Pushing the Boundaries of the Lab Diagnosis in Asia

Transcription

1 Pushing the Boundaries of the Lab Diagnosis in Asia Diana Lim MBBS, FRCPA, FRCPath (UK) Senior Consultant National University Health System and National University of Singapore Department of Pathology Singapore

2 Disclosures No financial relationships or conflict of interest to disclose

3 Area: km² Population: 5.61 million Ethnic groups: 74.1% Chinese 13.4% Malay 9.2% Indian 3.3% others GDP per capita: $53,053

4 Ten Most Frequent Cancers in Singaporean Females (%) ( ) 998 Singapore Cancer Registry

5 Age-Standardized Incidence Rates (ASIR) for Cervical Cancer ( ) Singapore Cancer Registry

6 Age-Standardized Mortality Rates (ASMR) for Cervical Cancer ( ) Singapore Cancer Registry

7 Cervical Cancer Screening in Singapore Started in 2002 Cytology only based guideline Largely opportunistic; recall mechanism limited only to those who registered and have their Pap smears at polyclinics

8 Cervical Cancer Screening in Singapore Where is it available? At polyclinics, healthcare institutions (public and private) and at General Practitioners (GPs) clinics How much does it cost? Polyclinic: $15 (Singapore citizens), $22.50 (Permanent Residents)

9 Cervical Screen Singapore: Minimum Standards for Laboratories Providing Cytology Services A Joint Cytology Committee (JCC) was formed to advise on the professional conduct of the laboratory aspect of the cervical cancer screening program Organization and Quality Management Procedure Manual Personnel Premises and environment Equipment, Information Systems and Materials Pre-examination, examination and post-examination processes Evaluation and Quality Assurance

10 Cervical Cytology Conventional Pap Smear Cervical sample manually smeared onto slide

11 Cervical Cytology Conventional Pap Smear Cervical sample manually smeared onto slide Liquid-Based Pap Test Cervical cell sample suspended in liquid medium before automated thin layer/monolayer slide preparation SurePath ThinPrep

12

13 Liquid-Based Pap Test SurePath Thinprep 13mm diameter typically containing K cells 19mm diameter typically containing K cells

14 Reporting Cervical Cytology Negative for intraepithelial lesion or malignancy Epithelial cell abnormalities Squamous cell Atypical squamous cells - of undetermined significance (ASC-US) - cannot exclude HSIL (ASC-H) Low grade squamous intraepithelial lesion (LSIL) High grade squamous intraepithelial lesion (HSIL) - with features suspicious for invasion (if invasion suspected) Squamous cell carcinoma Glandular cell Atypical (AGUS-NOS) - endocervical cells (NOS or specify in comment) - endometrial cells (NOS or specify in comment) - glandular cells (NOS or specify in comment) Atypical (AGUS-N) - endocervical cells, favor neoplastic - glandular cells, favor neoplastic Endocervical Adenocarcinoma in situ (AIS) Adenocarcinoma - endocervical - endometrial - extrauterine - not otherwise specified (NOS)

15 Screening Cervical Cytology Manual Screening Normal smears with no significant past medical history - Screened by 2 cytotechs - Case signed out by cytotech Automated Screening Lesional smears or those with significant past medical history - Screened by 2 cytotechs and pathologist - Case signed out by pathologist

16 Automation-Assisted Versus Manual Reading of Cervical Cytology (MAVARIC): A Randomised Controlled Trial Kitchener H et al. Lancet Oncol 2011;12: liquidbased cytology samples Manual screening arm Paired-reading arm Automation assisted reading is 8% less sensitive than manual reading Specificity of automation-assisted reading relative to manual reading increased by 0 6%

17 Era of HPV Testing

18 HPV and Cervical Cancer Early 1980s: Link between HPV and cervical cancer discovered Wright TC. N Engl J Med 2003; 348: Mid-1990s: First available test to detect HPV

19 Utility of HPV Testing ASCUS Triage Test of cure Co-testing with cervical cytology Primary Screening

20 Meta-analysis of ASCUS-HPV Triage Studies Arbyn et al. Gynecol Oncol 2005;99:S7 S11 Using HC2 with a disease threshold of CIN2+: Sensitivity: 94% (CI 92-96%) Specificity: 62.4% (CI 56-68%) Sensitivity of HC2 was 14% higher than repeat cytology with almost equal specificity

