2 with DSRCT, and 1 with ESFT) with metastatic disease, micrometastases in bone marrow (4) and other sites (2) were detected by RT-PCR alone. Importantly, none of the patients with localized disease diagnosed had micrometastases detected by RT-PCR in bone marrow.

CONCLUSIONS: The high sensitivity and specificity of RT-PCR for the characteristic fusion transcripts of pediatric sarcomas make it an ideal method to aid in the routine staging of these patients. In addition, the 100% sensitivity of RT-PCR in detection of micrometastasis makes it useful for follow-up and detection of minimal residual disease. However, the clinical significance of molecularly-detectable disease remains unknown. Further studies should aim to elucidate the therapeutic and prognostic implications of micrometastases detected by RT-PCR alone.

4. A practical approach to the clinical diagnosis of Ewing’s sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrange-ment using new fluorescence in situ hybridi-sation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue.

Yamaguchi U, Hasegawa T, Morimoto Y, et al. J Clin Pathol 2005; 58:1051-6.

BACKGROUND: Over 90% of Ewing’s sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. AIMS: To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS.

METHODS: Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. RESULTS: One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls.

CONCLUSIONS: Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

5. Molecular diagnosis of Ewing sarcoma/ primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT-PCR in malignant round cell tumors.

Bridge RS, Rajaram V, Dehner LP, Pfeifer JD, Perry A. Mod Pathol 2006; 19:1-8.
Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET) is a diagnostically challenging malignant round cell tumor with signature translocations involving the EWS gene. These translocations are detectable with both reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded tissue. However, RT-PCR is less sensitive in formalin-fixed paraffin-embedded than frozen tissue. Similarly, commercial FISH probes have recently become available, but have yet to be rigorously tested in the clinical setting. Therefore, we have compared RT-PCR with FISH using ‘home brew’ fusion probes for Ewing sarcoma (EWS)-FLI1 and a commercial EWS break apart probe set in 67 archival round cell tumors, including 27 EWS/ PNETs. Sensitivities and specificities for both FISH assays were 91 and 100%, respectively, whereas RT-PCR had a sensitivity of 54% and a specificity of 85%. The break apart strategy was easier to interpret than probe fusion approach. We conclude that FISH is a more sensitive and reliable ancillary technique than RT-PCR for the diagnosis of EWS/PNET in formalin-fixed paraffin-embedded tissue, although the latter provides additional information regarding fusion transcript subtype and prognosis. The commercial break apart probe set is both readily available and easy to interpret, making it particularly attractive. Nonetheless, complex round cell tumors often benefit from molecular testing with multiple methods.

6. Diagnosis of the small round blue cell tumors using multiplex polymerase chain reaction.

Chen QR, Vansant G, Oades K, et al. J Mol Diagn 2007; 9: 80-8.
The small round blue cell tumors of childhood, which include neuroblastoma, rhabdomyosarcoma, non-Hodgkin’s lymphoma, and the Ewing’s family of tumors, are so called because of their similar appearance on routine histology. Using cDNA microarray gene expression profiles and artificial neural networks (ANNs), we previously identified 93 genes capable of diagnosing these cancers. Using a subset of these, together with some additional genes (total 39), we developed a multiplex polymerase chain reaction (PCR) assay to diagnose these cancer types. Blinded testing of 96 new samples (26 Ewing’s family of tumors, 29 rhabdomyosarcomas, 24 neuroblastomas, and 17 lymphomas) using ANNs in a complete leave-one-out analysis demonstrated that all except one sample were accurately diagnosed as their respective category. Moreover, using an ANN-based gene minimization strategy in a separate analysis, we found that the top 31 genes could correctly diagnose all 96 tumors. Our results suggest that this molecular test based on a multiplex PCR reaction may assist the physician in the rapid confirmation of the diagnosis of these cancers.

7. Differentiating Ewing’s sarcoma from other round blue cell tumors using a RT-PCR translocation panel on formalin-fixed paraffin-embedded tissues.

Lewis TB, Coffin CM, Bernard PS. Mod Pathol 2007; 20:397-404.
Ewing’s sarcoma is a common malignancy of bone and soft tissue that occurs most often in children and young adults. Differentiating Ewing’s sarcoma from other round blue cell tumors can be a diagnostic challenge because of their similarity in histology and clinical presentation. Thus, ancillary molecular tests for detecting disease-defining translocations are important for confirming the diagnosis. We analyzed 65 round blue cell tumors, including 53 Ewing’s sarcoma samples from 50 unique cases. Samples were processed for RNA from archived formalin-fixed paraffin-embedded tissue blocks. Real-time RT-PCR assays specific for Ewing’s sarcoma (EWS-FLI1, EWS-ERG, EWS-ETV1, EWS-ETV4, and EWS-FEV), synovial sarcoma (SYT-SSX1 and SYT-SSX2), and rhabdomyosarcoma (PAX3-FKHR and PAX7-FKHR) were tested across the samples. The translocation panel had a sensitivity of 81% (43 out of 53 samples) for diagnosing Ewing’s sarcoma when using the histological criteria as the ‘gold’ standard. None of the Ewing’s specific translocations were found in the non-Ewing’s samples (100% specificity). Of the 43 samples with translocations detected, 26 (60%) had an EWS-FLI1 type 1 translocation, 13 (30%) had an EWS-FLI1 type 2 translocation, 3 (7%) had an EWS-ERG translocation, 1 had an EWS-ETV1 translocation, and 1 sample had both an EWS-FLI1 type 1 and type 2 translocation. Our real-time RT-PCR assay for detecting sarcoma translocations has high sensitivity and specificity for Ewing’s sarcoma and has clinical utility in differentiating small round blue cell tumors in the clinical lab.

8. Undifferentiated Small Round Cell Sarcomas with Rare EWS Gene Fusions- Identification of a Novel EWS-SP3 Fusion and of Additional Cases with the EWS-ETV1 and EWS-FEV Fusions

Wang L, Bhargava R, Zheng T, et al. J Mol Diagn 2007; 9: 498-509.

Ewing family tumors (EFTs) are prototypical primitive small round blue cell sarcomas arising in bone or extraskeletal soft tissues in children or adolescents. EFTs show fusions of EWS with a gene of the ETS family of transcription factors, either EWS-FLI1 (90 to 95%) or EWS-ERG (5 to 10%). Rare cases with fusions of EWS to other ETS family genes, such as ETV1, E1AF, and FEV, have been identified, but their clinicopathological similarity to classic EFTs remains unclear. We report four new cases of EFT-like tumors with rare EWS fusions, including two with EWS-ETV1, one with EWS-FEV, and a fourth case in which we cloned a novel EWS-SP3 fusion, the first known cancer gene fusion involving a gene of the Sp zinc finger family. Analysis of these three new cases along with data on nine previously reported cases with fusions of EWS to ETV1, E1AF, or FEV suggest a strong predilection for extraskeletal primary sites. EFT-like cases with fusions of EWS to non-ETS translocation partners are also uncommon but involve the same amino-terminal portion of EWS, which in our novel EWS-SP3 fusion is joined to the SP3 zinc-finger DNA-binding domain. As these data further support, these types of EWS fusions are associated with primitive extraskeletal small round cell sarcomas of uncertain lineage arising mainly in the pediatric population.

9. Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: analysis of conventional RT-PCR, qRT-PCR and dual color FISH as diagnostic tools for synovial sarcoma.

Amary MF, Berisha F, Bernardi Fdel C, et al. Mod Pathol 2007; 20: 482-96.
Synovial Sarcoma consistently harbors t(X;18) resulting in SS18-SSX1, SS18-SSX2 and rarely SS18-SSX4 fusion transcripts. Of 328 cases included in our study, synovial sarcoma was either the primary diagnosis or was very high in the differential diagnosis in 134 cases: of these, amplifiable cDNA was obtained from 131. SS18-SSX fusion products were found in 126 (96%) cases (74 SS18-SSX1, 52 SS18-SSX2), using quantitative and 120 by conventional reverse transcriptase-polymerase chain reaction (RT-PCR). One hundred and one cases in a tissue microarray, analyzed by fluorescence in situ hybridization (FISH), revealed that 87 (86%) showed SS18 rearrangement: four RT-PCR positive cases, reported as negative for FISH, showed loss of one spectrum green signal, and 15 cases had multiple copies of the SS18 gene: both findings are potentially problematic when interpreting results. One of three cases, not analyzed by RT-PCR reaction owing to poor quality RNA, was positive by FISH. SS18-SSX1 was present in 56 monophasic and 18 biphasic synovial sarcoma: SS18-SSX2 was detected in 41 monophasic and 11 biphasic synovial sarcoma. Poorly differentiated areas were identified in 44 cases (31%). There was no statistically significant association between biphasic, monophasic and fusion type. Five cases were negative for SS18 rearrangement by all methods, three of which were pleural-sited neoplasms. Following clinical input, a diagnosis of mesothelioma was favored in one case, a sarcoma, not otherwise specified in another and a solitary fibrous tumor in the third case. The possibility of a malignant peripheral nerve sheath tumor could not be excluded in the other two cases. We concluded that the employment of a combination of molecular approaches is a powerful aid to diagnosing synovial sarcoma giving at least 96% sensitivity and 100% specificity but results must be interpreted in the light of other modalities such as clinical findings and immunohistochemical data.

10. Prognostic implication of SYT-SSX fusion type in synovial sarcoma: A multi-institutional retrospective analysis in Japan.

Takenaka S, Ueda T, Naka N, et al. Oncol Rep 2008; 19: 467-76.

The prognostic implication of SYT-SSX fusion type in synovial sarcomas is still controversial. The aim of this study is to clarify the prognostic impact of fusion type, in association with other clinical factors, in patients with synovial sarcoma in Japan. Data on 108 SYT-SSX fusion transcript-positive patients with synovial sarcoma, treated in 11 tertiary referral cancer centers in Japan, were retrospectively analyzed. The following parameters were examined for their potential prognostic impact: SYT-SSX fusion type, patient age at presentation, sex, primary tumor location, tumor size, histological subtype, histological grade, treatment modalities and disease stage at presentation. Among the patients with localized disease at presentation, 5-year overall survival (OS) for SYT-SSX1 and -2 subgroups were 84.4 and 74.9%, respectively (P=0.244). Five-year metastasis-free survival (MFS) rates were 67.8% for SYT-SSX1 and 68.5% for SYT-SSX2 (P=0.949). Univariate survival analyses for 91 patients with localized disease at presentation showed that tumor size was the only significant prognostic factor for OS (P=0.0033) and MFS (P=0.0029) and the histological grade was marginally significant for MFS (P=0.0785), whereas the SYT-SSX fusion type and other variables were not. Multivariate survival analyses further indicated that tumor size was the most significant independent prognostic factor for OS and MFS and the histological grade was also significant for MFS. In conclusion, the SYT-SSX fusion type is not a significant prognostic factor unlike tumor size, followed by histological grade for patients with localized synovial sarcoma in Japan.

11. Malignant peripheral nerve sheath tumors with t (X;18). A pathologic and molecular genetic study.

O’Sullivan MJ, Kyriakos M, Zhu X, et al. Mod Pathol 2000; 13: 1253-63.
Spindle cell sarcomas often present the surgical pathologist with a considerable diagnostic challenge. Malignant peripheral nerve sheath tumor, leiomyosarcoma, fibrosarcoma, and monophasic synovial sarcoma may all appear similar histologically. The application of ancillary diagnostic modalities, such as immunohistochemistry and electron microscopy, may be helpful in the differentiation of these tumors, but in cases in which these adjunctive techniques fail to demonstrate any more definitive evidence of differentiation, tumor categorization may remain difficult. Cytogenetic and molecular genetic characterization of tumors has provided the basis for the application of molecular assays as the newest components of the diagnostic armamentarium. Because the chromosomal translocation t(X;18) has been observed repeatedly in many synovial sarcomas, it has been heralded as he specificity of this translocation for the diagnosis of synovial sarcoma, RNA extracted from formalin-fixed, paraffin-embedded tissue from a variety of soft tissue and spindle cell tumors was evaluated for the presence of t(X;18) by reverse transcriptase-polymerase chain reaction. Although 85% of the synovial sarcomas studied demonstrated t(X;18), 75% of the malignant peripheral nerve sheath tumors in our cohort also demonstrated this translocation. We conclude that the translocation t(X;18) is not specific to synovial sarcoma and discuss the implications of the demonstration of t(X;18) in a majority of malignant peripheral nerve sheath tumors.

Molecular classification of breast cancer: Ready for clinical application?

Tanuja Shet

Introduction

Breast cancer incidence is rising in the developing countries and assuming significant proportions. With targeted treatment being made available in the adjuvant setting for treatment of breast cancer, the clinician is often faced with a problem of choosing the best possible regimen. Though conventional prognostic factors like nodal status etc. are available for valuable decision making, there is always a small group of patients especially in the node negative category where crucial therapeutic dilemmas arise. Hence there is a need to add relevant biomarkers for appropriate patient selection. The widely expanding realm of gene profiling offers such opportunities and is being envisaged as the key for future therapeutic decision making in breast cancer.

With availability of these techniques in a developing country like India, there will be a section of patients who may wish to opt for these advances in technology. Before this, it would be worthwhile checking whether the world is ready to apply the vast data from these studies for routine

What is the molecular classification of breast cancerhe first landmark in gene profiling analysis in breast was to highlight that there are four types of breast cancer: luminal-like, basal-like, HER 2 positive and normal-like (1). Soon information regarding more genes emerged and the luminal subgroup was further divided into luminal - A, B, C. (2, 3).

Molecular classification vs histological /Immunohistochemical classification of breast cancer

Did molecular classification tell us something new? How do these studies translate into routine morphology? Before the advent of gene profiling, pathologists had recorded what was then labeled as poorly differentiated duct carcinoma with myoepithelial differentiation with a lower incidence of node metastasis but with propensity to involve brain and lungs more frequently (4). On the other hand, it was always intriguing to deal with tumors like medullary carcinomas which did well despite their high nuclear grade.

While molecular studies did not tell us something new altogether, they did offer us explanation for the subtype of basal like cancers regarding variable behavior of the myoepithelial rich carcinomas and the medullary carcinomas. In continuation to the molecular studies, it is now confirmed that basal-like cancers form the ‘triple negative’ cancers, i.e. ER–/PR–/HER2– cancers while luminal-like tumors are ER+ tumors. Studies also compared the morphology of these tumors with gene microarray data and found that most of these would fit into the category of atypical medullary carcinoma with circumscribed borders, nuclear grade III, areas of geographic or central necrosis, a central scar and negative nodes (5).

However, for this molecular classification to be translated to diagnostic and clinical use there is no concordance between immunohistochemical markers and gene microarray data (6). Though basal like cancers express CK5/6 and/or CK14, these cytokeratins are not expressed in all tumors classified as basal-like by gene microarray.

Molecular classification vs traditional prognostic factors

Histological type, grade, tumor size, lymph-node involvement, estrogen receptor (ER) and HER2 receptor status all influence prognosis and probability of response to systemic therapies, but they do not fully capture the varied clinical course of breast cancer (3). All of these have been incorporated into prognostic models like Nottingham prognostic index (NPI) and Adjuvant-Online. However, within the ER positive group still there are patients who do not benefit from adjuvant therapy equally well and the molecular classification has highlighted that this is related to the subtype of luminal cells and association with other sets of genes.

For application to a clinically relevant prognostic marker, this molecular classification needs to be translated into or incorporated into existing predictors / biomarkers. Though such studies are still emerging, one study reported a high level of correlation between molecular sub-classification and age, ER expression, tumor grade and p53 mutations. The basal-like tumors are more likely to affect younger women and are ER-negative, high grade, with a p53 mutation in 65% of patients (3, 7).

The 70 gene prognostic signature proposed by van’t Veer et al (8) was associated with some of the traditional prognostic markers like the age, the histological grade of the tumor, and ER status, but no correlation was found between the sordid predictor in breast cancer viz. nodal status and the poor prognostic signature (9).

Initial results of the TRANSBIG study documented that the 70 gene signature outperformed both NPI & St Gallen criteria (10) in predicting time to distant metastasis. However, studies have shown that the NPI was similar to the 70-gene predictor in prognostication of breast cancer (11, 12). When analyzing subgroups separately, according to the ER status both approaches could predict clinical outcome more easily for the ER-positive than for the ER-negative cohort. It will take time for microarray technology to be used worldwide, particularly due to the costs, the lack of standards and expertise required for analysis of data (12). Quintessentially, microarrays still hold the promise to add information and improve rather than replace current prognostic and treatment predictive tools (3).

Impact of molecular classification on management of patients

a. Prognostication of patients

It is increasingly clear that molecular characteristics determine the tumor interactions and its prognosis. The major break through came in the form of the 70 gene signature proposed by the Netherlands group on gene microarray in breast cancer (8). This 70 gene model divided tumors into those with good prognosis signature (GPS) and those with poor prognostic signatures (PPS) and it significantly predicted disease free survival (8).

This was subsequently validated in 295 patients younger than 50 years with 94.5% 10 year survival in patients with GPS & 54.6% in PPS. PPS was strongest predictor of likelihood of distant metastasis (13). Soon the 70 gene model was narrowed down to a 21 gene signature as a predictive as well as prognostic factor (14).

b. Predicting response to adjuvant therapy

The prognosis and chemotherapy sensitivity associated with the different molecular subgroups as per the molecular classification also appears to be different. Luminal-type cancers tend to have the most favorable long-term survival, whereas the basal-like and HER2 positive tumors are most sensitive to chemotherapy but have the worst overall prognosis (2, 3, 15).

Various studies have prospectively evaluated gene profiling in assessment of response to docetaxel in locally advanced breast cancer. Univariate analysis revealed 86 genes that were associated with response to sequential doxorubicin and paclitaxel based chemotherapy (16). Multigene predictors of preoperative response to single-agent paclitaxel, doxorubicin plus cyclophosphamide, and epirubicin plus cyclophosphamide have also been reported (17). These results represent exciting but preliminary data indicating that gene signatures might identify patients sensitive to a given chemotherapy regimen.

The Oncotype DX® RT-PCR-based assay represents another diagnostic advance for ER-positive breast cancers and is also the first multigene diagnostic assay that has become commercially available in the US (18). Studies have shown that tumors with high Oncotype DX® Recurrence Score® derive greater benefit from adjuvant CMF chemotherapy (18, 19).

c. Familial breast cancers

Microarrays have also been successful in demystifying familial cancers and in identifying distinct subgroups among familial breast cancers (11).

Tumors derived from individuals with BRCA1 or BRCA2 mutations, each display characteristic gene expression profile which opens the possibility for novel screening strategies (e.g. a BRCA1 / 2 diagnostic gene chip). The pathway for the heterogeneous group of non-BRCA1 & BRCA2 ( BRCAx) tumors is also opened up for further investigations (20,21).

