HPV LABORATORIO FRANCESCA CAROZZI

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1 HPV LABORATORIO FRANCESCA CAROZZI S.C. Laboratorio Prevenzione Oncologica Responsabile Programma Regionale HPV in Toscana Istituto per lo Studio e La prevenzione Oncologica (ISPO) Firenze 1"

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3 Lo"Screening"Cervicale"in"Italia" " Età Screening Intervallo Da 25y a y Pap-test con HPV di triage per ASC-US 3 anni Da > 30-35y a 64 HPV con Pap di Traige 5 anni HPV test nel the follow-up di donne : - Trattate per lesioni CIN2 - Con alterazioni citologiche e negative alla colposcopia

4 HPV test used Primary in HPV Screening screening with HPV test in programs Italy in Italy Hc2 Trento, new tender Piemonte TP HC2 HC2 Veneto TP,Roche Emilia Romagna: TP, Roche Liguria pap conv + STM, HC2 HPV test and citology Triage centralized in 1-2 Labs for Regions HPV Regional Programs HPV Locals Programs Toscana: Pap conv+hc2 TP+ HC2 Roma G pap conv, STMAbbott Latina, surepath Pap conv +stm Hc2+? Cobas Roche Abruzzo TP, Hc2 Basilicata Pap Conv+STM, Hc2 HPV Programs in the activation phase

5 HPV"Laboratorio:"" I"test"valida:"sono"tu<"uguali?"Hanno"le"stesse"problema:che"o"pun:"di"forza?" Quali"da:""anali:ci"raccogliere"nei"sistemi"informa:vi"?"" Come"refertare"il"test"HPV"di"screening?"(vedi"tool"box,"vedi"WHO,"vedi"raccomandazioni" Gisci)" I"controlli"interni""Come"raccogliere"i"da:"dei"controlli"?"" Quali"VEQ?"È"opportuno"fare"un"progeMo"Gisci"in"questa"fase"cruciale?"" Ges:one"e""della"qualità"del"nuovo"percorso"di"centralizzazione,"qualità"assurance"," valutazione"in"i:nere"di"performance"e"impamo" Quali"criteri"per"la"centralizzazione""?" Qual"è"il"workflow"delle"della"strumentazione"disponibile"sul"mercato?" Qual"è"il"carico"di"lavoro?"" Il"test"HPV"viene"dalla"ricerca,"abbiamo"finito?"Quale"ricerca"implemntare"?"Biomarctori?"Altri" test?"" Quale"test"HPV"nelle"vaccinate?"" Quali"le"problema:che"che"si""incontrano"tu<"i"giorni?" Quale"formazione?"Come"validare"l entrata"nella"rou:ne?"sono"u:li"le"check"list?"" Le"modalità"di"risposta"dei"non"valutabili"post"anali:ci"e"preXanali:ci"(tuMo"chiaro?)" 5"

6 Il"percorso"di"aMuazione"del"cambiamento:"la"centralizzazione" Il"ruolo"complesso"del"laboratorio"! " L "introduzione"del"codice"a"barre"come"sistema"iden:fi"ca:vo"del"prelievo"della"donna":" CoXesistenza"di"due"percorsi"(PAP"primario"e"HPV"primario" Ges:one"di"più":pologie"di"campioni"HPV"(screening,"Triage,"FollowXup"con"cito"o"senza"cito)" Adeguamento"dei"servizi"di"acceMazione"campioni"locali"e"del"lab"centrale" Definizione"flussi"e"comunicazioni"per"non"conformità"in"fase"di"presa"in"carico" " Gli"aspe<"logis:ci"lega:"alla"centralizzazione:"" impostazione"dei"flussi"di"trasporto"dei"campioni"dalle"unità"di"prelievo"dai"centri"di"riferimento,"con" eventuali"centri"di"raccolta"all interno"dela"singola"azienda"" RispeMo"delle"norme"per"il"trasporto"su"strada"(contenitore"triplo)" L ""alles:mento"del""laboratorio"regionale"di"riferimento"" aumento"del"numero"di"test"molecolari"e"il"passaggio"da"ricerca""ad"a<vità"clinica"con" numeri"eleva:" creazione"di"nuove"procedure"per"la"ges:one"di"tumo"il"fl"usso"di"lavoro." "creazione"check"list"di"lavoro"" Formazione"ed"Addestramento"del"personale"tecnico"da"colorazione"a"test"molecolari"" "Alles:mento"di"controlli"di"qualità"molecolari"interni"ed"esterni" " Oltre"la"VEQ"e"il"CI,"Ges:one"del"monitoraggio,"qualità"assurance"e"valutazione"di"performance"e"impaMo"con:nuo,"" ".."

7 ." Diagramma di flusso " screening ACCESSO AL PROGRAMMA REALIZZAZIONE TEST PERCORSO DIAGNOSTICO FOLLOW UP localizzata Laboratorio centrale Convocazione Accettazione accoglienza Esecuzione test Valutazione Valutazione diagnostica diagnostica Positivi al test Potenziale alto rischio Ulteriore Ulteriore accertamento accertamento diagnostico diagnostico Esito positivo Intervento chirurgico/ trattamento terapeutico Follow up Richiamo precoce Negativi al test Rischio basso Esito negativo

8 CRITERI"per"la"centralizzazione" Il"report"di"HTA"non"prevede"una"valutazione"di"costo"efficacia" in"senso"stremo"(per"la"definizione"dei"criteri"di" centralizzazione"),"ma"fa"una"previsione"degli"scenari"fino"agli" "test/anno:" "l obie<vo"era"dimostrare"l en:tà"della"riduzione"dei"cos:" all aumentare"del"numero"di"esami"fa<" "non"stabilire"un"temo"massimo"o<male" 8"

