HPV: cytology and molecular testing

Transcription

1 HPV: cytology and molecular testing Human Papillomavirus and how we test for it at Medlab Central Palmerston North for Cervical Cancer prevention and management. Developed by Reem Mustafa Cytology and Molecular departments, Medlab Central 2018

2 Introduction Human Papillomavirus 8000 bp circular double-stranded DNA within a protein capsid from Papillomaviridae family 40 of the 150 genotypes infect the human anogenital tract moist epithelium 15 types established high risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 Persistent (>2years) infection by high risk HPV are the cause of cervical cancer Cervical squamous cell carcinoma is an epithelial invasive cancer that affects the ectocervix Skin-to-skin sexually transmitted and cofactor required Cervical cancer is a leading cause of death worldwide National Cervical Screening Program established in 1990 Vaccine provides high and sustained immune protection Current practise: cervical screening in women aged years every 3 years using cytology and HPV reflex testing for high risk triage/ colposcopy for certain groups. Photo taken from VRPCC workbook

3 Cervical Intraepithelial Neoplasia CIN I = LSIL= mild dysplasia low grade CIN II = HSIL = moderate dysplasia CIN III = HSIL = severe dysplasia/carcinoma in situ (CIS) high grade Follow-up of all low or high grade by one or a combination of smear, colposcopy, biopsy, HPVDNA testing NCSP management algorithms Note: Atypical glandular cells can also cause cervical adenocarcinoma Invasive Squamous Carcinoma

4 Liquid-based Cytology (thin-layer technology) ThinPrep Pap Test Sample from endo and ectocervix, release into Preservcyt vial Filtration system 50,000 to 75, 000 cells for analysis Further testing can be carried out on the same sample Computerised imaging is used Normal superficial and intermediate squamous cells Photo taken from VRPCC workbook

5 Slide preparation ThinPrep vial Polycarbonate membrane ThinPrep Slide 1. dispersion 2. cell collection 3. transfer 20 mm fixed cells in a thin layer T5000 T2000

6 PAP Staining Papanicolaou Staining progressive Haematoxylin (ripened) with mordant stains nuclear chromatin Orange G and eosin stain cytoplasm Dehydration, clearing and mounting QC after each stain change Automated stainer Robotic cover slipping machine

7 ThinPrep Imaging System 14 digit numeric accession ID Fiducial marks (3) Efficiency Sensitivity Specificity Cassette Imaging station Image processor Server Review scope Imaging station scans--- Image processor analyses images and identifies FOV (22)--- Server stores slide data for review scope (10x magnification)

8 Manual Screening and Reporting Primary screen: look at all cells Identifying abnormal changes of cells or abnormal patterns. Bethesda 2001 New Zealand modified reporting system is used. Negative primary screened slides step screened rapidly by another screener. Full re-screen if: Minor discrepancies, if cell abnormality difference to pathologist. Abnormal/reactive/inflammatory or patient has symptoms. Certain Abnormal smear histories. Unsatisfactory gynaecological cytology. Authorization of results never by primary screener. For imaged slides: Negative for intraepithelial lesion or malignancy slides result can be authorised without rapid rescreen. Full re-screen required if symptoms, abnormal history, higher risk of abnormality than total screening population or If abnormalities identified. move one width of in-focus focal view Taken from VRPCC workbook

9 Cytology of HPV infection Koilocyte 95% specificity to HPV Large clear zone around nucleus Edge of the vacuole is sharp and distinct Nucleus abnormal (big and dark) dysplastic One, two or three nuclei (commonly two) Koilocyte Koilocyte Koilocyte Photos taken from VRPCC workbook

10 Other indicators of HPV infection Dyskeratocytes Condylomatous parabasal cells Spindle cells Have been described to be associated with HPV change. Spindle cells Dyskeratocytes Photos taken from VRPCC workbook