Problems with use of molecular classification in routine use

Problems in the technique of gene microarray

a. Use of Pooled reference sample (11): Expression profiles derived from printed cDNA and oligonucleotide (25–80-mer) microarrays, are based on the comparison between a sample of interest and a common reference sample. Hence, results are ratio-based and the output depends on the reference sample used.

b. Data analysis: The major problem with gene microarray studies is that thousands of genes are evaluated in a set wise manner; consequently data that emerge are extremely complex and require equally complex methods to analyze. To evaluate such complex data two methods are used viz: supervised and unsupervised clustering method. Supervised analysis requires prior knowledge, biological or clinical, to discriminate genes distributed between sample groups with statistical probability (11). Unsupervised methods are used to reveal the underlying variance structure of a dataset, typically utilizing pattern-recognition algorithms to define groups of samples with similar global molecular profiles and thus minimize a priori assumptions about the data. While unsupervised analysis are effective in classifying a large number of genes, they are less effective in identifying altered profiles of small numbers of discriminatory genes. In supervised classification analysis, data overfitting or non-generalizability is a typical problem in underpowered study designs with too few training samples and complex classification models (11).

c. The issue of nonmatching gene signature: A comparison of the 76-gene predictor by Wang et al (22) and 70-gene predictor by van’t Veer et al (8) revealed only a three gene overlap. This is mainly related to the use of different gene chips and reference samples.

d. Classifier accuracy: Michiels et al (23) using a multiple random sampling approach showed that of seven molecular profiles with proposed high classifier accuracy, five really should not have had classifier accuracy better than chance, if the training and validation had been performed truly without any bias. The other two had only modest classifier accuracy. A molecular profile for clinical use should be standardized, developed in an unbiased manner, and validated to have high classifier accuracy.

d. Errors in sample processing: mRNA is not a very stable molecule, and differences in the sampling and processing of the specimens could introduce considerable noise (24). This is worse than the noise introduced by the same factors in the discovery phase, in which highly expert teams are involved (24).

e. Applicability to paraffin embedded tissue: For use in routine practice, gene microarrays, and classification have to be translated to application in paraffin material. Though promising efforts are on in that direction, results are eagerly awaited (16).

Are we ready to use them in clinical practice? (24)

For molecular classification to be used in routine clinical practice the issues that need to be solved are as follows:

• Assay standardization and validation
• Demonstration of diagnostic, prognostic, and predictive performance as opposed to robust traditional factors
• Nonselective and transparent accumulation of evidence
• Demonstration of clinical effect (efficacy)

Cost-effectiveness: While in the long run, sparing patients of unnecessary therapy will be more cost effective, the present exorbitant costs need to be evaluated critically.

Looking at these issues critically, the answer to the above question is clear - In the current state we cannot use the molecular classification routinely. Nevertheless it holds great promise and we need to wait for the phase III trial results.

Efforts to standardize technology related issues

The initial enthusiasm for the application of microarray technology was tempered when the microarray quality control (MAQC) project reported contradictory results on the analysis of the same RNA samples hybridized on different microarray platforms (25). Finally, technologies are coming together sophisticated microarray databases, analysis tools, and pathway, and gene regulatory network software are now available. The minimum information about a microarray experiment (MIAME) guidelines also serves this purpose (24).

The MAQC project involving 137 participants from 51 academic institutions and industrial partners is trying to generate useful best practice protocols for microarray experiments.

Clinical trials based on gene microarray and molecular classification of breast cancer and their application to routine use

The immediate need to validate gene microarray data has led way to several studies. Two clinical trials are underway to asses the clinical value of using the Oncotype DX® recurrence score and the MammaPrint® (Agendia, Amsterdam, The Netherlands) prognostic signature in decision-making, and therefore could be considered phase III marker validation trials. The North American TAILORx study will randomize patients with intermediate recurrence score values to receive hormonal therapy alone or hormonal therapy plus chemotherapy and aims to find out whether adjuvant chemotherapy improves survival in this subset of patients.

The European MINDACT (Microarray In Node negative Disease may Avoid Chemo Therapy) trial will directly compare a gene-signature-based decision with an Adjuvant-Online-based decision and compare prognostic prediction results. The MINDACT trial is looking at whether we could avoid giving chemotherapy in node negative patients based on the gene signature. The TRANSBIG prospective trial is seeking to retrospectively validate 70 gene signatures from different subcontinents and countries. The preliminary results of this trial have been promising. The number of available profiles may escalate geometrically in the future and a major drawback from these large, randomized prospective trials is that their results will not be available for many years. How can one evaluate the use of rapidly evolving technologies in a shorter time frame, and how can one accelerate their clinical availability? (19).

The future

The immediate need is to achieve uniformity in microarray technology across all platforms. Once the stabilized results are standardized, the need will be to develop integrative models that combine clinical and complex multilevel molecular factors, such as gene expression patterns, traditional clinico-pathological risk factors and treatment information. Such a model should be tested against the impact of the molecular profile plus classic routine risk factors versus a model with classic risk factors alone in patient management.

References

1. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 406: 747–52.

2. Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinicalmplications. Proc Natl Acad Sci USA 2001; 98: 10869–74.

3. André F, Domont J, Delaloge S. What can breast cancer molecular sub-classification add to conventional diagnostic tools? Ann Oncol 2007; 18 (Suppl 9) :ix 33-6.

4. Tsuda H, Takarabe T, Hasegawa T, Murata T, Hirohashi S. Myoepithelial differentiation in high-grade invasive ductal carcinomas with large central acellular zones. Hum Pathol 1999; 30:1134-9.

5. Fulford LG, Easton DF, Reis-Filho JS et al. Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast. Histopathology 2006 ; 49: 22-34

6. Lerma E, Peiro G, Ramón T, et al. Immunohistochemical heterogeneity of breast carcinomas negative for estrogen receptors, progesterone receptors and Her2/neu (basal-like breast carcinomas). Mod Pathol 2007; 20:1200-7

7. Calza S, Hall P, Auer G, et al. Intrinsic molecular signature of breast cancer in a population-based cohort of 412 patients. Breast Cancer Res 2006; 8: R34.

8. van’t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002; 415: 530-6.

9. Andre F, Conforti R, Tomasic G. Predictive values of estrogen receptor (ER) expression and molecular subclassification for the benefit of adjuvant anthracycline-based chemotherapy (CT) in two randomized trials. San Antonio Breast Cancer Symposium 2006; 1028A.

10. Goldhirsch A, Glick JH, Gelber RD, Coates AS, Senn HJ International consensus panel on the treatment of primary breast cancer. Seventh international conference on adjuvant therapy of primary breast cancer. J Clin Oncol 2001; 19: 3817–27.

11. Gruvberger-Saal SK, Cunliffe HE, Carr KM, Hedenfalk IA. Microarrays in breast cancer research and clinical practice—the future lies ahead. Endocr Relat Cancer. 2006; 13:1017-31.

12. Eden P, Ritz C, Rose C, Ferno M, Peterson C. ‘Good Old’ clinical markers have similar power in breast cancer prognosis as microarray gene expression profilers. Eur J Cancer 2004; 40: 1837–41.

13. van de Vijver MJ, He Y D, van’t Veer LJ, et al. A gene expression signature as a predictor of survival in breast cancer. N Engl J Med 2003; 347: 1999-2009

14. Korkola JE, Blaveri E, DeVries S, et al. Identification of a robust gene signature that predicts breast cancer outcome in independent data sets. BMC Cancer 2007; 7: 61.

15. Rouzier R, Perou CM, Symmans WF, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 2005; 11: 5678–85.

16. Gianni L, Zambetti M, Clark K, et al. Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol 2005; 23: 7265–77.expression profile associated with response to doxorubicin-based therapy in breast cancer. Clin Cancer Res 2005; 11: 7434–43.

18. Paik S, Shak S, Tang G, et al. A multi gene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 2004; 351: 2817–6.

19. Sotiriou C, Piccart MJ. Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care? Nat Rev Cancer 2007; 7: 545-53.

20. Hedenfalk I, Duggan D, Chen Y, et al. Gene-expression profiles in hereditary breast cancer. N Engl J Med 2001; 344: 539–48.

21. Hedenfalk I, Ringner M, Ben-Dor A, et al. Molecular classification of familial non- BRCA1/BRCA2 breast cancer. PNAS 2003; 100: 2532–7.

22. Wang Y, Klijn JG, ZhangY, Sieuwerts AM, et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 2005; 365: 671–9.

23. Michiels S, Koscielny S, Hill C. Prediction of cancer outcome with microarrays: A multiple random validation strategy. Lancet 2005; 365: 488–92.

24. Ioannidis JP. Is molecular profiling ready for use in clinical decision making? Oncologist 2007;12: 301-11.

25. MAQC Consortium, Shi L, Reid LH, Jones WD, et al. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnology 2006; 24: 1151–61.

Circulating tumour cells as predictive and prognostic marker of breast cancer

Rajendra Badwe

Tumour cells outside the primary organ augur ominous thoughts in any patient with a solid tumour. This has been most investigated in breast cancer patients. However, for a scientist it throws a lot more questions than answers to clinical problems. Following questions haunt a clinician / scientist:

Where to look for tumour cells?
When to look?
How to look?
What to look for?
What to make of the outcome?

Where to look for tumour cells?
Tumour cells can be looked for in peripheral blood or in bone marrow aspirates or bone biopsy. The peripheral blood for circulating tumour cells (CTCs) could be collected from effluent vein (subclavian vein or superior vena cava) before it passes through the first filter (pulmonary circuit) or from a peripheral vein after it passes through two filters (pulmonary and peripheral tissue bed). The yield is likely to be better if one is to look for cells in bone marrow where cells once filtered would remain trapped and hence could be termed disseminated tumour cells (DTCs). It must be understood that the number of cells in peripheral blood are much fewer compared to those in the bone marrow aspirate.

When to look?
The answer to this question is determined by the research/clinical question one poses. The timing of testing is before or after adjuvant systemic therapy if one is looking for MRD and prognostication in early breast cancer. As against that, after treatment of advanced disease one could run the test for MRD for prognostication or early detection of a relapse. In a research setting of surgical dissemination, sample could be drawn before and after surgical removal of the primary tumour.

How to look?
Concentration methods (ficoll hypaque followed by immuno-magnetic separation) and detection methods (antibody based or PCR based) (1).

What to look for?
There could be two end points. The first more commonly used is quantitative or semi-quantitative estimate of number of cells per unit of peripheral blood or bone marrow aspirate and second a qualitative estimate of what are the characteristics of the CTCs or DTCs. The latter could allow one to draw the similarities or contrast between CTCs / DTCs vs the primary tumour cells. It would also allow one to assess the ‘stemness’ of CTCs/DTCs.

What to make of the outcome?
For a clinician prognostication remains the most important application of these results. There are now very reliable long term results of DTCs available making it an attractive independent determinant of prognosis in early breast cancer (2). A similar test in advanced disease has allowed US FDA to approve Veridex system for peripheral blood for prognostication (3). There are plenty of PCR based studies that have shown reliable prognostic discrimination on disease free and overall survival (3, 4).

In a research setting depending upon the type of antigens expressed on the surface of DTCs/CTCs one could plan systemic therapy. If surgical dissemination is confirmed then one could use that as a surrogate marker for prognostication as well as to test the effect of its modulators. This could be established as a rapid throughput in drug testing with effect on MRD as a surrogate marker.

References
1. Ring A, Smith IE, Dowsett M. Circulating tumour cells in breast cancer. Lancet Oncol 2004; 5: 79-88.
2. Braun S, Vogl FD, Schneitter A, et al. Disseminated tumor cells: are they ready for clinical use? Breast 2007; 16 Suppl 2: S51-4.
3. Riethdorf S, Fritsche H, Müller V, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res 2007; 13: 920-8.
4. Cristofanilli M, Broglio KR, Guarneri V, et al. Circulating tumor cells in metastatic breast cancer: biologic staging beyond tumor burden. Clin Breast Cancer 2007; 7: 471-9.

Evaluation of Prognostic and Predictive Role of HER 2 in Breast Cancer

Roshni Chinoy, Poonam Panjwani,
Tanuja Shet, Preetha Rajasekharan,
Sangeeta Desai

HER 2
HER2, a member of the growth factor receptor family of the tyrosine kinases, is involved in the regulation and differentiation of normal cell growth and is amplified in 18-20% of all breast cancers (1). HER2 amplification is found to be associated with aggressive tumour behavior, increased metastatic potential, increased resistance to tamoxifen and better response to anthracycline-based chemotherapy. All these factors make HER2 an important independent biologic marker for breast cancer. HER2 is over expressed with a high frequency in high grade ductal carcinomas, especially in tumors with unfavorable histology (2).

Role of HER2 as a prognostic and predictive factor in breast cancer

In 1999, HER2 was included as a category II factor (factor requiring more validation) by the CAP (3). However, at the 9th St. Gallen International Breast Cancer Treatment Consensus Conference in 2005, HER2 jumped ahead and was listed as an important factor associated with an increased risk of recurrence in both node-negative and node-positive breast cancers (4).

Role of HER2 in breast cancer

a. As a predictive factor
The expression, amplification, or both, of HER2 by a breast cancer is associated with benefit from the addition of paclitaxel after adjuvant treatment with doxorubicin plus cyclophosphamide in node-positive breast cancer, regardless of estrogen-receptor status (5). It is also associated with a better response to anthracyclines and a relative resistance to hormone based therapy and cyclophosphamide, methotrexate and 5-fluorouracil (CMF) based chemotherapy regimes (6-8).

b. As an agent for targeted therapy

Development of anti HER-2 monoclonal antibody in the form of trastuzumab is a major turning point in the history of molecular targeted therapy (1, 9). Trastuzumab therapy improves the response rate, time to progression with a longer disease free interval and overall survival in HER2/neu positive metastatic breast cancer (10-14). Trastuzumab was initially licensed for the treatment of patients with metastatic disease and HER2 positive cancer. However, the results of clinical trials in patients with early breast cancer viz. overall survival benefit and reduction in risk of recurrence are compelling us to consider its use in the adjuvant setting (14).

The clinical effects of trastuzumab, either alone or in combination with anthracyclines are seen only in patients who truly have the amplification of the HER2 gene which underscores the need for an effective reproducible test for the determination of the HER2 status. This is vital because the therapy is expensive and patients without amplification do not benefit from the treatment.

Recently Lapatinib a selective inhibitor of both epidermal growth factor receptor (EGFR) and HER2 tyrosine kinases (i.e. a dual HER1/HER2 receptor inhibitor) is available as an oral preparation and has been shown to be more potent in inhibiting cell growth in human breast cancer cell lines than trastuzumab, however this drug is still being evaluated (1,15)

Methods for estimation of HER2/neu amplification

Two popular methods that are used for Her2/neu estimation in breast cancer are IHC and FISH mainly due to their convenience for testing on a paraffin block. However, FISH has gained popularity as a reliable method for the purpose of confirming the gene amplification.

IHC
As IHC is almost universally available in large hospitals, it is the most popular method for estimation of the HER2 status in breast cancer. The first IHC assay to be approved for HER2 testing by the US FDA in September 1998, in response to indication for Herceptin therapy was the HercepTest (DAKO Corp, Carpentaria, CA), which uses a polyclonal antibody AO85.

At present, there are a large number of anti-HER2 antibodies like A085, CB11, TAB250, 3B5, CBE356 etc available in market. Though the US FDA has approved only 2 assays (DAKO HercepTest and the Pathway CB11 assays) for routine testing for HER2 protein by IHC, a large number of other assays continue to be used widely and many of these show comparable results with the standard HercepTest (16,17).

Numerous factors, however, impact the sensitivity of IHC like the use of different antibodies, differences in the fixation of tissues, different reagents used, the use of antigen retrieval methods, inter-observer variability. The HercepTest kit has overcome some of these problems by using standard methodology / reagents and by the inclusion of cell line controls. It is recommended that every antibody used in a given laboratory should be standardized with the US FDA approved assays (1, 18).

The validation of a standardized assay can be achieved by the following ways:
a. Dual IHC and FISH assay on a contemporary series of breast carcinomas (minimum 100 cases). FISH can be restricted to those cases demonstrating some evidence of membrane reactivity.
b. Validation on tumour tissue array blocks already scored for IHC and FISH from a research laboratory or reference source.
c. Laboratories should aim to achieve high degrees of concordance between IHC 0/1+ results and FISH negativity and between IHC 3+ results and FISH positivity.
d. Participation in appropriate internal and external quality assurance (QA) programs is also recommended for validating the results obtained with the assay in use.
The ASCO / CAP have laid down the following ‘Sample Exclusion Criteria’ to perform or interpret a HER2 IHC Assay (1):

• Tissues fixed in fixatives other than neutral buffered formalin.

• Needle biopsies fixed less than 1 hour in neutral buffered formalin.
• Excision biopsies fixed in formalin for less than 6 or longer than 48 hours.l Core needle biopsies with edge, retraction or crush artifact involving entire core.
• Tissues with strong membrane staining of internal normal ducts or lobules.
• Tissues where controls exhibit unexpected results.
  The Task-Force Guidelines also state the following ‘IHC Interpretation Criteria:
• Review controls: If not as expected, test should be repeated.
• More than 30% of tumor must show circumferential membrane staining for positive result.
• Membrane staining must be intense and uniform. A homogeneous, dark circumferential (chicken wire) staining pattern should be seen.
• Ignore incomplete or pale membrane staining.
• Quantitative image analysis is encouraged for cases with weak membrane staining (1+/2+) to improve consistency of interpretation.
• If cytoplasmic staining obscures membrane staining, repeat assay or do FISH.
• Reject sample if normal ducts and lobules show obvious staining or if there are obscuring artifacts.

Avoid scoring DCIS; score only the infiltrating component of the carcinoma.
The scoring for HER2 IHC is as follows:

1. Positive HER2 test (score 3+): Crisp membrane staining in >30% of cells ( as per revised ASCO/CAP guidelines).
2. Equivocal HER2 test (score 2+): Moderate or weak complete membrane staining in 10-30% of tumour cells or complete, non-uniform membrane staining in >10% of tumour cells.
3. Negative HER2 test (score 0 or 1+): Tumour cells showing no staining (score 0) or weak, incomplete membrane staining in any proportion of tumour cells (score 1+).
The main problems related to IHC are those related to standardization, the varied antibodies used, and its inability to detect chromosome 17 polysomy, which can lead to protein over-expression without true gene amplification.

FISH

FISH is a molecular tool, which detects and localizes specific DNA and RNA sequences in a tissue or on a chromosome by using a fluorescent labeled probe within a cellular preparation. Specifically, DNA FISH involves the precise annealing of a single stranded fluorescently labeled DNA probe to complementary target sequences. The hybridization of the probe with the cellular DNA site is visible by direct detection using fluorescence microscopy. FISH testing for HER2 uses a labeled probe to enumerate the HER2 gene copy number (19). Two FISH kits are commercially available that have been approved by the US FDA for HER2 testing (1).

1. The INFORM HER2/neu probe (Ventana) determines the absolute level of HER2 gene signals. It is a single biotin-labeled DNA probe to HER2 gene and fluorescently labeled avidin. It helps detect the HER2 gene copies/nucleus. No internal control probe is used. Detection is performed by indirect labeling of fluorescein tagged avidin, which binds to the DNA probe.

The results of the test are interpreted after counting of at least 60 cells (1) as follows:
1. Positive HER2 test: An average of > 6 gene copies per nucleus.
2. Equivocal HER2 test: An average of 4 to 6 gene copies per nucleus.
3. Negative HER2 test: An average of < 4 gene copies per nucleus.

The disadvantage of using a single probe for the HER2 gene is that it does not recognize the potential for cells to be polysomic for chromosome 17. This drawback can be overcome with the use of a dual probe kit which is incorporated by the PathVysion HER2 DNA Probe kit. Another advantage of the PathVysion dual probe kit is that the use of direct probe labeling by the PathVysion significantly reduces non-specific cellular staining, which is commonly seen as a result of the indirect labeling used in the INFORM assay (20).

