Welcome. THE ROLE OF oncofish cervical ASSESSMENT OF CERVICAL DYSPLASIA. March 26, 2013
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1 THE ROLE OF oncofish cervical IN THE ASSESSMENT OF CERVICAL DYSPLASIA The phone lines will open, 15 minutes prior to the start of the webinar. Toll Free: Entry Code: You may download a copy of the handout by clicking on the handout icon, located in the upper right hand corner of your screen. THE ROLE OF oncofish cervical IN THE ASSESSMENT OF CERVICAL DYSPLASIA March 26, 2013 Welcome Your Host: Karen Riba Handout is available by clicking on the handout icon the upper right-hand corner of your screen For technical difficulties please kchandler@paml.com. For questions you have during the presentation use the Q & A link at the top of your screen Questions will be answered at the end of the presentation in 1
2 Welcome P.A.C.E. credit may be obtained by submitting your completed evaluation form at the end of the webinar CE credit may be obtained by downloading the Certificate of Completion PAML employees will be able to receive one hour of continuing education. Robert J. Walat, M.D.,FCAP Dr. Walat is the Medical Director for Ikonisys Clinical Laboratory (New Haven, CT). He received his medical degree and postdoctoral training in anatomical and clinical pathology from the Yale School of Medicine and the Yale Medical Center. Learning Objectives At the end of this presentation participants will be able to: Describe the problems and shortcomings of current tests used in cervical screening Recognize the limitations of HPV testing Discuss how 3q26 fits into the oncogenetic process Explain the benefits of oncofish cervical testing for their patients with LSIL or ASCUS/HPV+ 2
3 CURRENT TESTS Thin-layer cytology (ThinPrep; SurePath) Computer-assisted screening Quality control manual rescreening High risk HPV DNA detection Genotyping (HPV16/18) Immunohistochemistry and molecular pathology (p16, ki67,hpv CISH, E6-E7 mrna, etc.) Cervical Cancer Prevention: Where have we been and where are we going? Widespread introduction of the Pap begins Markers Conventional Pap smear LBC HPV testing Vaccine s IMPORTANCE OF EARLY RISK STRATIFICATION It is very important for the cervical screening program to identify, as early as possible, which women with abnormal pap smears are not at risk for advancing to serious pre-malignant or malignant lesions (90%) from those who are at risk for progression to serious lesions (10%) for the following reasons: To relieve patient anxiety To prevent unnecessary testing (colposcopy and biopsy) To prevent over-treatment with subsequent pregnancy complications To save money 3
4 CERVICAL SCREENING - CURRENT STATUS Current cytological screening tests and HPV tests lack sufficient sensitivity or specificity to predict, at an early stage, which low grade lesions are destined to regress or to progress to HSIL or CA. CURRENT CERVICAL SCREENING TESTS PROBLEMS Cervical smear- too many false negatives (poor sensitivity), meaning cytology reported as normal when cervical disease is present. HPV testing - too many false positives (poor specificity), meaning HPV test is positive for the presence of virus, but cervical disease is not present.* *Most high risk HPV infections of the cervix do not even cause a cytological abnormality (cervical disease). CURRENT CERVICAL SCREENING TESTS Conventional pap smear: sensitivity 51% (Duke Study) Thin-layer cervical smear: sensitivity 82% (Duke Study) ASC with Digene hrhpv reflex: PPV for HSIL 20% NPV for HSIL - >99% Colposcopy: Initial colpoposcopy identified only 58% CIN2+ lesions. 28% LSIL cytology have CIN2+ on colposcopic biopsy. (ALTS Study) 4