11 Other indicators of HPV infection Low grade cells High grade cells CIN I CIN II CIN III Photos taken from VRPCC workbook

12 Other indicators of HPV infection Squamous cell carcinomas Well differentiated SQCC

13 Squamous cell carcinoma Well differentiated, Keratinized Poorly differentiated, Retroplasia Well differentiated, Keratinized Photos taken from VRPCC workbook Well differentiated, Keratinized Poorly differentiated, Pleomorphic Tadpole/caudate cells

14 Squamous cell carcinoma Pearls Spindle/ fibre cells Photos taken from VRPCC workbook We use PCR to amplify target DNA in-vitro in order to detect HrHPV in clinical specimens.

15 HrHPV PCR: Specimen preparation Prep room PCR suite. Remaining specimen in PreserveCyt vial HrHPV testing: Prevents unnecessary colposcopy and biopsy Better management of patients at higher risk In NCSP screening guidelines since 1 st Oct 2009 AmpErase enzyme

16 HrHPV PCR: Cobas x 480 Automated multi-channel pipetting instrument Control unit: runs software, controls x and z modules, set up work order file and manages workflow Load Sample racks LIS confirms work order Start new run Load consumables, reagents, controls Start preparation run Extraction and Purification Extraction: Lysis and denaturing: elevated temperatures and choatrophic reagent and hydrogen bonds broken. Purification: HPV DNA and β-globin adsorbed to magnetic glass particles, washed and separated from particles.

17 HrHPV PCR: Cobas z 480 Rapid Thermal Block Cycler Review results Seal microwell plate Amplification and Detection Check QCs Accept results Send to LIS Print report for sign out Inspect and dispose Results are released with cytology results

18 Real-time PCR background principles Thermocycled We need: Polymerase Template DNA Primers Nucleotides Buffer Hot-start thermophilic Eagle Z05 DNA Polymerase Labelled reporter probes bind Cleaved by DNA polymerase fluorescence Figure 1. Three steps of PCR (Taken from thermofisher Scientific website)

19 Real-time Multiplex PCR Controls & CT values Negative control ( buffer). Positive control (bacterial plasmid DNA containing β-globin HPV-16, 18, etc sequences). Internal control detects human β-globin in patients cells. External QCs run quarterly. Multiplex PCR reaction with 4 different dyes one for the 12 high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68), one for each: HPV 16, HPV 18 and β-globin. Amplified DNA fluorescent dye signals are measured as reaction proceeds, data on exponential phase allows us to quantitate. CT is the cycle number where the fluorescent signal crosses the pre-defined threshold. PCR phases Figure 1. PCR phases (Taken from Thermofisher Scientific website)

20 Cytology vs HrHPV PCR Cytology More specific Less rejected samples due to potential contamination HPV PCR More sensitive More efficient (avoids repeat cytology) Earlier detection (treatment before invasion), 2x as long protection No reading error (due to lack of expertise) Costs less Sensitivity: Cytology 55-94% HPV testing 90%

21 The future Optimal strategy for NZ: 5 yearly HPV screening (women ages years) with partial genotyping for HPV 16 and 18 and referral to colposcopy, and cytological triage of other high risk types. Strong evidence from 4 European randomized trials: Sweden (Swedescreen), Italy (NTCC), UK (ARTISTIC) and Netherlands (POBASCAM) demonstrated significant reduction in incidence of cervical cancer amongst women screened with HPV compared with cytology. HPV primary testing is more effective (predicted to reduce cervical cancer incidence and mortality by 12-16%) and costs less (save 4-13% per year in program costs) than the current cyto-screening based program. Netherlands, Australia, Italy are/have now transitioned to primary HPV screening. Vaccination of boys prevents infection and spread of HPV disease which can lead to cancer. Vaccine can prevent most HPV disease and cancer. HPV 1 screen with LBC triage drivers: better protection more efficient cost reduced vaccination programme screening intervals can be safely extended less referral to colposcopy