2. The PathVysion HER2 DNA Probe kit (Abbott Molecular) is based on hybridization of dual colored fluorescent DNA probes to HER2 gene (orange-red) and chromosome 17 centromere (green). The Locus Specific Identifier (LSI) is the HER2/neu DNA probe, which is a 190kb Spectrum Orange directly labeled fluorescent DNA probe specific for the HER2 gene locus (17q11.2-q12). The Centromere Enumeration Probe 17 (CEP17) DNA probe is a 5.4kb Spectrum Green directly labeled fluorescent DNA probe specific for the alpha satellite DNA sequence at the centromeric region of chromosome 17 (17p11.1-q11.1). Enumeration of the LSI HER2/neu and CEP17 signals is conducted by microscopic visualization of the nucleus, which yields a ratio of the HER2 gene to chromosome 17 copy number.

The results are interpreted as follows after counting at least 60 cells (2):

1. Positive HER2 test: HER2/CEP17 ratio of > 2.2.
2. Equivocal HER2 test: HER2/CEP17 ratio between 1.8-2.2.
3. Negative HER2 test: HER2/CEP17 ratio <1.8.

Non-amplified and amplified control slides (MDA-MB-231 and Hs578T cell lines respectively, fixed and embedded in paraffin) are provided along with the PathVysion kit.

The ASCO/ CAP Task-Force guidelines (1) have laid down the following ‘Sample Exclusion Criteria’ to perform or interpret a HER2 FISH Assay:

• Samples with only limited invasive cancer difficult to define under UV light.
• Tissue fixed in fixatives other than buffered formalin.
• Tissue fixed for prolonged intervals in formalin (greater than 48 hours).
• Controls with unexpected results.
• FISH signals non-uniform (< 75% identifiable).
• Background obscures signal (> 10% of signals over cytoplasm).

Non-optimal enzymatic digestion (poor nuclear resolution, persistent auto fluorescence).
The ASCO/CAP Task-Force guidelines (1) also state the following ‘FISH Interpretation Criteria’:

• Review corresponding hematoxylin and eosin and/or IHC slide to localize invasive cancer. Carcinoma in situ should not be scored.
• Review controls; if not as expected, test should be repeated.
• Count at least 20 non-overlapping cells in two separate areas of invasive cancer.
• Reject if signals are non-uniform (> 25%) or if there is high auto fluorescence or nuclear resolution poor.
• Reject if background obscures signal resolution (> 10% over cytoplasm).
• If HER2/CEP17 ratio between 1.8 and 2.2, have additional person recount

• If heterogeneous expression, have additional person recount.

Counting can be done by a trained technologist, but pathologist must confirm that result (count) is correct and that invasive tumor was counted. FISH may also be a reliable procedure for assessment of HER2 in cytological specimens (21). FNA material obtained from metastatic tissue can also be used for testing of the HER2 gene status (22). There are various disadvantages of FISH too, the chief being the high cost, labor intensive procedure, need for fluorescence microscope, specialist training, and the loss of fluorescence signals with time.

CISH

Though CISH is not commonly used for HER2/neu estimation we wish to highlight its promising role. CISH replaces the need for fluorescence evaluation by using chromogen instead of fluorophores. CISH was first described as an alternative to FISH for testing the HER2 gene by Tanner et al in 2000 (23). CISH detects the HER2/neu gene copies by conventional peroxidase reaction (24, 25). In CISH, the formalin fixed paraffin embedded tumour tissue sections are pretreated by heating in microwave oven and using enzyme digestion and hybridized with a digoxigenin labeled DNA probe. The probe is selected by antidigoxigenin fluorescein, antifluorescein peroxidase and diaminobenzidine according to the manufacturer’s instructions. Gene copies are visualized easily by 40x objective. HER2/neu amplification typically appears as large peroxidase positive intranuclear gene copy clusters. Concordance values of up to 93% have been observed between CISH and FISH. Advantages of CISH over FISH are:

1. The results can be read on a standard light microscope with a 40x objective, thus eliminating the need for a fluorescence microscope.
2. The sections can be counterstained for simultaneous histopathologic examination of the tumor. This feature is lost in FISH.
3. The staining is permanent and quality checks are possible.
4. CISH is less expensive as compared to FISH.

The main disadvantage of CISH similar to single color FISH is that it cannot distinguish chromosomal amplification from aneuploidy. FISH is also considered a more sensitive test than CISH in picking up low-levels of gene amplification. Also, IHC 2+ cases need to be confirmed by FISH and cannot be subjected to single color CISH alone (25, 26).

Conclusions
Therapeutic decisions in breast carcinoma treatment are often governed by biological markers. HER2 has emerged as one of the strongest predictive factors for breast carcinoma. The discovery of the anti-HER2 antibody has changed the management of breast cancer, in the metastatic as well as in the adjuvant setting.

References

1. Wolff AC, Hammond E, Schwartz J, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. J Clin Oncol 2007; 25: 118-45.

2. Schnitt SJ, Millis RR, Hanby AM, et al. The Breast. In: Mills SE eds. Sternberg’s Diagnostic Surgical Pathology. Philadelphia, PA: Lippincott Williams & Wilkins; 2004; 321-95.

3. Fitzgibbons PL, Page DL, Weaver D, et al. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 2000; 124: 966-78.

4. Tsuda H. HER-2 (c-erbB-2) Test update: Present status and problems. Breast Cancer 2006; 13: 236-48.

5. Hayes DF, Thor AD, Dressler LG, et al. HER2 and Response to Paclitaxel in Node-Positive Breast Cancer. N Engl J Med 2007; 357: 1496-506.

6. Petit T, Borel C, Ghnassia JP, et al. Chemotherapy response of breast cancer depends on HER-2 status and anthracycline dose intensity in the neoadjuvant setting. Clin Cancer Res 2001; 7: 1577-81.

7. Cianfrocca M, Goldstein LJ. Prognostic and predictive factors in early-stage breast cancer. Oncologist 2004; 9: 606-16.

8. Dowsett M, Cooke T, Ellis I, et al. Assessment of HER2 status in breast cancer: why, when and how? Eur J Cancer 2000; 36: 170-6.

9. Hortobagyi GN. Trastuzumab in the treatment of breast cancer. N Engl J Med 2005; 353: 1734-6.

10. Elkin EB, Weinstein MC, Winer EP, et al. HER-2 testing and Trastuzumab therapy for metastatic breast cancer: A cost-effectiveness analysis. J Clin Oncol 2004; 22: 854-63.

11. Fornier M, Risio M, Van Poznak C, et al. HER2 testing and correlation with efficacy of trastuzumab therapy . Oncology 2002; 16: 1340-52.

12. Slamon DJ, Brian LJ, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metatstatic breast cancer that overexpress HER2. N Engl J Med 2001; 344: 783-92

13. Henry NL, Hayes DF: Uses and abuses of tumour markers in the diagnosis, monitoring and treatment of primary and metastatic breast cancer. Oncologist 2006; 11: 541-52.

14. Baselga J, Perez EA, Pienkowski T, et al. Adjuvant trastuzumab : A milestone in the treatment of HER-2 positive early breast cancer. Oncologist 2006; 11: 4-12.

15. Konecny G, Pegram MD, Venkatesan N, et al. Activity of the dual kinase inhibitor Lapatinib (GW572016) against HER-2 overexpressing and Trastuzumab-treated breast cancer cells. Cancer Res 2006; 66: 1630-9.

16. Press MF, Slamon DJ, Flom KJ, et al. Evaluation of HER-2/neu gene amplification and overexpression: Comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 2002; 20: 3095-105.

17. Thomson TA, Hayes MM, Spinelli JJ, et al. HER-2/neu in breast cancer: interobserver variability and performance of immunohistochemistry with four antibodies compared with fluorescence in situ hybridization. Mod Pathol 2001; 14: 1079-86.

18. Ellis IO, Bartlett J, Dowsett M, et al. Best practice no 176: Updated recommendations for HER2 testing in the UK. J Clin Pathol 2004; 57: 233-7.

19. Hicks DG, Tubbs RR. Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: a technical review with interpretive guidelines. Hum Pathol 2005; 36: 250-61.

20. Wang S, Saboorian MH, Frenkel E, et al. Laboratory assessment of the status of the HER-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assays with fluorescence in situ hybridization assays. J Clin Pathol 2000; 53: 374-81.

21. Bozetti C, Nizzoli R, Guazzi A, et al. HER-2/neu amplification detected by Fluorescence in situ hybridization in fine needle aspirates from primary breast cancer. Ann Oncol 2002; 13: 1398-403.

22. Bozzetti C, Personeni N, Nizzoli R, et al. HER-2/neu amplification by fluorescence in situ hybridization in cytologic samples from distant metastatic sites of breast carcinoma. Cancer (Cancer Cytopathol) 2003; 99: 310-6.

23. Tanner M, Gancberg D, ba Angelo Di Leo, et al. Chromogenic in situ hybridization- A practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. Am J Clin Pathol 2000; 157: 1467-72.

24. Isola J, Tanner M, Forsyth A, et al. Interlaboratory comparison of HER-2 oncogene amplification as detected by Chromogenic and Fluorescence in situ hybridization. Clin Cancer Res 2004; 10: 4793-8.

25. Lambros M, Natarajan R, Reis-Filho JS. Chromogenic and fluorescent in situ hybridization in breast cancer. Hum Pathol 2007; 38: 1105-22.

26. Cayre A, Mishellany F, Lagarde N, Penault-Llorca F. Comparison of different commercial kits for HER2 testing in breast cancer: looking for the accurate cut-off for amplification. Breast Cancer Res 2007; 9: R64.

Prognostic and Predictive Markers of Gliomas

Chitra Sarkar, Prasenjit Das

Glioma constitutes the most common primary brain neoplasm in adults where advances in molecular genetics over the last decade have added considerable knowledge regarding the genesis and progression of these tumors. Interest in genetic and molecular background of brain tumors, not only provides proof of evidence in conventional morphological diagnosis, but has immense role in determination of prognosis, implementation of targeted treatment, determination of response to therapy and participation in global clinical trials. In this context, oligodendroglioma represents the first central nervous system (CNS) neoplasm in which the genetic signature has been shown to correlate with improved outcome. Genetic abnormalities associated with prognosis in astrocytic tumors still remain controversial.

1. Oligodendroglial tumors

1.1 Genetic / molecular alterations in Oligodendroglial tumors
The genetic/molecular abnormalities in WHO grade II and grade III oligodendrogliomas vary substantially (1). LOH for chromosomes 1p, 19q and 4q, over expression of EGFR, platelet derived growth factor receptor (PDGFR) and platelet derived growth factor (PDGF) have been described in WHO grade II oligodendrogliomas, while in grade III oligodendrogliomas mutations/ deletions of cyclin dependent kinase inhibitors, CDKN2A and CDKN2C, amplification of cyclin dependent kinase 4 (CDK4), EGFR, MYC, LOH for chromosomes 1p, 19q, 9p and 10q and over expression of vascular endothelial growth factor receptor (VEGFR) have been described (2). Amongst all these genetic/ molecular alterations, LOH of chromosome 1p and 19q have been studied most.

1.2 Loss of chromosomes 1p/19q in Oligodendroglial tumors: Diagnostic and prognostic implications
Combined LOH of chromosome 19q and 1p locus in grade II oligodendrogliomas was first described in 1994 (3). In adults, combined 1p/ 19q LOH has been observed in up to 83% of pure oligodendrogliomas, 63% of anaplastic oligodendroglioma, 56% of mixed low grade oligoastrocytoma and 52% of anaplastic mixed oligoastrocytomas (4, 5). Various studies showed a positive correlation between the presence of combined loss of 1p and 19q with the therapeutic outcome (6, 7). At present combined loss of 1p/ 19q is a well accepted molecular diagnostic hallmark and chemo & radiation response marker for oligodendroglial tumors (7, 8).

1.3 Other genetic abnormalities
p53 mutations are infrequent in oligodendroglial tumors and have been almost exclusively seen in oligodendrogliomas with intact 1p. Similarly, chromosome 10q, which is frequently deleted in glioblastomas, has also been associated with poor prognosis in oligodendroglioma, as 58% of anaplastic oligodendrogliomas show LOH at 10q. Alterations of 9p, p16 and p27, over expression of VEGF and CDKN2A/ p16 deletions have also been correlated with poor patient survival (9, 10). Promoter hypermethylation of MGMT (O6-methylguanine-DNA methyl-transferase) on 10q26.3 has been observed in 60–80% of oligodendroglial tumors, and an association with 1p/19q status and tumor grade is reported (11).

2 Astrocytic tumors
Identification of genetic and epigenetic alterations and their impact on the pathobiology of astrocytomas have not yet gained a significant impact.

2.1 Various genetic/molecular alterations and their role in prognosis

2.1.1 p53 mutations
Knowledge of the presence of p53 mutations in diffuse astrocytomas appears to be of controversial clinical value (12). Both Sarkar et al (13) and Chattopadhyay P et al (14) demonstrated p53 mutations as an early event in astrocytic tumorigenesis and also in the malignant progression of low grade to higher grade tumors. p53 immunopositivity was shown to have correlation with both recurrence interval and proliferation indices and can therefore be used as a surrogate marker of p53 mutations to predict biological behavior in astrocytic tumors (15, 16).

2.1.2 EGFR amplification/ over expression
Meta-analysis of the previous studies on the predictive value of EGFR amplification in astrocytoma has been unclear (17). Subsequent studies have reported EGFR overexpression and/or gains of chromosome 7 in low-grade (WHO II) and anaplastic astrocytomas (WHO III) to be related to shortened survival, especially with the small cell histologic phenotype of the latter (18).

EGFRvIII variant likewise is more frequent in the small cell variant; as opposed to non-small cell astrocytomas and its over expression has been associated with poorer survival. Inactivation of EGFR in GBMs is also postulated to enhance their responsiveness to combination treatment with radiation and chemotherapy (19).
To date, drug trial reports indicate that identification of the presence of the EGFRvIII variant and measurement of the activated downstream targets, phospho-Akt, phospho-S6, and phospho-MAPK, may be useful in predicting sensitivity to some of the EGFR kinase inhibitors. No studies to date have identified prognostic significance related to immunoreactivity status among any of these markers that is independent of histologic grade (20).

2.1.3 Other genetic abnormalities 10q/ PTEN mutations, RB/ p16 alterations, mdm2 overexpression, MAPK, Geminin

LOH of 10q, is the most frequent genetic alteration in glioblastomas and occurs in 60-80% of cases. 10q loss/monosomy 10 has been identified as an independent predictor of shorter patient survival (median survival time being 4.4 months vs. 34.4 months for cases with and without PTEN mutation, respectively) (21, 22). Several other candidate tumor suppressor genes have been mapped to 10q, including PTEN (10q23), DMBT1 (10q25.3–26.1), and more recently annexin VII (ANX7; 10q21) (23).

The p16/RB/CDK4 pathway is quite frequently disrupted in astrocytomas. p16 gene mutations and deletion or decreased p16 protein expression has been associated with increased sensitivity to antimetabolite chemotherapeutic agents and described as one of the independent predictors of poor patient survival (24, 25).
mdm2 is a regulator of p53 and its over expression has been found to be a negative prognostic indicator in some studies (26).

Mitogen-activated protein kinase (p-MAPK), also known as p42/44, is a member of RAS kinase family. Recent studies showed elevated p-MAPK expression is strongly associated with poor response to radiotherapy (27).
Geminin, is a nuclear protein and its labeling index (LI) is a significant predictive factor in patients with high-grade astrocytoma, with higher expression indicating a good prognosis (28).

2.2. Genetic / molecular markers in astrocytomas for prediction of response to treatment

2.2.1 O6-alkylguanine-DNA alkyltransferase (AGT)/ O6- methylguanine-DNA-methyltransferase (MGMT)
The landmark study of Stupp R et al (29), demonstrated the distinct survival advantage of radiotherapy + Temozolamide (TMZ) vs radiotherapy alone and established TMZ as the treatment of choice for GBM patients.
O6-alkylguanine-DNA alkyltransferase (AGT). AGT, a critical DNA repair protein also referred to as O6- methylguanine-DNA-methyltransferase (MGMT), removes chloroethylation or methylation damage from the O6 position of DNA guanines, thereby protecting normal cells from exogenous carcinogens, and similarly protecting tumor cells from alkylating and methylating chemotherapeutic agents.

Methylation of MGMT gene promoter, a frequent event seen in GBM, leads to silencing of gene (30) and imparts a distinct survival advantage in patients on TMZ treatment compared to those with unmethylated promoter. The 2 year survival in GBM patients with methylated MGMT was 46% with TMZ+ RT vs 14% in cases with unmethylated promoter (31).
MGMT levels can be most readily measured by either immunohistochemistry or by a methylation specific PCR assay (32, 33). Friedman et al (32) initially implicated TMZ response in 60% patients with high-level AGT (detected by immunohistochemistry in 20% of cells) in comparison to only 9% patients with low-level AGT (present in 20% of tumor cells), which has been confirmed further in subsequent studies. However most believe that methylation specific PCR assay is most sensitive and specific, while MGMT determination by immunohistochemistry is unreliable, especially because it can be upregulated due to many factors, including administration of radio and chemotherapy.

Therefore a looming, fundamental question is whether tumor AGT status should ultimately direct treatment of newly diagnosed GBM patients. Although results to date suggest that AGT status is an important biomarker for TMZ responsiveness, these findings require further validation in additional prospective analyses.

2.3 Genetic / molecular alterations and prognosis in pediatric astrocytic tumors

In contrast to their adult counterparts, much less is known about the molecular events involved in the pathogenesis and progression of pediatric astrocytomas (34,35). Several of the molecular alterations common in adult astrocytomas are infrequent in the diffuse pediatric astrocytomas. EGFR amplifications and PTEN mutations are seen in a minority of cases, but are nonetheless associated with shorter survival. On the other hand, chromosome 17p LOH and p53 mutations and/or over expression are fairly common in high-grade astrocytomas (35).

Summary
From this discussion, it is evident that genetic analyses may not only help distinguish the different histological groups of astrocytic gliomas but may also provide prognostic information beyond the histological classification. In this direction, EGFR amplification, EGFRvIII mutant expression and losses involving 10q/ PTEN, p16, 19q, and p27 all show strong promise as prognostic markers and potential therapeutic targets in adult astrocytomas. It is likely that some of these markers will eventually get incorporated into developing clinical and molecular patient stratification schemes, necessitating their inclusion in pathology report. However, to evaluate the clinical significance of these genetic parameters in a comprehensive and dependable manner, it is important to carry out controlled prospective studies on large numbers of patients.

References

1. Reifenberger G, Kros JM, Burger PC, et al. Oligodendroglioma, in Kleihues P, Cavenee WK (ed): Pathology and Genetics, Tumours of the Nervous System. World Health Organization Classification of Tumours. Lyon, France, Lyon Press, 2000, pp 56-69.

2. Puduvalli VK, Hashmi M, McAllister LD, et al. Anaplastic oligodendrogliomas: prognostic factors for tumor recurrence and survival. Oncology 2003; 65: 259–66.

3. Reifenberger G, Reifenberger J, Liu L, et al. Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. Am J Pathol 1994; 145:1175- 90.