5 CURRENT CERVICAL SCREENING TESTS PROBLEMS CAP Today (College of American Pathologists, 10/2010) Unfortunately, knowledge gained from the existing tools - Pap, colposcopy, biopsy- cannot predict which lesions will progress to cancer. * Dr. Darragh (UCSF) - We need something more specific than the presence of virus (HPV), something downstream (showing progression) We are looking for that magic marker* Dr. Wilbur (MGH) - we need new markers that can detect events more associated with neoplastic transformation of cells, rather than just HPV infection. * Dr. Unger (CDC) - likes the triage potential of 3q amplifications. * *Comments and quotes from CAP Today (College of American Pathologists 10/2010 PROGRESSION OF CIN LESIONS (Cyto) Bx Regress/persist Progress to CIS Progress to CA (LSIL) CIN 1 89% 11% 1% (HSIL) CIN 2 78% 22% 5% (HSIL) CIN 3 88%.. >12% 64 studies: 274 carcinomas; 15,473 cases Follow-up:,1-12 years Oster, AG, Int J GYN Path 1991; CERVICAL ONCOGENESIS The development of cervical cancer involves both viral and host factors. Infection with high-risk HPV is necessary - but not sufficient by itself - for the initiation of cervical oncogenesis. Additional genetic events (3q gains) in the host s HPVinfected cells are essential for a pre-cancerous lesions to progress to malignancy. HPV-induced cervical epithelial cellular chromosomal instability is a hallmark of malignant transformation. Journal of Molecular Diagnostics, Vol. 9, No.5, November Irina Sokolova. 5
6 Chromosomal Biomarkers for Detection of HPV-Associated Genomic Instability in Epithelial Cells of Cervical Cytology Specimens It seems that the HPV screening by itself is not sufficient in predicting malignant transformation of dysplastic cervical lesions, because only a fraction of HPV-infected women will progress from atypical or mild dysplasia to high grade and subsequently to cancer. Chromosomal instability at a numerical or structural level is a hallmark of malignant tumors. Irina Sokolova Journal of Molecular Diagnostics, Vol. 9, No. 5, November 2007 oncofish cervical Automated FISH test uses liquid cytology specimens. Hybridization is performed by using two-color probe set: 3q26 (gold) Chromosome 7 centromere (green) Entire slide (every cell!) is scanned to identify nuclei with 3q gain. Data are collected from 800 nuclei, including assessment of nuclear morphology, number of aneuploid nuclei, and enumeration of nuclei with integers of 3q copies. oncofish cervical Deposit Cervical cells Hybridize FISH probes Analyze slides (automated) Cells classified and presented for review by Ikoniscope 6
7 CELL EXHIBITING 3q GAIN FISH analysis of a liquid cytology specimen using probes for chromosome 3q26 (Gold) and chromosome 7 (Aqua) CASE STUDY Index Cytology: ASCUS HPV52 positive Colposcopy negative 3 nuclei with 3q>4 1 yr Follow-up: ASC-H HPV52 positive 264 nuclei with 3q>4 Colposcopy positive CIN2/CIN3 3q GAIN Whole genome analysis (CGH) of cervical carcinoma demonstrated acquisition or loss of genetic material on 1q, 3q, 5p, 6p, 8q, 9p, 11q, 12p, 14q, 17q, 19q, 20p, and 20q. The most consistent chromosomal gain in aneuploid tumors was mapped to chromosome arm 3q % of invasive cancer showed gain of 3q. Heselmeyer K, et al. Genes Chromosomes Cancer. 1997, 19:233. Heselmeyer K, et al. Proc Natl Acad Sci U S A. 1996, 93:479. Anderson et al. AJP 2009, 175:1831 7
8 TERC (RNA component of the telomerase gene) Localized to 3q26 Putative mechanism of immortalization via increased telomerase activity PREVALENCE OF 3q GAIN Anderson et al. Am J Pathol 2009, 175:1831 CERVICAL SCREENING TESTS OPTIMAL PERFORMANCE Optimal performance for cervical screening tests would be achieved by a test or a combination of tests that would maximize test sensitivity and maximize test specificity for detecting (at an early stage) squamous intra-epithelial lesions, having the potential to progress to HSIL or CA, thereby giving the fewest false negative and the fewest false positive test results. 8