21 Comparison of HPV Testing vs Cytology Huh W et al. Gynecol Oncol 2015;136:

22 FDA Approved HPV Tests Assay Methodology No of HR-HPV detected Hybrid Capture 2 (Digene/Qiagen) Cervista (Hologic) Cobas (Roche) Aptima (GenProbe/Hologic) RNA probe hybridization Able to detect HPV 16 and 18 specifically Internal control FDA approved indication 13 No No ASCUS triage Co-testing Invader assay 14 Yes Yes ASCUS triage Co-testing Real-time PCR 14 Yes Yes ASCUS triage Co-testing, Primary screen E6/E7 mrna 14 No Yes ASCUS triage Co-testing

23 A Comparison of the Roche Cobas HPV Test With the Hybrid Capture 2 Test for the Detection of HR-HPV Genotypes Levi A et al. Arch Pathol Lab Med 2016;140: Objective To compare the performance of the 2 assays for the detection of HR-HPV using both ThinPrep and SurePath preparations Methods 1371 samples (1122 SurePath ThinPrep) were tested (~40% ASCUS triage, ~60% co-testing) Linear Array HPV testing was performed for cases with discrepant results

24 94% of specimens demonstrated concordant results K value = 0.72 (95% CI of )

25 Genotype Distribution of Discordant Samples with the Linear Array Test

26 Comparing HPV Assays in Primary Cervical Screening Rebolj M et al. PLoS One 2014;9(1):e86835 Primary samples, years: Disagreement between Hybrid Capture 2, cobas, CLART, and APTIMA HPV assays

27 HPV Testing: Quality Assurance Test Validation Analytic/clinical validation Lab evaluation to ensure that the test is working as expected Internal Quality Control Known positive and negative controls in each run Regular review of results to assess patterns/trends External Peer Comparison CAP or self-developed program

28 Potential Pitfalls of HPV Testing False positive results: Cross reactivity with non-hr HPV types False-negative results: Detection assay does not cover the specific HPV type Cervical samples with low titer or low copy number of HPV Inhibitors in the specimen Limited analytic sensitivity Inadequate cellularity in the specimen Presence of true HPV negative cervical cancers Rapidly developing cervical carcinoma may also account for subset of HPV negative cancers if testing is performed outside of HPV infection period

29 HPV Testing at NUH Real-time PCR (Hybridio) (Qualitative detection of HPV16, HPV types) Feb Feb 2015 HC 2 (Qiagen) (Qualitative detection of 13 HR HPV types) Cobas (Roche) (Qualitative detection of HPV16, HPV types)

30 No. of HPV tests performed in NUH from Jan 2013 to Sept 2015 ASCUS Triage Data courtesy of Dr Ida Ismail-Pratt

31 No. of HPV tests performed in NUH from Jan 2015 to Dec 2016 (Cobas) Jan Feb March April May June July Aug Sept Oct Nov Dec Data courtesy of Molecular Diagnostic Centre, NUH

32 Results of HPV Testing in NUH (Jan 2013 Sept 2015) HPV genotyping results No. of tests performed Percentage (%) ATHENA study (%) Negative HPV HPV Non-16/18 HR-HPV Indeterminate Total Data courtesy of Dr Ida Ismail-Pratt

33 Indications for HPV Testing Indication for HPV testing Number of cases Percentage (%) Co-testing (routine screening) Co-testing (post-treatment) ASCUS/LSIL Triage ASCH Triage AGUS Triage Others (vault smear) Total Data courtesy of Dr Ida Ismail-Pratt

34 Results of Co-Testing for Routine Screening Cytology results HPV 16 + HPV18 + Non-16/18 HR-HPV HR-HPV not detected Negative ASCUS LSIL HSIL AGUS AGUS, favor neoplastic AdenoCa SCC Unsatisfactory Total 17 (1.3%) 5 (0.4%) 137 (10.1%) 1192 (88.2%) 1351 Total Data courtesy of Dr Ida Ismail-Pratt

35 Society for Colposcopy and Cervical Pathology of Singapore Scientific Committee Position Paper on Primary HPV Screening for Cervical Cancer Prevention

36 No. of Cases Number of Cervical Smears from Cervical Smear

37 No. of Cases 1400 No. of Cervical Smears (Jan 16-Jan 17) Jan-16 Feb-16 Mar-16 Apr-16 May-16 Jun-16 Jul-16 Aug-16 Sep-16 Oct-16 Nov-16 Dec-16 Jan-17 Jan-16 Feb-16 Mar-16 Apr-16 May-16 Jun-16 Jul-16 Aug-16 Sep-16 Oct-16 Nov-16 Dec-16 Jan