4. Smith JS, Perry A, Borell TJ, et al. Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas. J Clin Oncol 2000; 18: 636-45.

5. Buckner JC, Ballman KV, Scheithauer RM, et al. NCCTG 94-72-53. Diagnostic and prognostic significance of 1p and 19q deletions in patients with low-grade oligodendroglioma and astrocytoma. J Clin Oncol 2005; 23(16s):1502.

6. Jaeckle KA, Ballman KV, Rao RD, et al. Current strategies in treatment of oligodendroglioma: Evolution of molecular signatures of response. J Clin Oncol 2006; 24:1246-52.

7. Fallon KB, Palmer CA, Roth KA, et al. Prognostic value of 1p, 19q, 9p, 10q, and EGFR-FISH analyses in recurrent oligodendrogliomas. J Neuropathol Exp Neurol 2004; 63: 314-22.

8. Buckner JC, Gesme D Jr, O’Fallon JR, et al. Phase II trial of procarbazine, lomustine, and vincristine as initial therapy for patients with low-grade oligodendroglioma or oligoastrocytoma: efficacy and associations with chromosomal abnormalities. J Clin Oncol 2003; 21:
251–5.

9. Sanson M, Leuraud P, Aguirre-Cruz L, et al. Analysis of loss of chromosome 10q, DMBT1 homozygous deletions, and PTEN mutations in oligodendrogliomas. J Neurosurg 2002; 97:1397–401.

10. Bissola L, Eoli M, Pollo B, et al. Association of chromosome 10 losses and negative prognosis in oligoastrocytomas. Ann Neurol 2002; 52:842–5.

11. Dong SM, Pang JC, Poon WS, et al. Concurrent hypermethylation of multiple genes is associated with grade of oligodendroglial tumors. J Neuropathol Exp Neurol 2001; 60: 808–16.

12. Peraud A, Kreth FW, Wiestler OD, et al. Prognostic impact of TP53 mutations and P53 protein over expression in supratentorial WHO grade II astrocytomas and oligoastrocytomas. Clin Cancer Res 2002; 8: 1117–24.

13. Sarkar C, Chattopadhyay P, Ralte AM, et al. Loss of heterozygosity of a locus in the chromosomal region 17p13.3 is associated with increased cell proliferation in astrocytic tumors. Cancer Genet Cytogenet 2003; 144: 156-64.

14. Chattopadhyay P, Rathore A, Mathur M, et al. Loss of heterozygosity of a locus on 17p13.3, independent of p53 is associated with higher grades of astrocytic tumors. Oncogene 1997; 15: 871-4.

15. Nayak A, Ralte A M, Sharma M C, Sarkar C. p53 protein alterations in adult astrocytic tumors and oligodendrogliomas. Neurology India 2004; 52: 228-32.

16. Sarkar C, Karak AK, Nath N, et al. Apoptosis and proliferation: correlation with p53 in astrocytic tumors. J Neurooncol 2005; 73: 93-100.

17. Hurtt MR, Moossy J, Donovan Peluso M, Locker J. Amplification of epidermal growth factor receptor gene in gliomas: Histopathology and prognosis. J Neuropathol Exp Neurol 1992; 51: 84–90.

18. Huncharek M, Kupelnick B. Epidermal growth factor receptor gene amplification as a prognostic marker in glioblastoma multiforme: results of a meta-analysis. Oncol Res 2000; 12: 107–12.

19. Wessels PH, Twijnstra A, Kessels AGH, et al. Gain of chromosome 7, as detected by in situ hybridization, strongly correlates with shorter survival in astrocytoma grade 2. Genes Chromosomes Cancer 2002; 33: 279– 84.

20. Aldape K, Ballman K, Furth A, et al. Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance. J Neuropathol Exp Neurol 2004; 63: 700–7.

21. McLendon R E, Turner K, Perkinson K, et al. Second Messenger Systems in Human Gliomas. Arch Pathol Lab Med 2007; 131: 1585–90.

22. Knobbe CB, Merlo A, Reifenberger G. PTEN signaling in gliomas. Neurooncol 2002; 4: 196–211.

23. Smith JS, Tachibana I, Passe SM, et al. PTEN mutation, EGFR amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J Natl Cancer Inst 2001; 93:1246– 56.

24. Ichimura K, Schmidt EE, Goike HM, Collins VP. Human glioblastomas with no alterations of the CDKN2A (p16INK4A, MTS1) and CDK4 genes have frequent mutations of the retinoblastoma gene. Oncogene 1996; 13:1065– 72.

25. Iwadate Y, Mochizuki S, Fujimoto S, et al. Alteration of CDKN2/p16 in human astrocytic tumors is related with increased susceptibility to antimetabolite anticancer agents. Int J Oncol 2000; 17: 501–5.

26. Korkolopoulou P, Christodoulou P, Kouzelis K, et al. MDM2 and p53 expression in gliomas: a multivariate survival analysis including proliferation markers and epidermal growth factor receptor. Br J Cancer 1997; 75:1269– 78.

27. Pelloski C E, Lin E, Zhang E, et al. Prognostic Associations of Activated Mitogen-Activated Protein Kinase and Akt Pathways in Glioblastoma. Clin Cancer Res 2006; 12: 3935-41.

28. Shrestha P, Saito T, Hama S, et al. Geminin: a good prognostic factor in high-grade astrocytic brain tumors. Cancer 2007; 109: 949-56.

29. Stupp R, Masson WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolamide for glioblastoma. N Engl J Med 2005; 352: 987-96.

30. Watts GS, Pieper RO, Costello JF, et al. Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene. Mol Cell Biol 1997; 17: 5612-9.

31. Hegi M, Diserens A, Gorlia T, et al. MGMT gene silencing and benefit from Temozolomide in glioblastoma. N Engl J Med 2005; 352: 997-1003.

32. Friedman HS, McLendon RE, Kerby T, et al. DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma. J Clin Oncol 1998; 16: 3851- 57.

33. Hegi ME, Diserens AC, Godard S, et al. Clinical trial substantiates the predictive value of O-6- methylguanine-DNA methyltransferase promoter methylation in glioblastoma patients treated with temozolomide. Clin Cancer Res 2004; 10: 1871-4.

34. Biegel JA. Genetics of pediatric central nervous system tumors. J Pediatr Hematol Oncol 1997; 19: 492–501.

35. Sure U, Ruedi D, Tachibana O, et al. Determination of p53 mutations, EGFR overexpression, and loss of p16 expression in pediatric glioblastomas. J Neuropathol Exp Neurol 1997; 56: 782– 9.

• To establish monoclonality and / or lineage when the same cannot be achieved by IHC and /or morphology. Unlike most B-cell lymphomas, T-cell lymphomas are often difficult to diagnose by morphology and IHC alone as the neoplastic T-cell infiltrates can be polymorphous and are sometimes difficult to separate from intermixed benign T cells. This diagnostic difficulty can be resolved by performing TCR rearrangement studies.
• Resolution of aberrant immunophenotype in a tumor or for detecting clonality in a polymorphous population. e.g. a lymphoma that shows admixture of both T and B cells.

   
• Distinguishing between subtypes of low grade B cell lymphomas (2) – With modern chemotherapy treatment it is essential to diagnose low grade lymphomas accurately. These tumors often have overlapping morphologic features. Thus a SLL or CLL should be distinguished from follicular and mantle cell lymphoma. t(11;14) which encodes for cyclin D1 is essential for diagnosis of mantle cell lymphoma.
• Distinguishing Hodgkin lymphoma (HL) Vs non-Hodgkin lymphoma (NHL) especially anaplastic large cell lymphoma (ALCL): demonstration of the NPM-ALK protein and t(2;5) helps distinguish ALCL from HL.

   

• Fine needle aspirate can be used for all methods but may prove insufficient for conventional cytogenetics.
  
• Bone marrow aspirate - RT-PCR & conventional cytogenetics
  
• Paraffin blocks – PCR, RT-PCR & FISH
  
• Fresh tissue biopsy – preferred for all molecular tests, currently gene profiling has been standardized on fresh tissue only.
  

• 1. Antigen receptor gene rearrangements (DNA based assays) for monoclonality
  2. Chromosomal translocations
  
• DNA based- t(14;18) in follicular lymphoma
  
• RNA based- RT-PCR for fusion gene transcripts – e.g. in t(2;5) in ALCL

   

• Nature of fixative: Formalin gives the best results, while mercuric fixatives and Bouin’s solution are least suitable.
  
• Method for decalcification of bone marrow for molecular studies: EDTA is superior to formic acid in preserving DNA quality and is preferred fixative for such studies.
  
• Assay design: PCR based tests are influenced by nature of primers and methods for detection of the amplified product. Single primer pair PCR is less sensitive than semi nested PCR, similarly monoplex PCR is less sensitive then multiplex reactions (multiple primer sets in a single assay (7).
  
• Detection systems: More complex gel systems afford greater separation of products and these include DGGE, TGGE, etc. Capillary electrophoresis with automated fluorescent DNA fragment analysis (Gene Scan) is rapidly becoming a method of choice, particularly in academic and research centers, because of its sensitivity and high throughput. However, these sophisticated methods are costly and the chance of them producing pseudoclonal products is high, increasing the frequency of detecting inappropriate AgR gene rearrangements, even in reactive conditions.
  
• Types of PCR: Most PCR assays for diagnostic purposes are qualitative in nature. Quantitative assay are done while assessing MRD. Two alternative PCR approaches, RT-PCR and long range PCR, can be used to assess for some translocations like the t(2;5) translocation, and both are routinely possible in diagnostic laboratories. A long PCR method is more technically demanding than standard PCR and requires high-quality DNA.

   

Minding our T’s and B’s! (9)

Problems in B cell clonality assessment
Reasons for lack of 100% sensitivity in IgH based assays are enumerated below:

a. Wide number of segments to be probed: Of the three Ig genes that rearrange (the heavy chain gene, the kappa and lambda light chain genes), the heavy chain gene IgH is most frequently rearranged and thus also most commonly evaluated for clonality assays. If the results of IgH rearrangement are uncertain than the Igk gene is also studied. The Igl locus is highly polymorphic, resulting in a number of germline bands and thus its assessment can complicate interpretation of patient specimens. The IgH gene rearranges before the light chain genes however some B cell malignancies, typically precursor lymphoblastic leukemias and lymphomas, have not yet rearranged their light chain genes and hence can yield false negative clonality results.

Within the IgH gene, the VDJ region is probed for rearrangement. In a normal cell V & J are far away but with rearrangement they are brought closer and the intervening DNA is amplified. PCR analysis of the IgH gene typically involves the use of a consensus primer pair, with the upstream primer being homologous to a V segment and the downstream primer annealing to one of the J segments.

Usually, a single J region primer is sufficient to recognize all six possible J segments, but as there are many V segments, no single V region primer recognizes all V segments. They are also more heterogeneous, as compared with J segments. This is the primary explanation for the lack of a 100% diagnostic sensitivity of single primer pair IgH PCR assays. The addition of Framework II & III primers besides the usual Framework I increases the sensitivity of detection of IgH even in a polymorphous tumor like follicular lymphomas (10).

Standard IgH PCR demonstrates monoclonality in 82% B cell lymphomas. The detection rate improves to 96% using heavy and light chain PCR (10).

b. Presence of somatic hypermutations: The B cells in germinal centers are constantly in a state of division. Somatic hypermutations are added onto the arrangement and thus resulting in additional alterations of this mutation. One cannot predict what these mutations could be, so subsequently a given mix of primers may fail to pick the rearrangement. This is responsible for a false negative IgH rearrangement test. Somatic hypermutation alters primer annealing; these small mutations do not bring V & J regions together (5).

Lymphomas that arise from B-cell precursors or mature cells e.g. mantle cell lymphoma have a very low rate of somatic mutation, and therefore false-negative results are not a problem.

On other hand, germinal centre tumors, follicular lymphoma / marginal zone lymphoma, HIV associated NHL arise from cells that commonly have ongoing somatic mutations in the V and J segments, therefore false negative rate by PCR is more.

c. Assay related problems as discussed above: The most important among these being an 8% false negative rate in paraffin processed tissue. B5 and Bouin’s fluid also negatively impact molecular results. The other problems have already been elucidated above.

d. Polymorphous lymphomas like follicular lymphoma yield false negatives mainly due to the fact that the PCR may be compounded by the presence of increased numbers of diluting polyclonal B cells, whose IgH genes would compete for the primers.

e. Method dependent variations: Pick up rate by RT-PCR is lower than SB as PCR assays are highly focused on the break points while SB always detects additional break points. A large multicentric study documented that there is remarkable inter-laboratory heterogeneity with regard to diagnostic sensitivity of IgH rearrangement by PCR, ranging from over 90% to as low as 20% (10).

Problems in T cell clonality assessment

The diagnosis of T cell lymphomas on histology is more complicated than the B cell proliferations given the polymorphous nature and the fact that non neoplastic T cell proliferations mimicking malignancy are seen very frequently. This is where the TCR rearrangement studies are of immense value. The reported efficacy of TCR rearrangements protocols ranges from 60% to greater than 90% (11).

Reasons for the high variability in TCR clonality assays are given below:

a. Wide number of segments to be probed and subsequently variability of assays due to use of different primer sets (5, 12): The TCR d gene, is routinely deleted in normal T cells and in most T-cell lymphomas. The TCRa gene is very large, with a J region of up to 80 kb, and therefore cannot be analyzed conveniently using routine SB method. Hence for clonality assessment TCRg is probed for rearrangement, it also rearranges early and is much simpler than B clonality assays. Of the 14 variable region segments of TCRg, 11 are functional and have been described as rearranged in T-cell lymphoproliferative disorders. Most variable region (V) rearrangements occur within the V1–8 subgroup (Group I) and most joining region (J) rearrangements involve the J1/2 segment. Thus most laboratories to devise TCR PCR assays that use only single primer sets for the V1–8 and J1/2 segments.

b. Polyclonal bands: The limited V region repertoire results in a number of germ line bands in polyclonal T-cell lesions (13). Distinguishing the bands of polyclonal T cells from a population of monoclonal T cells can be difficult, particularly when the polyclonal T cells are numerous (<30% of all cells). It is also important to repeat PCR assays due to pseudoclonal TCR g in inflammatory skin disorders for confirmation (14).

c. Assay related problems:
A recent multi-center study by Arber et al (15) noted a significant difference in true positive results in TCR rearrangements among laboratories that used multiple primer sets (84%) versus those that used only a single primer set (61.4%) directed against the V1–8 and J1/2 segments and emphasized the importance of using a comprehensive set of V and J primers for an optimal detection rate of TCR (15).
Perhaps one concern is potential false positive results associated with multiple primers (11).

d. Type of tumor: The gene rearrangement process is normal & usually occurs prior to neoplastic transformation. As neoplasms are derived from same clone they show the same rearrangement and a monoclonal peak. In immunocompetent patient, detection of a gene rearrangement that represents 1% to 5% of all cells in the biopsy specimen (the lower limit of the sensitivity of SB) highly correlates with malignant lymphoma

Presence of gene rearrangement cannot always be equated with malignancy, since the gene rearrangement process is normal and not involved in neoplastic transformation. Lymphomas that arise from lymphocytes at an early stage of maturation lack rearrangement while monoclonal gene rearrangements may be detected in immunocompromised patients (e.g. organ transplantation & AIDS).

Cross linkages (Neoplasms with both T & B!) (9)

There are a variety of distinct situations in which monoclonal IGH and TCR gene rearrangements may coexist, either in the same neoplastic cell or in different cells, one or both of which may be overtly neoplastic. e.g. Angioimmunoblastic lymphadenopathy type of T cell lymphoma often harbors IgH rearrangement.

Presence of “cross-lineage” receptor gene rearrangements provides potentially confusing diagnostic laboratory results that may be difficult to interpret for both uninitiated and experienced laboratories.

This is either due to lineage infidelity in same cell or due to presence of different populations of cells. This is interestingly more prevalent in immature precursor neoplasms. e.g. Seventy percent of (immature) precursor B-cell lymphoblastic leukemias/ lymphomas may contain monoclonal TRG rearrangements while only 5 to 10% of cases of (mature) B-cell CLL harbor such cross-lineage rearrangements. Understanding these cross lineages is helpful in evaluating tumors for MRD and before interpreting laboratory results.

Gene microarray technology
Studies in the past few years have shown quite clearly that gene expression profiling may indeed live up to the expectations and will have a significant impact on molecular diagnostics. DLBL was always considered as difficult to classify on histology but gene microarray data have shown that DLBL can be divided into two distinct subsets of B cells viz. Germinal center (GC) and activated B cell type (ABC) and 76% of GC signature patients alive at 5 years as opposed to 16% with activated B cell type irrespective of anthracycline based chemotherapy (16).

Patients with a low IPI score had better overall survival as compared to patients with a high IPI score. In patient with low risk IPI score the ABC subtype of DLBL did worse than GC type. Rosenwald et al (17) used 17 genes to construct a gene signature as predictor of overall survival after chemotherapy. This gene-based predictor and the IPI were independent prognostic indicators. Lossos et al (4) proposed a six gene model incorporating - LMO2, BCL6, FN1, CCND2, SCYA3, and BCL2- genes as being an important predictor of survival. IHC demonstration of the germinal centre markers (CD10, BCL6) was also predictor of survival. However; many studies have contested their reproducibility given the variability in techniques involved.

Though gene studies hold promise, the biostatistics/bioinformatics part is too complicated to apply this information to routine diagnosis currently.

Conclusion

• Rapid advances in molecular pathology make it essential for at least few surgical laboratories to integrate molecular information into histopathology
• The molecular diagnostic laboratory needs to incorporate both PCR and FISH in its routine diagnostic armamentarium for a diagnosis of lymphoma.

References

1. Kocjan G. Best Practice No 185. Cytological and molecular diagnosis of lymphoma. J Clin Pathol 2005; 58: 561.

2. Spagnolo DV, Ellis DW, Juneja S, et al. The role of molecular studies in lymphoma diagnosis: a review. Pathology 2004; 36:19-44.

3. Chan WC, Fu K. Molecular diagnostics on lymphoid malignancies. Arch Pathol Lab Med 2004; 128:1379–84.

4. Lossos IS, Czerwinski DK, Alizadeh AA, et al. Prediction of survival in diffuse large-B-cell lymphoma based on the expression of six genes. N Engl J Med 2004; 350: 1828-37.

5. Medeiros LJ, Carr J. Overview of the role of molecular methods in the diagnosis of malignant lymphomas. Arch Pathol Lab Med 1999; 123:1189–207.

6. Liu H, Huang X, Zhang Y, et al. Archival fixed histologic and cytologic specimens including stained and unstained materials are amenable to RT-PCR. Diagn Mol Pathol 2002; 11: 222-7.

7. Meier VS, Rufle A, Gudat F. Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates. Am J Pathol 2001; 159: 2031–43.

8. Langerak AW, Molina TJ, Lavender FL, et al. Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2007; 21: 222–9.

9. Bagg A. Immunoglobulin and T-cell receptor gene rearrangements: minding your B’s and T’s in assessing lineage and clonality in neoplastic lymphoproliferative disorders. J Mol Diagn 2006; 8: 426-9.

10. Bagg A, Braziel RM, Arber DA, Bijwaard KE, Chu AY. Immunoglobulin heavy chain gene analysis in lymphomas: a multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays. J Mol Diagn 2002; 4: 81-9.