9 NILM/CIN1 vs. CIN2/CIN3/CA NILM vs. CIN3 Sensitivity Specificity Sensitivity Specificity 3q Gain 79% 84% 91% 93% HR-HPV 97% 36% 96% 36% p16 68% 87% 83% 86% HPV viral load, Quantovir assay mrna E6/E7 HPV, NorChip Assay Anderson et al. AJP 2009, 175: % 43% 79% 31% 50% 79% 68% 92% oncofish cervical - A PREDICTIVE MARKER FOR LSIL PREDICTIVE VALUE OF NEGATIVE TEST >99% for no progression PREDICTIVE VALUE OF POSITIVE TEST 80% for progression to HSIL+ Ikonisys Clinical Laboratory New Haven, CT Lack OF 3q GAIN FOR PREDICTION OF NON-PROGRESSION/REGRESSION OF LSIL NPV Heselmeyer et al 100% Jalali et al 97% Rodolakis et al 100% Harper et al 100% Heselmeyer et al Genes, Am J Pathol 2005, 166:1229 Jalali et al Am J Obstet Gynecol 2010, 202: Rodolakis et al submitted Harper et al in preparation 9
10 Amplification of the 3q chromosomal region as a specific marker in cervical cancer HPV 2012 (28 th International Papillomavirus Conference Thomas C. Wright MD, et alia Cytology (-) hrhpv (-) CIN 2+ Total 3q Gain (+) q Gain (-) Total Sensitivity 94% Specificity 100% NPV 98% PPV 100% overexpression of E6/E7 p16 INK4a overexpression prb/p53 inactivation Host integration into host genome Virus p 3q gain OFC HPV infection q progression to invasive cancer POTENTIAL CLINICAL USES FOR oncofish cervical Should the primary screening system change to HR-HPV testing, a secondary triage test would be needed for those women who test positive. The triage test could revert to cytological screening or could encompass a biomarker, such as 3q gain, that measures the host s genetic response to HPV infection. Jalali et al. Am J Obstet Gynecol 2010;202:581.e
11 oncofish cervical does not compete with HPV testing ASC HPV Triaging of women with LSIL/ASCUS-HPV+ LSIL 3q gain ASC-HPV+ Clinical utility of oncofish cervical POTENTIAL UTILITY OF oncofish cervical IN MANAGEMENT OF HPV-INDUCED LESIONS Cervical: ASC, ASC-H, SIL, Vaginal: atypia, SIL, CA Vulvar: atypia, SIL, CA Anal: atypia, SIL, CA Oral: atypia, SIL, CA SUMMARY Importance of early risk stratification Problems of current screening tests: Cytology lacks sensitivity; HPV lacks specificity Cytology/HPV cannot predict low grade progression/regression in individual patients HPV tests indicate viral activity, not cervical cellular response HPV tests indicate risk for oncogenesis, not actual oncogenesis oncofish cervical Good sensitivity and specificity Predicts progression/regression of low grade lesions in individual patients (personalized medicine) Indicates host chromosomal cervical cellular response to HPV infection Measures 3q26 chromosomal cervical cellular response hallmark of malignant transformation Ikoniscope Sophisticated automated computerized microscope Examines every cell in cervical smear for 3q26 response 11
12 Questions Resources PAML - Jackie Modrow, Marketing Manager, jmodrow@paml.com 3q Gain in Cervical Cancer Bibliography you may download the document under the handouts icon in the upper right-hand corner Thank You for Attending P.A.C.E. credit may be obtained by submitting your completed evaluation form. You will find the form by clicking on the handouts icon in the upper right hand corner of your screen CE credit may be obtained by downloading the Certificate of Completion under the handouts icon PAML employees will be able to receive one hour of continuing education credit by submitting your attendance through CE Manager. 12
13 Thank You for Attending We will be leaving the webinar open for 15 minutes to allow you to download the handouts This webinar has been recorded and will be available by Friday, March 29 th, on PAML s next webinar is scheduled for May 14, 2013, 12:30 p.m. 1:30 p.m. (PT) Please send ideas for future webinar topics. 13
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