Guidelines for Molecular Diagnostics in Oncology
Volume VII, 2008 |
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Editors Published by
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Contents
1. Inherited Cancer Predisposition and Issues in Genetic Testing
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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.
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.
Dr. (Ms.) K.A. Dinshaw,
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Contributors
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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 Need for Mutation Analysis and Molecular Diagnostics in Hereditary Cancers Ethical, legal and social issues in genetic testing 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 Counseling, Screening and prophylactic treatments for high risk individuals Cancer genetics services in India Hereditary breast and ovarian cancer and BRCA1 and BRCA2 genes 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 Li-Fraumeni syndrome (LFS) and TP53 mutation
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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 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.
3.1 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%. 3.4 Evidence from randomized trials 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 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 6. Surveillance after treatment of CIN 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 8. Age group for 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 10. Conclusions 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: 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: 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.
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Translocation based diagnosis of bone and soft tissue sarcoma Selected Abstracts Pfeifer JD, Hill DA, O’Sullivan MJ, Dehner LP. Histopathology 2000; 37: 485-500. 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. 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. 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. 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. 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. 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. 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.
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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. 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:
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). 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 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.
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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? Where to look for tumour cells? When to look? How to look? What to look for? What to make of the outcome? 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 |
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Evaluation of Prognostic and Predictive Role of HER 2 in Breast Cancer Roshni Chinoy, Poonam Panjwani,
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).
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Role of HER2 in breast cancer a. As a predictive factor 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
Avoid scoring DCIS; score only the infiltrating component of the carcinoma. 1. Positive HER2 test (score 3+): Crisp membrane staining in >30% of cells ( as per revised ASCO/CAP guidelines).
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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). The results of the test are interpreted after counting of at least 60 cells (1) as follows: 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. 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:
Non-optimal enzymatic digestion (poor nuclear resolution, persistent auto fluorescence).
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.
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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. 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 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.
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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.
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1. Oligodendroglial tumors 1.1 Genetic / molecular alterations in Oligodendroglial tumors 1.2 Loss of chromosomes 1p/19q in Oligodendroglial tumors: Diagnostic and prognostic implications 1.3 Other genetic abnormalities
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2 Astrocytic tumors
2.1.1 p53 mutations 2.1.2 EGFR amplification/ over expression 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). 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) 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 Summary 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: 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.
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Evidence for Use of Molecular Methods in Lymphomas Tanuja Shet Role of molecular methods in lymphomas A. Diagnosis & classification of lymphomas: Today we are in the “Molecular era” and it is clear that ‘‘Morphology alone is often not sufficient for a definitive diagnosis’’ (1). While 90% of lymphomas can be diagnosed and managed based on morphology and IHC alone, the remainder pose a problem. Molecular methods help resolve the following diagnostic gray zones (2):
B. Prognostic markers & Predictors: With the advances and validation of molecular methods, molecular analysis has become an integral ancillary investigational component in clinical management. It is clear that pathologist needs to accurately classify, prognosticate and subtype lymphomas in view of wider choices of therapies now available (1). Molecular techniques also help us prognosticate lymphomas better. e.g. ALK 1 in ALCL is a prognostic marker. Likewise demonstration of t(1;14) and t(11;18) translocations and overexpression of BCL10 in gastric mucosa associated lymphoid tissue lymphoma convey adverse prognosis (3). In B cell CLL, ZAP-70 is a strong predictor of the need for treatment (1). The six gene signature by gene profiling is a predictor of better response to treatment in DLBL (4). With the advent of targeted therapies, it is now becoming important to carry the tests for expression of certain molecules e.g. NFkappaB etc. C. Staging: The stage of tumor cell differentiation can be detected using molecular methods e.g. Germinal Vs post germinal centre differentiation in DLBL and this will help us stratify, treat and prognosticate DLBL better. Molecular methods are also used for detection of MRD in bone marrow in follicular and MCL. D. Biology of disease: Advances in molecular medicine have contributed to fundamental insights into the genetic basis of pathogenesis of these diseases and thus have helped us understand nature and behavior of certain lymphomas. e.g. It is clear now that HL is a B cell neoplasm. Molecular studies have also illustrated pathways of action and role of viruses in various lymphomas. e.g. role of Epstein Barr virus in the etiopathogenesis of HL (5). Material for molecular studies (6)
Which technique and why? (2) Targets to be assessed
Techniques Three most common techniques used in lymphomas are – PCR, SB & FISH. While RT-PCR based assays are used for both clonality assessment and chromosomal translocation, FISH is mainly used to assess amplification and translocation. SB is not commonly used for routine laboratory molecular diagnosis due to its tedious nature. Each test has its own merits and demerits and Table 1 gives a brief overview of which test is popularly used for a given translocation (2). 1. PCR & RT PCR based assays Disadvantages & factors affecting PCR results are
There is a critical need to standardize PCR technique given the variability of procedure followed in different laboratories. A large-scale interinstitutional European collaborative effort (BIOMED-2 Concerted Action) has suggested optimal primer set combinations to increase the sensitivity of clonal detection and to achieve some standardization in the methodology (5,8). It is important to optimize each assay in individual laboratory, to follow accepted guidelines for the performance and interpretation of tests and to be aware of the sensitivity and limitations of each assay being used. 2. Southern blot (SB) 3. FISH The FISH test involves detection of chromosomal translocations in tumors by using specific fluorescent labeled probes.
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Minding our T’s and B’s! (9) Problems in B cell clonality assessment 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. 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). 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).
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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: 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 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
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.
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An Algorithm for molecular monitoring of Chronic Myeloproliferative Disorders Hari Menon, Purvish Parikh Introduction 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 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 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?
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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.
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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 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 JAK2 inhibitors – Is there a potential JAK2V617F monitoring 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: 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.
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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 A. MYCN 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 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 C. Unbalanced gain of 17q D. Deletion of 11q E. Tumor cell DNA content Other chromosomal abnormalities are 3p deletion, 4p, 9p, 14q and 19q associated abnormalities all of which have aggressive behavior. F. Expression of neurotropin receptors 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.
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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.
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.
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Molecular markers and selection of Targeted therapy Dr. Sudeep Gupta, Dr. Preetesh Jain Clinical applications of advances in molecular oncology
Some of the prime targets for therapy therefore include:
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).
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Drugs utilized for targeted cancer treatment
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). 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) IV Other drugs 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
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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? 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? Molecular markers in clinical oncology
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CML (9) 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 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).
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Lung Cancer 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.
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Colorectal Cancer 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.
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Head and Neck cancers (27) 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 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).
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