11. Lawnicki LC, Rubocki RJ, Chan WC, Lytle DM, Greiner TC. The distribution of gene segments in T-cell receptor gamma gene rearrangements demonstrates the need for multiple primer sets. J Mol Diagn 2003, 5: 82-7.

12. Wilkins BS. Molecular genetic analysis in the assessment of lymphomas. Current Diagnostic Pathology 2004; 10: 351–9.

13. Wan JH, Trainor KJ, Brisco MJ, Morley AA. Monoclonality in B cell lymphoma detected in paraffin wax-embedded sections using the polymerase chain reaction: J Clin Pathol 1990, 43:888–90.

14. Lee SC, Berg KD, Racke FK, Griffin CA, Eshleman JR. Pseudo-spikes are common in histologically benign lymphoid tissues. J Mol Diagn 2000, 2:145–52.

15. Arber DA, Braziel RM, Bagg A, Bijwaard KE. Evaluation of T-cell receptor testing in lymphoid neoplasms: results of a multi-center study of 29 extracted DNA and paraffin-embedded samples. J Mol Diagn 2001, 3: 133–40.

16. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000, 403 : 503.

17. Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 2002; 346: 1937-47.

An Algorithm for molecular monitoring of Chronic Myeloproliferative Disorders

Hari Menon, Purvish Parikh

Introduction
Chronic myeloproliferative diseases (CMD) are a heterogeneous group of myeloid disorders characterized by bone marrow hypercellularity with relatively normal myeloid maturation, clonal hemopoiesis and an overproduction of circulating myeloid elements. The main clinical features of these diseases are the overproduction of mature, functional blood cells and a protracted clinical course with an elevated risk for thrombosis and progression to acute leukemia. CML is a myeloproliferative disorder that is defined by its causative molecular lesion, the BCRABL fusion gene, which results from the translocation, t (9;22), referred to as the Philadelphia (Ph) translocation. CMD also include Ph negative entities which mainly include - polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IPF). Other less common conditions included under the classification used by the World Health Organization are systemic mastocytosis, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, and chronic neutrophilic leukemia. The recent identification and understanding of recurrent mutation in the JAK2 tyrosine kinase in most patients with PV, ET or IPF has been one of the landmark advances in the understanding of the pathogenesis of these entities.

Molecular Pathogenesis of CML

CML is a hematopoietic stem cell disorder characterized by an uncontrolled proliferation and accumulation of mature and immature myeloid cells. The hallmark of CML is the presence of a balanced translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), known as the Ph chromosome. Despite the constant Ph chromosome related molecular events in CML, the disease is heterogeneous in its presentation and clinical course.

The formation of the Ph chromosome results in a head to tail fusion of the BCR gene on chromosome 22 at band q11 with the Abelson gene possessing tyrosine kinase activity on chromosome 9 at band q34. The resultant chimeric gene results in the generation of a constitutively activated tyrosine kinase protein, p210 BCR-ABL, which stimulates uncontrolled proliferation. The BCR-ABL protein is central to the pathogenesis of CML. The oncogenic potential of the fusion proteins has been validated by their ability to transform hematopoietic progenitor cells both in vitro and in vivo. Studies have revealed that the BCR-ABL protein exerts its influence through various pathways which include, alteration in cellular adhesions, activation of mitogenic signaling pathways, inhibition of apoptosis, and induction of proteosomal degaradation of ABL interactor proteins causing uncontrolled activation and progression to blastic phase (1-8).

The understanding of the integral role of BCR-ABL induced pathogenesis in CML has resulted in agents that target the activity of this protein. This has spawned an entire new generation of drugs that have brought about a paradigm shift in the management of this condition. Inhibition of this protein results in a hematological response, cytogenetic response and finally a molecular response occurring in that sequence. However, the degree of such responses varies amongst patients and correlate with long term outcomes with such therapies.

Molecular techniques for monitoring CML
Molecular techniques have now been validated to monitor the rate and degree of such responses by precisely quantifying the degree of decline of the BCR-ABL protein while on such therapy, thereby allowing the clinician to take appropriate decisions pertaining to change of drugs or increasing drugs doses. Monitoring methods now include FISH, qualitative PCR, and QPCR. Molecular studies (i.e. PCR) quantify the amount of disease with an increased sensitivity of up to 10-8. Better molecular responses may correlate with improved outcome. The rapidity of molecular response may also have additional prognostic significance (9). Achievement of a major molecular response after 12 months of imatinib therapy has been associated with the best progression-free survival (10,11). Thus, many oncologists use today molecular studies in lieu of cytogenetic analysis to monitor response.

Resistance to imatinib and to other BCR-ABL tyrosine kinase inhibitors has been associated with development of mutations in the BCR-ABL kinase domain in 40% to 50% of instances. Mutations prevent the activity of imatinib in different ways. Some may prevent the binding of the tyrosine kinase inhibitor to BCR-ABL; others may favor the active conformation of the kinase, a conformation to which imatinib cannot bind. Different mutations may confer different levels of resistance, varying between absolute resistance, to minimal or no resistance, depending on their location and their effect on the kinase domain. Such resistance patterns can be identified through molecular techniques such as QPCR.

Monitoring the patient on CML on therapy
Monitoring of CML traditionally has focused on hematological and cytogenetic responses. Routine cytogenetic testing remains the “gold standard” for the diagnosis of CML. The major advantage lies in its ability to detect simultaneously additional chromosomal abnormalities that may suggest clonal evolution, a marker for disease progression. Molecular methods have been used only for patients achieving a complete cytogenetic response or after an allogeneic stem cell transplant (SCT). With greater rates of complete cytogenetic responses being achieved with imatinib therapy, molecular monitoring has been recommended as routine assessment for CML patients even at baseline.

Cytogenetic analysis has been the mainstay of disease monitoring in CML. Earlier response criteria based on the percentage of Ph positive cells in the bone marrow were established for patients on interferon-alpha. They have proved to be good predictors of long term response. However, over the past 12 years, several groups have developed QRTPCR assays to measure BCR–ABL transcript in the blood and marrow, an excellent target that enabled the dynamics of residual disease to be monitored over time. The transcript level correlates with the number of leukaemic cells present in the blood and marrow and can be used as an accurate barometer of the response to therapy. There is no need for patient-specific primers because nearly all CML patients have one of two transcripts types (some patients have both) which differ by just one BCR exon.

It is well established that the level of BCR-ABL measured by QPCR is predictive of length of remission in patients who achieve a complete cytogenetic response on interferon (12-14). Again rising levels of BCR-ABL transcripts post allogeneic transplantation are highly predictive of cytogenetic and hematological relapse (15-17). Studies of patients on imatinib therapy have also shown that the BCR-ABL level in the blood correlates with the percentage of Ph positive cells in the marrow and that molecular response can be related to the disease phase at start of imatinib treatment (17-18).

In patients who achieve a complete cytogenetic response, QPCR is the most sensitive way to continue to monitor their disease, to predict relapse and document imatinib resistance. A major molecular response has been defined as a >3-log reduction from baseline of the BCR-ABL/ABL ratio. Although there is evidence that some complete cytogenetic responders on interferon eventually become PCR negative if followed long enough, the value of PCR negativity in the setting of therapy with IFN-a is unclear. It has been observed that transcripts in patients who were PCR negative for BCR-ABL expressed BCR-ABL in myeloid and erythroid colonies.

There are no validated guidelines for monitoring of treatment in CML. Routine cytogenetic testing at diagnosis is recommended at diagnosis and should be repeated at intervals of 6 to 12 months to identify clonal evolution. After the number of Ph-positive metaphases has fallen below 10%, FISH and quantitative PCR should be applied for further monitoring of residual disease. Following SCT, it is recommended that QPCR be performed every 4 weeks for as long as BCR-ABL transcripts continue to be identified. If the levels become undetectable, then testing intervals can be increased to every 3 months or every 6 months if PCR testing remains negative. In the case of increasing levels, PCR testing should be done more frequently.

Mutational studies in CML-when do you do it?
The time for doing mutational studies are not defined. However, there is some consensus that mutational studies should be performed only with evidence of hematologic or cytogenetic resistance-relapse. Their role in patients responding to imatinib therapy if PCR transcript levels increase by 2-5 folds remains open to deliberation. However, if they are performed, the results should be interpreted with caution, particularly when it has an impact on any proposed change in therapy. Mutational studies may also be helpful any time a change of therapy is considered: identification of specific mutations may help in the choice of one treatment approach over another (e.g. allogeneic SCT if T315I mutation identified) or of a specific BCR-ABL inhibitor depending on the relative sensitivity of the particular mutation to these inhibitors.

Proposal for monitoring the CML patient

1. All patients should undergo conventional cytogenetic analysis on bone marrow aspirates for both identifying the Ph chromososme and any other cytogenetic abnormalities at the time of diagnosis.

2. FISH is useful in detecting Ph negative BCR-ABL positive disease and is helpful in detecting deletions of derivative chromosome 9 and in monitoring patients till cytogenetic remission

3. QPCR may be considered the method of choice for monitoring patients on imatinib by measuring BCR–ABL levels in peripheral blood based on its accuracy and sensitivity provided it is also being continued for subsequent monitoring. For any individual patient, QPCR studies can estimate degrees of molecular response that predict long-term stability, as well as identify patterns of response that indicate relapse and imatinib resistance.

4. On therapy may be monitored by cytogenetics every 3-6 months or FISH as an alternative to cytogenetics every 3 months or by QPCR alternative to cytogenetics every 3 months until cytogenetic complete remission (or equivalent disease level)

5. On achieving cytogenetic complete remission, cytogenetic analysis may be considered every 12-24 months while FISH or QPCR may be an alternative considered every 3 to 6 months.

6. In case of development of resistance to therapy, conventional cytogenetics to assess for clonal evolution is necessary and RTPCR may be resorted to assess for mutations in the BCR-ABL protein.

Molecular Pathogenesis of the Philadelphia negative CMDs

Clonal hemopoiesis has been demonstrated in several CMDs suggesting that these disorders arise from acquisition of somatic mutations and this was described in 1951 by William Dameshek (19). However, the molecular basis of these disorders was described only in 2005 with several independent groups identifying a recurrent mutation in the JAK2 tyrosine kinase in most patients with PV, ET or PMF (20-23).

The JAK2 tyrosine kinase
JAK2 is a member of the Janus family of cytoplasmic non-receptor tyrosine kinases, which also includes JAK1, JAK3 and TYK2. The mutation, guanine-to-thymidine substitution, which results in a substitution of valine for phenylalanine at codon 617 of JAK2 (JAK2V617F). is present in haematopoietic cells but not germline DNA in patients with CMDs, demonstrating that JAK2V617F is a somatic mutation that is acquired in the hematopoietic compartment (20-23).

Physiologically, the Jak kinases function through their association with cytokine receptors lacking intrinsic kinase activity. Jak kinase phosphorylation and activation occur when the ligand bind to their appropriate cytokine receptor followed by recruitment and phosphorylation of Stat proteins, and activation of downstream signaling proteins. The specificity of different cytokine receptors for one or more different Jak kinases accounts in part for their differential effects on signal transduction. Genetic deletion of JAK2 results in embryonic lethality owing to a lack of definitive erythropoiesis, and JAK2-deficient haematopoietic progenitors do not respond to erythropoietin (24). Alteration in the JAK2 through substitution of valine-for-phenylalanine at codon 617 might result in constitutive activation of kinase activity (25). This results in cytokine hypersensitivity and cytokine-independent growth for haematopoietic cells (26).

Although existing data indicate that acquisition of JAK2V617F mutations contributes to the pathogenesis of PV, ET and PMF, there are probably additional genetic events that contribute to the development of these MPD as indicated in the JAK2 negative CMDs. A significant proportion of patients with ET and PMF and a small number of patients with PV are JAK2V617F negative and clonal haematopoiesis is observed in these patients (22, 27- 29). These are distinct entities among the CMDs and their presence suggests alternate alleles accounting for myeloproliferation.

The clinical impact of JAK2V617F
Although JAK2V617F provides evidence for clonal haematopoiesis, presently it is not diagnostic of specific CMDs by conventional classification systems. There are negative JAK2 CMDs. However, its presence would give credence to the diagnosis of PV, ET or PMF along with incorporation of other clinical and lab parameters (30- 32). Even though testing for JAK2V617F has become widespread, it ought to be remembered that methods for AK2V617F detection are not currently standardized resulting in false negative or false positive results. Furthermore, there is no consensus as to which biological specimens (whole blood, peripheral blood-derived granulocytes or mononuclear cells, bone marrow cells or endogenous erythroid colonies) constitute the ideal source for mutation screening in MPD patients. All these limitations not withstanding in the JAK2V617F era, revisions to the World Health Organization diagnostic criteria for PV, ET and PMF have been proposed. Based on our current understanding, it is reasonable to incorporate JAK2V617F testing into the initial diagnostic work up for PV. Virtually all patients with overt or inapparent PV harbor this mutation which is absent in spurious PV. Unlike PV, the use of JAK2V617F mutation screening for the diagnosis of ET or PMF is limited by suboptimal negative predictive value and a lack of diagnostic specificity within the context of myeloid neoplasms (30). A bone marrow biopsy is required in the workup for ET to distinguish JAK2V617F-negative ET from reactive thrombocytosis. It is also important to distinguish ET from other causes of clonal thrombocytosis including cellular phase or pre-fibrotic PMF or MDS. In the setting of myelofibrosis, the presence of JAK2V617F effectively excludes the possibility of reactive bone marrow fibrosis associated with infections, inflammatory processes, toxins, hairy cell leukaemia, lymphoid disorders or metastatic cancer.

JAK2 inhibitors – Is there a potential
Although it may be possible to design selective inhibitors, most compounds that are undergoing preclinical and clinical testing inhibit both wild-type JAK2 and JAK2V617F. Given the importance of JAK2 signaling to many different cellular functions, and the embryonic lethality and lack of definitive erythropoiesis associated with JAK2 deficiency, inhibition of wild-type JAK2 might result in haematopoietic and non-haematopoietic toxicities, including dose-dependent cytopenias. In addition, JAK3 inhibition will also need to be minimized, as their loss-of-function mutations are associated with severe combined immune deficiency (33, 34). Selective JAK2 inhibitors may hold promise as in vitro studies reveal an increased sensitivity to inhibit cell cultures that harbor the mutations as compared to the wild type. The therapeutic window of tyrosine kinase inhibitors does not correlate with mutant or wild-type selectivity, and JAK2 inhibitors that potently inhibit both wild-type and mutated JAK2 might have efficacy.

JAK2V617F monitoring
It might seem premature to discuss this aspect even before such drugs are available for clinical use. However it will be important to develop molecular assays to assess response to therapy, and to determine whether abrogation of the JAK2V617F-positive clone correlates with clinical response. Quantitative assays exist to reliably measure AK2V617F mutational burden, and these assays have already been used to assess the molecular response to treatment of PV to interferon or allogeneic transplant (35- 38).

Conclusion

Understanding the molecular aspects of CMDs has resulted in effective ways of targeting pathways resulting in profound responses as seen with CML. Understanding the pathogenesis of other CMDs has yet again a potential for profound outcomes. Along with the advent of such therapies has emerged the need to monitor responses to such therapies through molecular monitoring effectively. While this has been more or less standardized with CML, the work for the future would be to ascertain a methodology for CMDs.

References

1. Verfaillie CM, Hurley R, Lundell BI, Zhao C, Bhatia R. Integrin-mediated regulation of hematopoiesis: do BCR/ABL-induced defects in integrin function underlie the abnormal circulation and proliferation of CML progenitors? Acta Haematol 1997; 97: 40–52.

2. Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature 1987; 328: 342– 4.

3. Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000; 96: 3343–56.

4. Daley GQ, Baltimore D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein. Proc Natl Acad Sci USA 1988; 85: 9312 –6.

5. Sirard C, Laneuville P, Dick JE. Expression of bcr-abl abrogates factor-dependent growth of human hematopoietic M07E cells by an autocrine mechanism. Blood 1994; 83:1575– 85.

6. Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ. Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia. Blood 1994; 83: 2038–44.

7. Bedi A, Barber JP, Bedi GC, et al. BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: a mechanism of resistance to multiple anticancer agents. Blood 1995; 86:1148– 58.

8. Dai Z, Quackenbush RC, Courtney KD, et al. Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway. Genes Dev 1998; 12:1415– 24.

9. Druker B, Guilhot F, O’Brien S, et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 2006; 355: 2408-17.

10. Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: 1423-32.

11. Kantarjian H, Talpaz M, O’Brien S, et al. Survival benefit with imatinib mesylate versus interferon alpha-based regimens in newly diagnosed chronic phase chronic myelogenous leukemia. Blood 2006; 108: 1835- 40.

12. Hochhaus A, Lin F, Reiter A, et al. Quantitative molecular methods to monitor the response of CML patients to interferon-alpha. Bone Marrow Transplant 1996; 17 (Suppl 3): S41– 4.

13. Hochhaus A, Lin F, Reiter A, et al. Variable numbers of BCR-ABL transcripts persist in CML patients who achieve complete cytogenetic remission with interferon-alpha. Br J Haematol 995; 91: 126– 31.

14. Kurzrock R, Estrov Z, Kantarjian H, Talpaz M. Conversion of interferon-induced, long-term cytogenetic remissions in chronic myelogenous leukemia to polymerase chain reaction negativity. J Clin Oncol 1998; 16: 1526– 31.

15. Olavarria E, Kanfer E, Szydlo R, et al. Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2001; 97: 1560– 5.

16. Radich JP, Gooley T, Bryant E, et al. The significance of bcr-abl molecular detection in chronic myeloid leukemia patients ‘‘late,’’ 18 months or more after transplantation. Blood 2001; 98: 1701–7.

17. Lin F, van Rhee F, Goldman JM, Cross NC. Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. Blood 1996; 87: 4473–8.

18. Merx K, Muller MC, Kreil S, et al. Early reduction of BCRABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha. Leukemia 2002; 16: 1579– 83.

19. Damashek W. Some speculationson the myelopro-liferative syndromes. Blood 1951; 6: 372-5.

20. Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–97.

21. James C, Ugo V, Le Couédic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144–8.

22. Baxter EJ et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–61.

23. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–90.

24. Pagans E, Wang D, Stravopodis D, et al. Jak2 is essential for signaling through a variety of cytokine receptors. Cell 1998; 93, 385–95.

25. Ihle JN, Gilliland DG. Jak2: normal function and role in hematopoietic disorders. Curr Opin Genet Dev 2007; 17: 8–14.

26. Prchal JF,Axelrad AA. Bone-marrow responses in polycythemia vera. N Engl J Med 1974; 290: 1382.

27. Jelinek J, Oki Y, Gharibyan V, et al. JAK2 mutation 1849G >T is rare in acute leukemias but can be found in CMML, Philadelphiachromosome negative CML and megakaryocytic leukemia. Blood 2005; 106: 3370–3.

28. Levine RL, Belisle C, Wadleigh M, et al. X-inactivation based clonality analysis and quantitative JAK2V617F assessment reveals a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F negative ET and MMM patients with clonal hematopoiesis. Blood 2006; 107: 4139– 41.

29. Jones AV Kreil S, Zoi K, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005; 106:
2162–8.

30. Tefferi A, Thiele J, Orazi A, Cervantes F, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood 2007; 110: 1092-7.