38 Impact of HPV Testing on Cytology Workflow Decrease in the number of cases of pap cytology Manpower issues (no. of cytotechs and pathologists) LBP will be increasingly used because of improved sensitivity and the convenience of using liquid specimens with automated screening technologies and molecular tests like HPV Decrease in numbers of conventional smears Cytology specimens processed may include a higher proportion of abnormal or difficult cases

39 What s Next?

40 Biomarkers in Cervical Cytology p16 Negative cell cycle regulator; anti-proliferative effect in normal cells Strong over-expression of p16 in precancerous and cancerous cervical lesions is associated with HR-HPV Surrogate marker for transforming HPV infection p16 can be overexpressed in some non-dysplastic cells: - Squamous metaplasia - Repair - Atrophy - Endocervical and endometrial glandular cells

41 Biomarkers in Cervical Cytology Ki67 protein is a marker of cell proliferation; can be detected within nuclei of proliferating cells Co-expression of p16 and Ki67 should be mutually exclusive in normal cells Indicates cell cycle deregulation

42 p16 and Ki-67 Dual Stain CINTec PLUS (Ventana Medical Systems, Roche) Ki67 p16 Highlights oncogenic cells, independent of cytomorphology and HPV genotype Can be performed on conventional or liquid based cytology slides Presence of dual-stain positive cells is strongly associated with established high-grade disease

43 CINtec PLUS Cytology: Objective Aid in Triage CINtec PLUS CINtec PLUS CINtec PLUS Pap Cytology Negative Negative Disease Subjective Expression of p16 (brown) signals = halting of cell division Expression of Ki- 67 (red) signals = progression of cell division Co-expression of p16 & Ki-67 (brown & red) = abnormality Reliant on interpretation of morphology only Slide courtesy of Roche

44 Performance of Dual Staining for CIN2+ in the Routine Screening Population Population (age range) Test Sensitivity (%) Dual Staining 89.4% ( %) 86.7% ( %) 84.7% ( %) Pap Cytology 71.9% ( %) 68.5% ( %) 65.9% ( %) Test Specificity (%) Dual Staining 92.0% ( %) 95.2% ( %) 96.3% ( %) Pap Cytology 92.6% ( %) 95.4% ( %) 96.3% ( %) Tjalma W. Eur J Obstet Gynecol Reprod Biol 2017;210:

45 Meta-Analysis Results for Dual Staining Sensitivity Specificity Population referred to colposcopy Dual Staining 89.7% ( %) 69.3% ( %) HPV testing 96.0% ( %) 43.6% ( %) Population referred to colposcopy and population with ASCUS/LSIL on Pap cytology Dual staining 89.5% ( %) 65.7% ( %) HPV Testing 94.4% ( %) 33.5% ( %) Specificity = false positives and in the number of inappropriate referrals to colposcopy Tjalma W. Eur J Obstet Gynecol Reprod Biol 2017;210:

46 Factors to Consider What is the target screening population? Which testing method to use? With or without Pap cytology? What should the screening frequency be? How to manage screen positives and negatives individuals? What can lead to false positive/negative results? Who will pay for the test? What is the overall cost-effectiveness?

47 GOOD Expensive Keep Dreaming Slow FAST Crappy CHEAP

48 Future Challenges

49 Effects of HPV Vaccination on Cervical Cancer Screening HPV vaccines may ultimately have the greatest impact on cervical cancer screening Although HPV panels include the hrhpv genotypes, many other HPV genotypes can also cause cervicovaginal lesions, including high-grade dysplasia Plausible that some HPV genotypes previously considered of unknown oncogenic potential or low-risk could be determined to be causative agents of high-grade cervicovaginal lesions The incidence of cervicovaginal lesions related to these less common HPV genotypes could increase with HPV vaccination against HPV-16 and HPV-18.

50 Acknowledgements Cytology Staff Dr Ida Ismail-Pratt (Consultant O&G) Gynaecology cancer screening and prevention Molecular Diagnostic Center, National University Hospital, Singapore - Dr Benedict Yan - Ms Lily Chu - Ms Sharah Mae - Ms Tracy Png