31. Tefferi, A,. Pardanani, A. Evaluation of increased hemoglobin in the JAK2 mutations era. Mayo Clin Proc 2007; 82: 559– 606.

32. Tefferi A, Vardiman JW. The diagnostic interface between histology and molecular tests in myeloproliferative disorders. Curr Opin Hematol 2007; 14: 115– 22.

33. Russell SM, Tayebi N, Nakajima H, et al.Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 1995; 270: 797–800.

34. Macchi P, Villa A, Giliani S, et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 1995; 377: 65–8.

35. Samuelsson J, Mutschler M, Birgegård G, et al.Limited effects on JAK2 mutational status after pegylated interferon a-2b therapy in polycythemia vera and essential thrombocythemia. Haematologica 2006; 91: 1281–2.

36. Kiladjian JJ, Cassinat B, Turlure P, et al. High molecular response rate of polycythemia vera patients treated with pegylated interferon alpha-2a. Blood 2006; 108: 2037–40.

37. Jones AV, Silver RT, Waghorn K, et al. Minimal molecular response in polycythemia vera patients treated with imatinib or interferon alpha. Blood 2006; 107: 3339– 41.

38. Ruiz-Argüelles GJ, Garcés-Eisele J, Reyes-Núñez V, et al. Clearance of the Janus kinase 2 (JAK2) V617F mutation after allogeneic stem cell transplantation in a patient with myelofibrosis with myeloid metaplasia. Am J Hematol 2006; 82: 400–2.

Role of molecular methods in prognostication of neuroblastoma

Seethalakshmi Viswanathan

Neuroblastoma is the most common extracranial solid tumor in children especially in infants, accounting for 8% to 10% of all childhood cancers (1). They are extremely heterogeneous in their biologic and clinical behavior varying from spontaneous regression, maturation to aggressive behavior with advancing age (1). This heterogeneity can be partly explained by differences in the molecular genetic profile. Neuroblastoma is a model of solid tumor in which molecular genetic analysis plays a vital role in prognostication which guides optimal patient management (2).

With advances in the field of medical oncology, focus in neuroblastoma treatment has shifted to risk stratification of patients based on clinicopathologic and molecular prognostic factors (1, 3, 4).

Although traditional histopathologic prognostic factors including degree of differentiation and mitotic karryorhectic index have proved useful prognostic indicators, emergent molecular markers identify tumors resistant to therapy. This defines criteria for selection of high risk patients who would benefit from aggressive chemotherapy for better survival in contrast to low and intermediate risk patients who can be spared of potentially toxic chemotherapy and deleterious side effects associated with better survival (1, 3, 4).

The most important molecular genetic factor implicated in prognostication of neuroblastomas is MYCN amplification; although many other genetic features have now been identified to correlate with clinical outcome. e.g. allelic loss on 1p, 11q, 14q; gain of 17q; TRK gene expression (2, 4).

Prognostic factors in Neuroblastoma
The following is a summary of prognostic factors evaluated in neuroblastoma.

A. MYCN
MYCN is located on the distal short arm of chromosome 2. In neuroblastomas a large region from 2p24 (including MYCN) locus gets amplified and this provides selective survival advantage to the cells (5).

As mentioned earlier, most pediatric oncology groups stratify patients into low, intermediate and high risk groups for patients on clinical trials. This system uses age at diagnosis, international neuroblastoma staging system (INSS stage), Shimada histology and most importantly MYCN amplified status. In this schema, MYCN amplification differentiates high risk patients from low / intermediate risk group as shown below (1, 3).

a. INSS stage II , older than 1 year with unfavourable histology with amplified MYCN
b. Any INSS stage III with amplified MYCN
c. Stage IVs with amplified MYCN

These patients need more intensive induction chemotherapy, myeloablative consolidation therapy with stem cell rescue and targeted therapy for minimal residual disease. They also require radical surgery and external beam radiotherapy. High risk patients have current survival rates of less than 15% with associated significant immediate and long term morbidity (1, 6). In contrast, children with equivalent tumors without MYCN amplification (intermediate risk group) have much less aggressive clinical course and respond to moderate less intense chemotherapy also avoiding radical surgery and RT (1).

It has been well recognized that other prognostic histopathological features, such as differentiation and MKI, are related to the MYCN copy number (2, 7). Thus amplification of MYCN is associated with advanced stages of disease and poor outcome associated with rapid tumor progression, risk for relapse and poor prognosis even in infants and lower stages of disease (2, 8). It is almost always present at the time of diagnosis and is reported to be 18% to 25% of all neuroblastomas in various series. This marker has been identified as an independent prognostic marker in various studies across the world and hence its detection is essential as part of routine diagnostic and prognostic work-up (1).

MYCN amplification can be detected by a variety of molecular techniques including SB, FISH, quantitative PCR, ICC and CGH. SB was the older standard method of detection. However, now most laboratories consider interphase FISH as the technique of choice for MYCN detection, (1, 9) as it has 21% greater sensitivity than the traditional SB. SB can cause a falsely low MYCN hybridization signal due to degraded DNA sample which is not seen with FISH (9).

FISH also allows (9, 10)

a. Morphological verification of MYCN signal in situ within tumor cell.

b. Identification of low level amplification especially in the presence of intratumoral heterogeneity.

c. To discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. FISH procedure allows one to visualize cell-to-cell differences in the MYCN copy number due to the unequal segregation of double minutes between daughter cells during mitosis, resulting in a heterogeneous distribution of amplified sequences within a defined cell population.

d. To analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH can be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies which can cause false-negative measurements by the SB.

e Distinguishing the source of extra copies of MYCN due to aneuploidy (that is increased number of the entire chromosome) in contrast to true amplification within the chromosome by using a biotin-labeled chromosome 2 centromere-specific probe in addition to the MYCN probe.

Thus, the FISH technique not only shows a high level of agreement with SB, but detects authentic gene amplification in a significant number of additional cases. SB should only be reserved for samples with intermediate to high fractions of tumor cells (9).

The definition of exact copy number to constitute MYCN amplification remains controversial in literature. However, most laboratories consider greater than 10 copies per diploid genome as evidence for MYCN amplification. This can be detected by QPCR (10 - 13). Both frozen and paraffin embedded tissues can be used. The advantages of QPCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. Determination of deletion or amplification is achieved by comparing the copy number of a target gene (TG from the region of interest) to an unaffected reference gene (RG) within the same chromosome. PCR raw data is normalized to a serial dilution standard curve and a ratio TG/RG is created. The ratio to define a deletion is set as 0.5 (= expected ratio 1 TG copy/2 RG copies), the amplification threshold is set as >10.0 (10 - 13).

B. Deletion of 1p
This is seen in 25 – 35% of tumors at diagnosis. This is seen with advanced stage disease associated with MYCN amplification. Current evidence suggests its presence to be associated with increased risk of disease relapse in localized tumors and is an independent predictor of unfavourable histology. Future trials plan to incorporate 1p deletion along with the presence of 11q allelic status to assess treatment planning for stage II & III disease (11, 13).

C. Unbalanced gain of 17q
The above abnormality is seen in 50% of neuroblastomas. Gain of 17q can occur independently or as part of an unbalanced translocation between chromosomes 1 & 17. This is associated with more aggressive neuroblastomas. Its prognostic significance awaits large prospective trials & multivariate analysis (1, 2, 14).

D. Deletion of 11q
This is present in 35-45% of newly diagnosed cases. This marker, though highly associated with advanced disease, older age, unfavourable histology is not seen associated with MYCN amplification or 1p deletion. Recent evidence strongly suggests that it is independently predictive of disease relapse. This will be incorporated in clinical trials for intermediate risk disease (11, 13, 14).

E. Tumor cell DNA content
Neuroblastomas can be near diploid or near triploid analyzed semi automatically by flow cytometry. This is known to be predictive of disease outcome in infants. This is currently used in clinical trials for infants with stage III, IV or IVS disease with near triploid tumors being associated with favorable outcome (1, 15).

Other chromosomal abnormalities are 3p deletion, 4p, 9p, 14q and 19q associated abnormalities all of which have aggressive behavior.

F. Expression of neurotropin receptors
These pathways signal differentiation of sympathetic neuroblasts to neuroblastoma cell lines and are responsible for the regulation of their malignant transformation. Three tyrosine kinase receptors (Trk) of neurotropin pathways have been cloned. The ligands for these receptors are NGF, BDNE and NT3 respectively. Activation of Trka leads to survival and differentiation and its inhibition leads to programmed cell death. High levels of Trka expression correlate with younger age, lower stage and absence of MYCN amplification and is correlated with favorable outcome, propensity to differentiate or regress.

In contrast, expression of Trkb is strongly associated with aggressive behavior and MYCN amplification representing an autocrine or paracrine pathway providing growth advantage to the tumor cells as well as contributing to drug resistance. Targeted inhibition of this pathway may provide therapeutic benefit (1, 14).

The challenge of the next decade is to translate all this information into more effective and less toxic therapy for these children and help predict outcome and optimizing strategies for appropriate management in neuroblastomas.

References

1. Brodeur GM, Maris JM. Neuroblastoma In: Pizzo PA & Poplack DG eds. Principles and Practice of Pediatric Oncology, fifth edition. Philadelphia: Lippincott Williams & Wilkins, 2006; 933-70.

2. Sebire NJ. Histopathological features of pretreatment neuroblastoma are of limited clinical significance following adjustment for clinical and biological marker status. Med Hypotheses 2006; 66:1078-81.

3. Perez CA, Matthay KK, Atkinson JB, et al. Biologic variables in the outcome of stages I and II neuroblastoma treated with surgery as primary therapy: a children’s cancer group study. J Clin Oncol 2000; 18: 18–26.

4. Maris JM. The biologic basis for neuroblastoma heterogeneity and risk stratification. Curr Opin Pediatr. 2005; 17: 7-13.

5. Corvi R, Amler LC, SavelyevaL, et al. MYCN is retained in single copy at chromosome 2 band p 23-24 during amplification in human neuroblastoma cells. Proc Natl Acad Sci USA 1994; 91: 5523 - 7.

6. Green AA, Hayes FA, Rao B. Disease control and toxicity of aggressive 4 drug therapy for children with disseminated neuroblastoma. Proc Am Soc Clin Oncol 1986; 5: 210.

7. Tajiri T, Tanaka S, Higashi M, et al. Biological diagnosis for neuroblastoma using the combination of highly sensitive analysis of prognostic factors. J Pediatr Surg 2006 ; 41: 560-6.

8. Seeger Rc, Brodeur GM, Sather H, et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 1985; 313: 1111–6.

9. Mathew P, Valentine MB, Bowman LC, et al. Detection of MYCN Gene Amplification in Neuroblastoma by Fluorescence In Situ Hybridization: A Pediatric Oncology Group Study. Neoplasia 2001; 3: 105–9.

10. Layfield LJ, Willmore-Payne C, Shimada H, Holden JA. Assessment of NMYC amplification: a comparison of FISH, quantitative PCR monoplexing and traditional blotting methods used with formalin-fixed, paraffin-embedded neuroblastomas. Anal Quant Cytol Histol 2005 ; 27 : 5-14.

11. Boensch M, Oberthuer A, Fischer M et al. Quantitative real time PCR for quick simultaneous determination of therapy stratifying markers MYCN amplification, deletion 1p and 11q. Diagn Mol Pathol 2005 ; 14: 177-82.

12. Melegh Z, Bálint I, Tóth E, et al. Detection of n-myc gene amplification in neuroblastoma by comparative, in situ, and real-time polymerase chain reaction. Pediatr Pathol Mol Med 2003; 22: 213-22.

13. Boensch M, Oberthuer A, Fischer M et al. Quantitative real time PCR for quick simultaneous determination of therapy stratifying markers MYCN amplification, deletion 1p and 11q. Diagn Mol Pathol 2005; 14: 177-82.

14. Vasudevan SA, Nuchtern JG, Shohet JM. Gene profiling of high risk neuroblastoma. World J Surg 2005; 29: 317-24.

15. Spitz R, Betts DR, Simon T, et al. Favorable outcome of triploid neuroblastomas: a contribution to the special oncogenesis of neuroblastoma. Cancer Genet Cytogenet 2006; 167: 51- 6.

Molecular Genetics of Colorectal Carcinoma

Mukta Ramadwar

Remarkable progress has been made in past 20 years in the understanding of genetic basis of cancer development and progression. Colorectal carcinoma (CRC) has become the paradigm on which our current theories of cancer genetics are based (1). Identification of specific syndromes such as FAP and HNPCC have helped delineate specific molecular pathways and mechanisms in CRC (2).

Colon cancer occurs in one of three clinical settings, each with different molecular characteristitics.

1. A vast majority (70%) are sporadic carcinomas seen in elderly individuals at age older than 50 years. Diet, environmental factors and normal aging are implicated as etiological factors (3).

2. About 10% of CRC patients have inherited predisposition. The inherited syndromes are broadly divided into polyposis (FAP and hamartomatous polyposis syndromes) and HNPCC syndromes. The familial, clinical, histological, and molecular features of inherited syndromes are well delineated.

3. Familial colon cancer affects families far too frequently for sporadic carcinoma and also do not show patterns of inherited syndromes.

A well accepted multi-step genetic model of CRC progression is proposed by several investigators (1). The model describes an accumulation of genetic events, each conferring a selective growth advantage to an affected colon cell, thus facilitating cell survival and subsequent cancer progression. The cumulative effect of these somatic mutations is the cause of sporadic colon cancer. It can be inferred from this model that CRC results from the mutational activation of oncogenes and inactivation of tumour suppressor genes. Accumulation of multiple genetic mutations rather than the sequence of mutations determine the biological behavior of the tumour. Several genes are involved in this stepwise carcinogenesis of sporadic CRC. e.g. Activation of oncogene RAS leads to constitutive activity of the protein, which results in a continuous growth stimulus. Inactivation of tumour suppressor genes APC, p53, and DCC plays an important role in sporadic CRC. APC mutations occur early while p53 occur late in the process.

FAP is a dominantly inherited syndrome in which affected persons develop hundreds to thousands of colonic polyps with risk of cancer development approaching 100% by the age of 50 years. APC gene is a tumour suppressor gene. It controls the WNT signaling pathway via regulation of beta-catenin levels (1). Inactivation of APC leads to beta-catenin stabilization and subsequent activation of various other genes such as MYC, CCND1 etc. These mutations are directly associated with the development of thousands of colonic adenomas at a very young age. The cancer progression in FAP syndrome also follows the same steps. However, a major difference between sporadic cancer and FAP is the presence of germline APC mutation in FAP as against somatic mutation in sporadic CRC. A germline mutation is inherited and ispresent throughout the genome making more than one organ susceptible to development of tumours. When a tumour suppressor gene (e.g. APC gene in CRC) is inherited as a germ line mutation, only mutation of the remaining normal allele is required before the loss of function of the gene. When both copies of the genes are normal, two mutation events are needed before the gene function is lost. This two-hit hypothesis explains why FAP manifests at an earlier age than sporadic cancer and also emphasizes the mechanism of carcinogenesis mediated by tumour suppressor genes (4).

Inactivation of both APC alleles in even very small tubular adenomas and also in aberrant crypt foci provides critical evidence supporting the ‘gatekeeper’ role of APC. Almost all mutations of the APC gene cause premature termination or truncation of its protein product which provides basis for the most frequently used genetic screening test for FAP.

Familial colon cancer results due to an I1307K APC germline mutation. This mutation causes a predisposition to sporadic mutations at distant sites of the gene resulting in protein structural abnormalities.

HNPCC syndrome

The criteria for this syndrome were established in 1991 in Amsterdam by the International Collaborative Group on HNPCC (4). These criteria were rather stringent to ensure studying individuals and families with the same syndrome. Later, mutations of MMR genes genes as a genetic basis of HNPCC became apparent and the clinical criteria were modified in 1996. These modified criteria are called Bethesda criteria.

Understanding of functions of MMR genes is central to the carcinogenesis of HNPCC. MMR genes are involved in correcting errors of DNA replication. Mutations of these genes result in abnormal sequences in parts of the DNA known as microsatellites. Microsatellites consist of small sequences of nucleotide bases repeated several times. The resulting abnormalities of these microsatellites are referred to as MSI. Depending on the number of loci detected, MSI is referred either as absent (microsatellite stable MSS), low (MSI- L), or high (MSI-H). Six MMR genes are identified: hMSH2, hMLH1, hMSH 3, hPMS1, hPMS2 and hMSH6 (GTBP). Out of these six genes, mutation of hMLH-1 and hMSH2 mutation are the commonest genetic abnormalities associated with HNPCC, seen in 95% of cases. Both are considered as MSI-H. hMSH6 mutations are uncommonly seen.

It is important to recognize that 10 to 15% of sporadic colon cancers can exhibit MSI (usually hMLH 1). These sporadic CRCs are believed to have better prognosis. MSI positive sporadic CRCs lack APC or KRAS mutations and show hypermethylation of MLH 1 along with BRAF mutations.

Epigenetic mechanisms are heritable changes in DNA other than nucleotide changes. They are now known to be involved in evolution of CRC. Hypermethylation of promoter CpG islands is one of the examples of epigenetic mechanisms involved in CRC which leads to silencing or loss of function of the gene (5). Hypermethylation followed by transcriptional inactivation of MLH 1 gene is found in MSI positive sporadic CRCs. Knowledge of the heritable changes in gene expression as a result of epigenetic events is of increasing relevance to clinical practice, particularly in terms of diagnostic and prognostic molecular markers as well as novel therapeutic targets (6). There is growing evidence from many studies that suggest MSI positive tumours respond differently to traditional chemotherapeutic agents and the outcomes with standard treatment may be worse (7). This might reflect fundamental differences in drug responsiveness, possibly due to epigenetic mechanisms. Inhibitors of DNA methylation are being tried as a part of treatment regime for CRC.

Much progress has been made in our understanding of HNPCC which has led to subdivision of this syndrome on the basis of the underlying germline mutation (8,9). Compared to families with germline mutations in MSH2 and MLH1, families with MSH6 germline mutations have a later age of onset of CRC (10). Moreover, women in these families have a lower risk of CRC, but higher risk of endometrial cancer. Molecular genetic characteristics can be used to classify CRC, sporadic or hereditary (11). A different subset of CRCs called as CpG island methylator phenotype (CIMP) is also identified. These tumours are characterized by many of the features typical of MSI tumours. However, half of these tumours are microsatellite stable. Jass (12) has proposed a new classification of CRC based on type of genetic instability and the presence of DNA methylation.

Five molecular subtypes of CRC are suggested:

1. CIMP- high, methylation of MLH 1, BRAF mutation, chromosomally stable, MSS or MSI-H, origin in serrated polyps, known generally as sporadic MSI-H (12%).

2. CIMP-high, partial methylation of MLH 1, BRAF mutation, chromosome stable, MSS or MSI-L, origin in serrated polyps (8%).

3. CIMP-low, KRAS mutation, MGMT methylation, chromosomal instability, MSS or MSI-L origin in adenomas or serrated polyps (20%).

4. CIMP negative, chromosomal instability, mainly MSS, origin in adenomas (may be sporadic, FAP- associated or MutYH-associated polyposis coli (57%).

5. Lynch syndrome: CIMP negative, BRAF mutation negative, chromosomally stable, MSI-H, origin in adenomas (3%)

Detection techniques for MSI include IHC, PCR and germline testing for DNA MMR gene mutations. The germ line testing can be done by identification of truncated protein products. However, gene sequencing is considered to be the gold standard for testing MMR mutations. Recently, newer and more sophisticated mutation detection techniques such as multiplex ligation dependent probe amplification and conversion technology have shown MMR mutations in the form of genomic rearrangements which were not detected by DNA sequencing earlier. This finding now explains the presence of HNPCC families with MSI positive tumours but no detectable germline mutations.

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant condition characterized by hamartomatous polyps which can occur throughout the GI tract with melanin pigmentation on the lips and buccal mucosa. In addition to GI tract, tumours occur in the pancreas, breast, ovary and testis. Recently, a gene defect leading to PJS has been identified. The STK11 / LKB1 gene appears to play a crucial role in tumours development in PJS. This pathway and mechanism of carcinogenesis is very different in PJS where the polyps are hamartomatous and ‘adenoma-carcinoma sequence’ does not play a major role (13).

Serrated adenomas (SAs) of the colorectum show architectural features of hyperplastic polyps and cytological features of classical adenomas. Molecular studies comparing SAs and classical adenomas suggest that each may be a distinct entity; in particular, it has been proposed that MSI distinguishes SAs from classical adenomas. SAs and the colorectal cancers arising from them develop along a pathway driven by low level microsatellite instability (MSI-L) (14).

To conclude, many molecular genetic processes in colorectal carcinogenesis are being unraveled and have helped define specific syndromes. This information is vital for diagnosis, prognosis and genetic counseling of the affected patients and families (15).

References

1. Redston M. Carcinogenesis in the GI tract: From morphology to genetics and back again. Mod Pathol 2001; 14: 236-45.

2. Konishi M, Kikuchi R, Tanaka K, et al. Molecular nature of colon tumours in Hereditary Nonpolyposis Colon Cancer, Familial Polyposis and sporadic colon cancer. Gastroenterology 1996; 111: 307-17.

3. Ogino S, Brahmandam M, Cantor M, et al. Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component. Mod Pathol 2006; 19: 59-68.

4. Calvert P, Frucht H. The genetics of Colorectal Cancer. Ann Intern Med 2002: 137: 603-12.

5. Baylin S, Herman J. DNA hypremethylation in tumourigenesis. Epigenetics joins genetics. Trends Genet 2000, 16:168-74.

6. Wong J, Hawkins N, Ward R. Colorectal cancer: a model for epigenetic tumourigenesis. Gut 2007; 56: 140-8.

7. Mueller-Koch Y, Vogelsang H, Kopp R, et al. Hereditary non-polyposis colon cancer: clinical and molecular evidence for a new entity of hereditary colorectal cancer. Gut 2005; 54: 1733-40.

8. Lynch H, Lynch J. Hereditary Cancer: family history, diagnosis, molecular genetics, ecogenetics, and management strategies. Biochimie 2002; 84: 3-17.

9. Gruber S. New developments in Lynch Syndrome (Hereditary non polyposis colorectal cancer) and mismatch repair gene testing. Gastroenterology 2006; 130: 577-87.

10. Valle L, Perea J, Carbonell P, et al. Clinicopathologic and pedigree differences in Amsterdam I positive hereditary nonpolyposis colorectal cancer families according to tumour microsatellite instability status. J Clin Oncol 2007; 25: 781-6.

11. Gardy W. Molecular basis for subdividing hereditary colon cancer ? Gut 2005; 97: 1676-8.

12. Jass J. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology 2007; 50: 113-30.

13. Entius M, Keller J, Westerman A, et al. Molecular genetic alterations in hamartomatous polyps and carcinomas of patients with Peutz-Jeghers syndrome. J Clin Pathol 2001; 54: 126-31.

14. Sawyer EJ, Cerar A, Hanby AM, et al. Molecular characteristics of serrated adenomas of the colorectum. Gut 2002; 51: 200-6.

15. Houlston R. What we could do now: molecular pathology of colorectal cancer. Mol Pathol 2001; 54: 206-14.

Molecular markers and selection of Targeted therapy

Dr. Sudeep Gupta, Dr. Preetesh Jain

Clinical applications of advances in molecular oncology
As advances in cancer therapy have already produced a new generation of innovative and powerful drugs that act on clinically relevant targets, it is now believed that more careful selection of patients using appropriate biomarkers will further improve the therapeutic benefits. All the impressive data generated by sophisticated tools have resulted in better characterization of the signaling pathways involved in tumor growth, insensitivity to anti-growth and apoptotic signals, resistance to radiation and in mechanism of invasion or metastasis using sustained angiogenesis. These advances revealed a myriad of potential candidates for targeted drugs (1).

Basic principles of cancer biology
Cancer growth and metastasis are modulated by the interplay of tumor cells, their interaction with the tumor stroma and systemic influences. The interaction of growth factor ligands with receptors on the malignant cell surface initiates a cascade of enzymatic reactions aimed at transducing a dual signal that stimulates cell proliferation and inhibits apoptosis (programmed cell death) (2). Growth factors may be generated by the tumor cells (autocrine production), stromal cells (paracrine production) or by distant organs (endocrine- e.g., insulin-like growth factor 1 or erythropoietin may act as tumor growth stimulating factors).The production of growth factors in the tumor stroma may be under endocrine control (e.g., the production of insulin-like growth factor 1 is under control of the growth hormone; the production of transforming growth factors a and b may be stimulated by estrogen and other sex hormones).

Some of the prime targets for therapy therefore include:

• Receptor bound tyrosine kinases that are responsible in initiating the signal transduction cascade.
• Cytoplasmic tyrosine kinases, of which the best characterized is the one encoded by the BCRABL gene found in CML.
• Farnesyl transferases that modulate signal transduction.
• Mammalian Target of Rapamycin (mTOR), that plays a key role in stimulating the reproduction of the cell and impeding apoptosis.
• Growth factors produced by both the neoplastic cells and the stroma responsible for stimulating the growth of endothelial cells needed for neo-angiogenesis.
• Structural components of the tumor cells that may be targeted by specific antibodies include for e.g. the CD20 antigen present in virtually all malignancies of B-cells, targeted by rituximab and the CD52 antigen expressed in most indolent lymphoid malignancies targeted by alemtuzumab (3).
• Epigenetic changes that allow the unlimited proliferation of the tumor cells by silencing tumor-suppressor genes.

These include DNA hypermethylation that is reversed by 5-azacitidine and decitabine and histone deacetylase that is reversed by a number of substances, including valproic acid (4).

The proteosome responsible in catabolizing substances that reverse inhibition of apoptosis (5).

Metalloproteinases that are lytic enzymes that allows tumor infiltration to surrounding tissues and metastasis to distant tissues (6).

Drugs utilized for targeted cancer treatment

I. Monoclonal antibodies may be used in at least three capacities:

• Promote the destruction of the neoplastic cells by the immune system. Rituximab, is targeted on the CD20 antigen present in B lymphocytes, and alemtuzumab on the CD52 in B and T cells. Rituximab has improved the prognosis of virtually all B cell malignancies, alemtuzumab has proven effective both in B and T cell malignancies, and is being used also as immunosuppressant prior to bone marrow transplant.
• Carry radioisotope or cytotoxic substances within the neoplastic cells. Tositumomab, and ibritumomab tiuxetan, are monoclonal antibodies directed to CD20 and tagged with radioactive iodine and yttrium, respectively. They have proven very effective in follicular lymphomas resistant to chemotherapy. Gemtuzumab ozogamicin is a monoclonal antibody targeted to the myeloid antigen CD33 that is combined with calicheamycin, a powerful cytotoxic antibiotic. This drug has some activity in AML.

Interfere with the activation of growth factor receptors. The best known of these antibodies is trastuzumab that prevents the dimerization of the epidermal growth factor receptor 2 (EGFR 2) in breast cancer and has led to improved survival of patients whose tumor over-expresses this receptor. Cetuximab and panitumab target the EGFR 1 and have proven effective in patients with colorectal cancer CRC. Cetuximab has also improved the prognosis of SCCHN patients on radiotherapy (RT). Bevacizumab is targeted to the vascular endothelial growth factor (VEGF) and prevents neo-angiogenesis. This compound has some single agent activity in several cancers, but its most important effect has been synergism with cytotoxic chemotherapy. By preventing neo-angiogenesis, it prevents the increase in interstitial pressure in the tumor and favors the diffusion of chemotherapy to the tumor cells. In combination with chemotherapy it has improved the response rate and survival of patients with CRC, and the response rate of breast and non-small cell lung cancer (1).

II. Inhibitors of the signal transduction cascade
The enzyme tyrosine kinase plays a key role in signal transduction. At least two important types of tyrosine kinase have been recognized: one is cytoplasmic and unrelated to a receptor; the other is part of the receptor complex and becomes active in response to receptor–growth factor interaction. The best known of the cytoplasmic tyrosine kinases is the one encoded by the BCRABL oncogene resulting from the 9-22 chromosomal translocation in CML. Inhibition of this enzyme by imatinib or dasatinib has prolonged hematological and cytogenetic remission in patients with CML. A number of small molecules such as gefitinib and erlotinib inhibit the tyrosine kinases that are part of the EGFR. Both drugs show activity in a small group of non small cell lung cancer patients, including women with no smoking history and Asians. Erlotinib also has some activity in pancreatic cancer.

An inhibitor of the EGFR2 tyrosine kinase, lapatinib, was recently reported to be very effective in HER-2 positive breast cancer resistant to trastuzumab. Sunitinib and sorafenib, inhibitors of different receptor bound kinases, including those related to the VEGF and platelet-derived growth factor (PDGF) receptors have doubled the survival of patients with metastatic renal cell carcinoma. The enzyme farnesyl transferase has also an important role in signal transduction. Its inhibition by the experimental agent tipifarnib showed some activity in AML.

mTOR is a serine/ threonine protein kinase and represents a cross road in the signal transduction pathways. Its inhibition by temsirolimus has induced therapeutic responses in renal cell carcinoma, mantle cell lymphoma, lung and breast cancer.

III Drugs that reverse epigenetic changes (4)
DNA hypermethylation and histone deacetylation are responsible for silencing tumor suppressor genes. The inhibition of these processes may lead to differentiation of the neoplastic cells. Two analogs of citidine: azacitidine and decitibine inhibit the DNA methyl-transferase and are effective in inducing a remission in patients with advanced myelodysplastic syndromes (MDS). Drugs inhibiting histone-deacetylase are undergoing clinical trials. “In vitro” they appear synergistic with the methyltransferase inhibitors.

IV Other drugs
A number of agents do not fit any particular category, yet represent a form of targeted therapy. Thalidomide and its derivative lenalidomide have multiple actions including inhibition of angiogenesis and of the production of inflammatory cytokines. It is not clear at this moment which of these actions is responsible for the antineoplastic activity. Both drugs are effective in the management of multiple myeloma and MDS. In MDS, lenalidomide induced a cytogenetic response rate close to 80% in patients with the 5q-mutation, and might have prolonged the survival of these individuals (7). Bortezomib inhibits proteosome 26S responsible for the lysis of ubiquitinated proteins that are waste product of cell metabolism. The inhibition of proteosome activity favors the apoptosis of the cancer cells. This drug has proven effective in multiple myeloma and in a number of lymphomas (8).

Antisense oligonucleotides are small DNA sequence complementary to the mRNA carrying the message of oncogenes. By blocking the expression of the oncogenes these agents may inhibit tumor growth and restore apoptosis. Though promising, none of these agents have found a clear clinical application yet. Likewise, metalloproteinase inhibitors promise to block local invasion and metastasis, but have not yet demonstrated clear clinical activity. A special type of targeted therapy is the oral cytotoxic agent capecitabine. This is a prodrug of the antimetabolite 5- fluoro-uracil that becomes activated in the tumor tissue by thymidine phosphorylase and cytidine deaminase, because neoplastic tissues are richer in these enzymes than normal tissues. As a consequence the effectiveness of the drug is enhanced and the toxicity reduced. This drug represents a model of targeting cytotoxic agents on the tumor and limiting the damage to surrounding tissues.

Undoubtedly, targeted therapy has improved the prognosis of several malignancies. At this time it is important to outline potential limitations related both to the construct of targeted therapy and to its clinical applications.

Criticism of targeted therapy
At least three lines of criticism of this new form of cancer treatment, viz. the novelty of the concept of targeted therapy, the limitations of its activity, and its safety, are legitimate.

Is targeted therapy really a new concept?

For more than four decades cytotoxic chemotherapy has been the mainstay treatment of metastatic cancer. One may argue that even cytotoxic chemotherapy was a form of targeted treatment, on at least three accounts. First, cytotoxic chemotherapy was targeted on proliferating cells. As the growth fraction of neoplastic tissues is generally higher than that of normal tissues, cytotoxic chemotherapy was expected to destroy the tumor with reversible toxicity to the normal tissues. Second, many cytotoxic agents, such as the antimetabolites, inhibit specific enzymes involved in DNA- or RNA-synthesis, that may be considered their specific target. Third, a number of cytotoxic treatments were designed to target the tumor and protect the normal tissues.

For example, high dose methotrexate with leucovorin rescue was designed as a marrow-sparing treatment; pegylated liposomal doxorubicin maintains the activity of doxorubicin with negligible myelosuppression or cardiotoxicity by allowing a slow release of the drug from the liposomal involucres. Thus cytotoxic chemotherapy may certainly be considered a legitimate, albeit rudimentary form of targeted chemotherapy.

At the same time some of the modern targeted agents such as rituximab or alemtuzumab do destroy a number of normal cells, as the antigens that they target are not exclusive to lymphoid neoplastic cells. It is reasonable to conclude that what we call targeted treatment refers to more specific molecular targets that were largely unknown when cytotoxic chemotherapy was first introduced.

Has targeted therapy improved the cancer cure rates?
Undoubtedly, the survival of patients with CML and different forms of lymphoma has improved since the introduction of imatinib, and rituximab respectively, and the recurrence rate of HER2 / neu rich breast cancer has decreased since the introduction of trastuzumab in the adjuvant therapy.

Most targeted therapies, seek to transform cancer into a chronic disease in the metastatic setting rather than to cure cancer.

Even in the case of CML, discontinuance of imatinib has been associated with rapid recurrence of the disease. However, dramatic direct results are obtained when the molecular target is key to the continuous growth of the neoplasm (cytoplasmic tyrosine kinase in CML, HER2 in breast cancers over-expressing this receptor) or when the target is an antigen expressed by all the neoplastic cells (CD20 in B cell lymphomas). In addition, lenalidomide has been very effective in multiple myeloma and MDS, though the exact mechanism of action is still unknown.

In all other cases targeted therapy has led to a modest, albeit significant survival improvement. This may be due both to the diversity of the tumor cells and to the presence of several salvage pathways that allow the signal transduction to proceed despite the inhibition of some key reactions. In these situations the combination of different targeted agents or of targeted agents and chemotherapy appear as reasonable strategies to improve the results. For example, bevacizumab alone has negligible activity in cancer of the large bowel, lung, and breast, but it improves significantly the results of cytotoxic chemotherapy.

Is the targeted therapy safe?
It is now clear that virtually all forms of targeted therapy are not perfect missiles and some form of collateral damage to the normal tissues is unavoidable. Examples of serious forms of toxicity related to targeted therapy include myelodepression, immune suppression, cardiotoxicity, severe acneiform skin reactions, deep vein thrombosis, and magnesium losing nephropathy. Despite these complications in general targeted treatment is safer and better tolerated than cytotoxic chemotherapy.

Molecular markers in clinical oncology
Biochemical markers may serve for early prediction of tumor recurrence, progression and development of metastasis including bone metastasis and for prediction of response to therapy. They are different types of molecular tumor markers including DNA, mRNA, proteins, antigens, or hormones measured quantitatively and/or qualitatively by appropriate assays. Tumor marker assays comprise IHC, quantitative immunoassays, PCR, western or NB and more recently, micro arrays (genomic and proteomic) and mass spectrometry.

CML (9)
The BCR-ABL product displays a constitutively active tyrosine kinase (ABL) that boosts the expansion of undifferentiated myeloid precursors and impinges on normal hematopoiesis. A recent study evaluating imatinib in treatment-naive CML patients versus standard interferon/ cytarabine combinations clearly demonstrates a significant higher efficacy, with a largely superior toxicity profile, making imatinib the new gold standard in this disease. Interestingly, imatinib has a broader spectrum of action by inhibiting other tyrosine kinases (c-Kit and PDGFR) playing a dominant role in the pathogenesis of diseases such as gastrointestinal stromal tumor, chronic myelomonocytic leukemia, hypereosinophilic syndrome, and dermatofibrosarcomas protuberans.

However, despite this success, resistance to imatinib does occur in CML patients, mostly by over expression of BCRABL or mutations within the ABL kinase domain that prevent effective binding of the drug. Interestingly some of these mutations exist before the introduction of the drug. Obviously, detection of these mutations by RT-PCR might play an important role in the clinical management of CML patients as biomarkers predicting treatment failure. The search for independent inhibitors of ABL kinase has been a priority and novel ABL kinase inhibitors Dasatinib and Nilotinib retain activity against many of these mutants.

QPCR provides an accurate measure of the total leukemia cell mass and the degree to which BCR-ABL transcripts are reduced by therapy correlates with progression-free survival. Because a rising level of BCR-ABL is an early indication of loss of response and thus the need to reassess therapeutic strategy, regular molecular monitoring of individual patients is desirable. A consensus meeting at the NIH in Bethesda in October 2005 made the following suggestions (10):

a. Harmonizing the differing methodologies for measuring BCR-ABL transcripts in CML patients undergoing treatment and using a conversion factor whereby individual laboratories can express BCR-ABL transcript levels on an internationally agreed scale

b. Using serial QPCR results rather than bone marrow cytogenetics or FISH for the BCRABL gene to monitor individual patients responding to treatment.

C. Detecting and reporting Ph-positive sub-populations bearing BCRABL kinase domain mutations.

Breast Cancer
A paradigm shift from empirical treatment to an individually tailored approach based on specific molecular profiles may soon become an established option for the treatment of breast cancer patients. RNA expression of individual genes can be detected and quantified by a variety of techniques, such as NB, serial analysis of gene expression (SAGE), and more recently, microarray analysis. Microarray analysis is a method that compares gene expressions between normal and cancerous cells, and is stimulating the discovery of new targets in the treatment of breast cancer. One of the most important advances in understanding the biology of breast cancer has been itsclassification using DNA microarray into subtypes – luminal A & B, basal, HER2 and normal-like types (11). It is now becoming clear that these subtypes are not only prognostic markers but also predictive of benefit from different therapies – luminal A & B from endocrine and basal and HER2 from chemotherapy especially taxane and anthracycline based therapy.

A specific gene-expression profile that reveals significant prognostic information about clinical outcomes in these patients was utilized in a statistical analysis termed “supervised clustering” to identify a set of 70 genes with an expression pattern that allowed highly accurate classification to identify those with a poor prognosis and those with a good prognosis. Mammaprint, the custom-made microarray assay from this 70-gene tumor expression profile, was approved by the US Food and Drug Administration (FDA) in February 2007. This assay yields a score that indicates whether the patient is in a “low risk” or a “high-risk” category for recurrence of cancer. The Mammaprint assay is currently being validated in the MINDACT Trial (Microarray in Node negative Disease may Avoid Chemotherapy) (12).

Twenty years ago, amplification of the ErbB2 tyrosine kinase was identified as a major oncogenic event in 20– 30% of breast cancers that correlate with a high rate of metastatic relapse and poor survival. Not only considered as an interesting biomarker for drug response, ErbB2 is also a target for a humanized monoclonal antibody, trastuzumab. The selection of patients according to the ErbB2 status has a significant impact on the management of the disease. Trastuzumab response is detected in about 15% of heavily pretreated metastatic breast cancer patients, a moderate response rate that could have been missed in an unselected population. As a first-line strategy in the treatment of naive metastatic breast cancers, trastuzumab alone induces roughly a 30% response rate and a better response, progression-free survival and overall survival when combined with chemotherapy (preferentially taxanes). Still based on ErbB2 over expression, trastuzumab is currently under intense evaluation in the adjuvant setting for treatment of early breast cancer.

Detection of ErbB2 over expression by IHC or FISH analysis is therefore crucial to predict the response to trastuzumab but not sufficient because only a fraction of patients respond to treatment. More reliable biomarkers including protein and transcriptional profiles associated with ErbB2 over expression would greatly optimize trastuzumab-based therapy (12). It has been appreciated for sometime that there is considerable variability in the estimation of ErbB2 by both IHC and FISH. The ASCO and CAP convened an expert panel, which conducted a systematic review of the literature and developed recommendations for optimal HER2 testing performance (13). It was found that approximately 20% of current HER2 testing may be inaccurate. When carefully validated testing was performed, available data did not clearly demonstrate the superiority of either IHC or FISH as a predictor of benefit from anti-HER2 therapy. The panel recommended that HER2 status should be determined for all invasive breast cancer. A testing algorithm that relies on accurate, reproducible assay performance, including newly available types of brightfield FISH was proposed. An algorithm defining positive, equivocal, and negative values for both HER2 protein expression and gene amplification was recommended: a positive HER2 result is IHC staining of 3 (uniform, intense membrane staining of 30% of invasive tumor cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2; a negative result is an IHC staining of 0 or 1, a FISH result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8. Equivocal results require additional action for final determination.

Another interesting and potentially useful corollary of the HER2 status has been its interaction with anthracycline chemotherapy. It is now known that the enzyme Topoisomerase 2A (TOP2) exists on the same amplicon as HER2 on chromosome 17. TOP2 is also the target for anthracyclines that have been the mainstay of treatment for breast cancer for several decades. However, anthracyclines also have significant potential for long term cardiac toxicity. Therefore it is of great interest to know which patients preferentially benefit from anthracyclines. Two recent observations have elucidated this matter. In recent BCIRG 006 study of adjuvant trastuzumab in HER2 positive patients it was found that 35% of patients also had co-amplified TOP2 and had an overall better disease free survival compared to the non-coamplified patients. Subset analysis showed that it was this subgroup that derived the greatest benefit from anthracycline chemotherapy. In the non TOP2 coamplified group the non-anthracycline regimen conferred equal benefit with lesser cardiac toxicity (14). A metanalysis of adjuvant studies comparing anthracycline with non-anthracycline regimens showed that the benefit of adjuvant anthracyclines was confined to the patients who overexpressed HER-2 (15). The speculative correlation is with coamplification of TOP2 in a fraction of these latter patients. On January 14, 2008, the US FDA announced the approval of a Premarket Application for a genetic test (TOP2A FISH pharmDx assay, Dako Denmark A/S) to determine the risk for tumor recurrence and long-term survival in patients with relatively high-risk breast cancer.

Although patients diagnosed with axillary node–negative estrogen receptor–positive breast cancer have an excellent prognosis, about 15% of them fail after 5 years of tamoxifen treatment. Clinical trials have provided evidence that there is a significant benefit from chemotherapy for these patients, but it would be significant over treatment if all of them were treated with chemotherapy. Therefore, context-specific prognostic assays that can identify those who need chemotherapy in addition to tamoxifen, or those who are essentially cured by tamoxifen alone, and can be performed using routinely processed tumor biopsy tissue would be clinically useful. Using a stepwise approach of going through independent model-building and validation sets, a 21-gene recurrence score (RS), based on monitoring of mRNA expression levels of 16 cancer-related genes in relation to five reference genes has been developed (16). The RS identified approximately 50% of the patients who had excellent prognosis after tamoxifen alone. Subsequent study of patients in the NSABP-B20 trial suggested that high-risk patients identified with the RS preferentially benefit from chemotherapy (17). Ideally the RS should be used as a continuous variable. A prospective study—the Trial Assigning Individualized Options for Treatment (Rx) (TAILORx)—to examine whether chemotherapy is required for the intermediate-risk group defined by the RS is accruing in North America. The above mentioned 21-gene expression-based recurrence score assay, Oncotype DXTM, was also found to strongly correlate with pathologic complete response in patients treated with neoadjuvant paclitaxel and doxorubicin.

That prognostic and predictive abilities of a marker do not always go hand in hand is exemplified by the recent study that showed that Ki-67 was an independent adverse prognostic marker in node negative patients but not predictive of benefit from adjuvant chemotherapy (18).

Lung Cancer

In a similar manner, discovery of over expression of EGFR, in several human malignancies has made this receptor one of the preferred targets of the pharmaceutical industry. EGFR is one of four transmembrane growth factor receptors that share structural and functional similarities, including EGFR (¼HER1, c-erbB-1), HER-2/neu (c-erbB-2), HER3 (c-erbB-3) and HER4 (c-erbB-4). The EGFR, a 170 kDa glycoprotein, consisting of an extracellular domain, a transmembrane region and an intracellular domain with tyrosine kinase function, responds to numerous ligands, such as transforming growth factor alpha (TGFa), betacellulin, amphiregulin, epiregulin, EGF and heparin binding EGF. Deregulation of EGFR function is a common feature in several human malignancies including lung, breast, colorectal, prostate and HNC. Mechanisms of EGFR activation include -1) receptor over expression in most epithelial malignancies (EGFR in up to 90% of SCCHN; ErbB2 in 3–29%; ErbB3 in 21% and ErbB4 in 26%), 2) constitutively activated EGFR mutants, 3) autocrine activation by ligand over expression (e.g. TGF-a), 4) ligand independent activation through other receptor systems (e.g.ErbB2/HER2), 5) EGFR transactivation by GPCR-induced processing of transmembrane growth factor precursors by ADAM family metalloproteases, 6) gene amplification and/or 7) loss of negative regulatory mechanisms.

Molecular resistance mechanisms include (1) specific EGFR mutations (e.g. EGFRvIII, T790M), (2) constitutive activation of downstream effectors (e.g. loss/inactivation of PTEN, activation of Src, RAS, and STAT3/5), (3) increased angiogenesis (up regulation of VEGF) and (4) the presence of redundant tyrosine kinase receptors (e.g. HER-2, c-MET, IGF-1R). Many anti-EGFR Mabs are unable to bind the aberrant extracellular domain of EGFRvIII and thus fail to inhibit ligand-induced receptor activation. Interestingly, resistance caused by the T790M mutation can be overcome by CL-387785, a specific and irreversible anilinoquinazoline EGFR inhibitor.

Clinical developments of EGFR antagonists (gefitinib and erlotinib tyrosine kinase inhibitors or monoclonal antibodies) were carried out in an unselected population of patients because EGFR is highly expressed in many tumors. Despite the authentic anti-tumor effects (between a 10 and 20% response rate) of these inhibitors in heavily pre-treated patients, evaluation of a combination of standard chemotherapy and small-molecule tyrosine kinase inhibitors in randomized studies in advanced NSCLC patients did not identify any benefit over chemotherapy alone (19, 20). Even though the hypothesis of an antagonistic effect between tyrosine kinase inhibitors and chemotherapy was discussed, it was rapidly suspected that the absence of biomarkers able to identify EGFR-dependent tumors impaired the success of this therapy. Such a hypothesis was recently confirmed by the identification of activating mutations within the kinase domain of EGFR of gefitinib-responding patients with lung cancers that sensitize tumors to treatment (21, 22). EGFR mutations were predominantly found in female subjects, nonsmokers, adenocarcinomas, and Japanese patients. These patient characteristics precisely coincide with those with a high response rate to EGFR-TKIs. EGFR mutation and high gene copy number were associated with better objective response in univariate analysis. The main core of recent research has centered on EGFR mutations and gene copy numbers. For the first time, EGFR mutations have been shown to predict dramatic responses in metastatic lung adenocarcinomas, with threefold increase in time to progression and survival in patients receiving EGFR tyrosine kinase inhibitors. In contrast, K-ras mutations confer a negative effect in these patients.

Abundant pre-clinical and clinical data indicate that BRCA1 mRNA expression is a differential modulator of chemotherapy sensitivity. Low levels predict cisplatin sensitivity and antimicrotubule drug resistance, and the opposite occurs with high levels. Secondly, SNPs in the excision repair cross-complementing (ERCC) 1 gene influence survival and toxicity with cisplatin based chemotherapy. Cisplatin resistance is associated with increased expression of the ERCC1 gene (23, 24).

Evidence has also been accumulating on the crosstalk between estrogen and EGFR receptor pathways, paving the way for clinical trials of EGFR tyrosine kinase inhibitors plus aromatase inhibitors. MicroRNAs control the expression of cognate target genes, and down regulation of Dicer (ribonuclease in RNase III family) has been shown to be a strong predictor of relapse in surgically resected non-small-cell lung cancer patients.

Colorectal Cancer

Despite growing insights into molecular biology of CRC the reality to date is that no molecular marker has made it into routine clinical use with the possible exception of MSI. Most molecular markers have not yet even successfully cleared the stage of replication in independent patient sets, hampering any initiatives at prospective validation. Approximately 15% of CRCs are characterised by MSI, reflecting inactivation of the MMR genes. The remaining 85% of CRCs develop from the chromosomal instability pathway, characterized by aneuploidy, allelic losses, amplifications, and translocations. Cancers developing from each of these pathways show a number of specific differences. Tumors with high frequency MSI tend to be more proximal, poorly differentiated, mucinous, and show marked lymphocyte infiltration. A number of studies have investigated the relationship between MSI status and survival in CRC patients. Although many have reported better survival with MSI, estimates of the prognostic value of MSI between studies have differed considerably. In one study the presence of 18q LOH was associated with poorer overall survival. They also studied MSI, which on its own was not related to significantly improved survival. However, MSI together with the transforming growth factor-beta 1 RII mutation was associated with improved outcome when compared with presence of MSI without the mutation (25).

In patients with chemotherapy-refractory metastatic colorectal cancer, the clinical efficacy of panitumumab (Vectibix) monotherapy appears to be restricted to patients with wild-type KRAS tumors, according to data presented at the 2008 Gastrointestinal Cancers Symposium (GCS). Panitumumab was able to induce a partial response in patients with nonmutated KRAS tumors, but had limited efficacy in patients whose tumors contained the mutant form of the KRAS gene (26). In this phase 3 randomized controlled trial, KRAS status was evaluated among patients with metastatic CRC who received either panitumumab 6 mg/kg every 2 weeks plus best supportive care, or best supportive care alone. The primary objective of the study was to examine whether the efficacy of panitumumab on progression-free survival was significantly greater in patients with wild-type KRAS than in those with tumors containing the mutated gene. They found that median progression-free survival for patients treated with panitumumab who had wild-type KRAS was 12.3 weeks; for those with the mutated gene, median progression-free survival was 7.4 weeks. The median progression-free survival for patients in both KRAS groups who received best supportive care only was 7.3 weeks. Among patients with wild-type KRAS who were treated with panitumumab, 17% responded and 34% had stable disease. Among patients with the mutated KRAS gene, 0% responded and 12% had stable disease. When the 2 treatment groups were combined, the overall survival was longer in patients with wild-type KRAS than in those with mutated KRAS. A similar finding has been reported for cetuximab. Both drugs are antibodies that inhibit EGFR, and they are expensive. Patients with KRAS mutations have zero response to these drugs. Not using these expensive products in such patients would save money, and restricting the use of these drugs to patients who have tumors without KRAS mutations enriches the likelihood that these patients will respond to therapy.

Head and Neck cancers (27)

The EGFR is a particularly interesting target as it plays an important role in regulation of cellular proliferation, differentiation and survival of epithelial cells and tumors of epithelial cell origin. SCCHN overexpress EGFR when compared to normal mucosa. Additionally, aberrant EGFR signaling imparts SCCHN cells with classic tumor cell characteristics, including decreased apoptosis, enhanced invasiveness, migration, angiogenesis and metastasis. Furthermore, EGFR is over expressed in approximately 90–100% of SCCHN specimens and has been associated with worse prognosis, including advanced stage, poorly differentiated tumors and poor survival. Across the board, high EGFR expression on the cell membrane of head and neck cancer cells as well as its nuclear localization have been shown to be markers of poor prognosis, with inferior progression-free and overall survival.

Cetuximab, a recombinant human/mouse chimeric EGFR monoclonal antibody is a targeted agent that has seen expanding indications in recent years. After first gaining US FDA approval for CRC patients with irinotecan-refractory disease, cetuximab earned an expanded indication in 2006 for head and neck cancer. In phase II Trials of second line therapy in platinum refractory disease response rates of 2.6%, 10%, 6 to 20%, and 13% with median survival of 3.5,6, 4.3 to 6.1 and 5.9 months respectively have been noted. A large, international, multicenter, phase III study examining the effect of cetuximab and concurrent radiation therapy on locoregional control for locally advanced, unresectable SCCHN has been recently reported (28). Patients with stage III and stage IV non-metastatic disease were randomized to receive radiation therapy or radiation with weekly cetuximab. Median duration of locoregional control was 24.4 months for combined therapy versus 14.9 months for RT alone (p=0.005); this translated into a 32% reduction in the risk of locoregional progression. Median survival was also significantly improved: 49 months for patients receiving cetuximab and RT versus 29.3 months for those receiving radiotherapy alone (p = 0.05).

It must be emphasized that the expression levels of EGFR have not been found to correlate with response to EGFR directed therapies in multiple tumor types (29). Therefore currently there is no role for EGFR estimation by any technique prior to administration of these therapies.

Conclusions
An increasing number of molecularly targeted drugs have been investigated and introduced for routine clinical use in the recent years. The molecular correlates of benefit (or otherwise) from these drugs in selected patient subgroups is only beginning to be understood. It is likely that in the near future patients within a broad diagnostic category (e.g. breast cancer) would be evaluated and treated based on the molecular portrait of more homogeneous subgroups. The development, validation and integration of robust, reproducible and affordable tests to reliably define these subgroups remain a challenge for clinical pathologists and oncologists.

References

1. Balducci L. Molecular insight in cancer treatment and prevention. Int J Biochem Cell Biol 2007; 39: 1329–36.

2. Faivre S, Djelloul S, Raymond E. New paradigms in anticancer therapy: Targeting multiple signaling pathways with kinase inhibitors. Semin Oncol 2006; 33: 407–20.

3. Rai KH. Novel therapeutic strategies with alemtuzumab for chronic lymphocytic leukemia. Semin Oncol 2006; 33 (2 Suppl. 5): S15–S22.

4. Gore SD. Six (or more) drugs in search of a mechanism: DNA methyltransferase and histone deacetylase inhibitors in the treatment of myelodysplastic syndromes. J Natl Compr Canc Netw 2006; 4: 83–90.

5. Nalepa G, Rolfe M, Harper AG. Drug discovery in the ubiquitin–proteasome system. Nat Rev Drug Discov 2006; 5: 596–613.

6. Sang QX, Jin Y, Newcomer PG, et al. Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases. Curr Top Med Chem 2006; 6: 289–316.

7. Richardson PG, Mitsiades C, Hideshima T, et al. Lenalidomide in multiple myeloma. Expert Rev Anticancer Ther 2006; 6: 1165–73.

8. Cavo M. Proteasome inhibitor bortezomib for the treatment of multiple myeloma. Leukemia 2006; 20; 1341–52.

9. Guilhot F. Indications for imatinib mesylate therapy and clinical management. Oncologist 2004; 9: 271–81.

10. Hughes TP, Deininger MW, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors– review and recommendations for ‘harmonizing’ current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006; 108: 28–37

11. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast Tumors. Nature 2000; 406: 747–52.

12. Barginear MF, Bradley T, Shapira I, Budman DR. Implications of applied research for prognosis and therapy of breast cancer. Crit Rev Oncol Hematol 2008; 65: 223–34.

13. Wolff AC, Hammond MEH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. J Clin Oncol 2007; 25: 118-45.

14. Slamon D, Eiermann W, Robert N, et al. Phase III randomized trial comparing doxorubicin and cyclophosphamide followed by docetaxel (AC®T) with doxorubicin and cyclophosphamide followed by Docetaxel and trastuzumab (AC®TH) with docetaxel, carboplatin and trastuzumab (TCH) in HER2 positive early breast cancer patients: BCIRG 006 study. San Antonio Breast Cancer Symposium, Breast Cancer Research and Treatment Contents Vol. 94, Supplement 1, December 2005, Abstract 1.

15. Gennari A, Sormani MP, Pronzato P, et al. HER2 Status and Efficacy of Adjuvant Anthracyclines in Early Breast Cancer: A Pooled Analysis of Randomized Trials. J Natl Cancer Inst 2008; 100: 14–20.

16. Paik S. Development and Clinical Utility of a 21-Gene Recurrence Score Prognostic Assay in Patients with Early Breast Cancer Treated with Tamoxifen. Oncologist 2007; 12: 631-5.

17. Paik S, Tang G, Shak S, et al. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol 2006; 24: 3726-34.

18. Viale G, Regan MM, Mauro G, et al. Predictive Value of Tumor Ki-67 Expression in Two Randomized Trials of Adjuvant Chemoendocrine Therapy for Node-Negative Breast Cancer. J Natl Cancer Inst 2008; 100: 207-12.

19. Fukuoka M, Yano S, Giaccone G, et al. Multiinstitutional randomized phase II trial of gefitinib for previously treated patients with advanced nonsmall cell lung cancer (The IDEAL 1 Trial). J Clin Oncol 2003; 21:2237-46.

20. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: A randomized trial. JAMA 2003; 290: 2149-58.

21. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350: 2129-39.

22. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004; 304:1497-500.

23. Rosell R, Cuello M. Usefulness of predictive tests for cancer treatment. Bull Cancer 2006; 93: E101-8.

24. Giaccone G, Rodriguez JA. EGFR inhibitors: What have we learned from the treatment of lung cancer? Nat Clin Pract Oncol 2005; 2: 554–6.

25. Tejpar S. The use of molecular markers in the diagnosis and treatment of colorectal cancer. Best Pract Res Clin Gastroenterol 2007; 21:1071–87.

26. Amado RG, Wolf M, Freeman D. 2008 Gastrointestinal Cancers Symposium (GCS): Abstract 278. Presented on January 26, 2008.

27. Tourneaua CL, Faivre S, Siu LL. Molecular targeted therapy of head and neck cancer: Review and clinical development challenges. Eur J Cancer 2007; 43: 2457-66.

28. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006; 354: 567–78.

29. Bailey LR, Kris M, Wolf M, et al: Tumor epidermal growth factor receptor (EGFR) expression levels does not predict for response in patients (pts) receiving gefitinib (“Iressa,” ZD1839) monotherapy for pretreated advanced non-small-cell lung cancer (NSCLC): IDEAL 1 and 2. Proc Am Assoc Cancer Res 2003; 44: 1362 (Abstract LB-70).

Glossary