9 Quali criteri per la centralizzazione? Come definiamo un laboratorio di grandi dimensioni? Organizzazione dello screening (logistica del territorio, modalità di trasporto dei campioni) Grado di automazione e workload del sistema Tipologia di prelievo (fase liquida, quali sono i criteri di sicurezza da rispettare?) Modalità e tempi di processazione dei campioni ed esecuzione dei test! Volume minimo di campioni vs volume max: determinato da cosa?! Spazi?! Altro?! Gestione regionale

10 CDC, Atlanta USA

11 Variations in Cervical Samples " Collection Device - Brush, broom, spatula, swab, tampon " Collection Media - STM, PreservCyt, SurePath, alcohol, paper " Collection Method - Clinician collected - Self-collected

12 Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening? Marc Arbyn, Peter J.F. Snijders, Chris J.L.M. Meijer, Hans Berkhof, Kate Cuschieri, Bostjan J. Kocjan, Mario Poljak Clinical Microbiology and Infection 27 March April April 2015 " " " " The cobas 4800 HPV Test and RealTime High Risk HPV test were consistently validated in two and three studies, respectively, PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV Risk Assay were validated each in one study. Other tests which partially fulfil the 2009-guidelines are: Cervista HPV HR Test, PCR-LMNX, a homebrew E6/E7 RT qpcr and MALDI-TOF. The APTIMA HPV assay targeting E6/E7 mrna of hrhpv was also fully validated. However, the cross-sectional equivalency criteria of the 2009-guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is waited for to inform about its utility in cervical cancer screening at five year or longer intervals. 12"

13 The"HORIZON"Study" Four HPV assays four different technologies Cobas HPV test Real-time PCR assay, with co-detection of HPV HR and 16 and 18 HC2 Hybridization assay with HR HPV detection APTIMA RNA assay with HR HPV detection CLART PCR-Microarray assay with simultaneous detection of 35 genotypes QiaSymphon y"rcs"hc2 " work"flow" Roche cobas 4800 workflow Gen-Probe PANTHER APTIMA workflow Genomica! CLART HPV2"

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15 Sensi:vity"for"CIN"3+" 38 CIN 3+ in 2,869 women screened at age years* Screening test CIN 3+ Cytology Detection rate per 100 screened women Sensitivity % -- 1 (ref) 82% Hybrid Capture 2 cobas CLART APTIMA % % 89% % % 95% % % 95% % -- +6% 87% * Histological follow-up will be complete by end of 2013 [Rebolj et al., in preparation]

16 HPV e rischio cancerogeno: 2011 Gruppo 1 (cancerogeni per l uomo): 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59. Gruppo 2A (probabilmente cancerogeni per l uomo): 68. Gruppo 2B (possono essere cancerogeni per l uomo): 26, 53, 66, 67, 70, 73, 82. Gruppo 2B (possono essere cancerogeni per l uomo su base filogenetica): 30, 34, 69, 85 and 97. Gruppo 3 (non classificabili per la loro cancerogenicità nell uomo): 6 and 11 Fonte: Monografia 100B IARC 2011

17 Elenco test hrhpv clinicamente validati Come mantenerlo aggiornato? Chi lo fa? Con quale frequenza? Come renderlo disponibile? Come valutare i dati che saranno pubblicati?

18 Considerations before implementing an assay "What"type"of"assay"is"it?"Can"the"laboratory"provide"the" infrastructure"and"environment"to"deliver"the"assay"robustly."" Is"the"laboratory"accredited"to"perform"clinical"microbiology" tes:ng"or"to"perform"hpv"test"within"screening"program?" InXhouse"valida:on,"to"show"assay"performs"to"expected" standard"with"local"operators"and"systems"" Training"and"demonstra:on"of"competence"of"single"" specialist""or"techinician""which"is"monitored"through"regular" competency"assessment"" Set"up"of"quality"system"and"framework"to"monitor" performance"longitudinally""

19 Example of quality framework for HPV Testing within screening program " " Accreditation through Regional Audit ; HPV tests within screening program listed in their repertoire of services " Participation in accredited external quality assurance scheme for HPV and good performance " Each member of staff undertaking HPV testing must first pass company training procedures, with training recorded in individual s training logs " Competency of staff/laboratory should be demonstrated through testing of an HPV Validation Panel " Competency should be assessed regularly for as long as staff has responsibility for the function " Laboratories undertaking HPV testing must include in every run known positive and negative IQC samples validated by repeated testing, in addition to required kit controls " " Laboratories undertaking HPV testing should check regularly (eg weekly; 1% of samples) for systemic errors and perform environmental testing of the laboratory areas used.

20 Quality control endogenous, within-sample controls """"""To control for cellularity or PCR reaction inhibition? " - Common targets, human gene (beta globin, GAPDH) - Detected in human cells, not just epithelial cells - What cellular content is adequate/sufficient for HPV testing, what type of cellular content? - Primer sequences can be differentially affected by inhibition*"" Does this become an increasingly pertinant question for HPV primary screening? *Lefevre et al Journal of Virological Methods 114 (2003)

21 Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening? Marc Arbyn, Peter J.F. Snijders, Chris J.L.M. Meijer, Hans Berkhof, Kate Cuschieri, Bostjan J. Kocjan, Mario Poljak Clinical Microbiology and Infection 27 March April April 2015

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23 Assurance quality in programme changes " We need to ensure that any change to screening methodology is at least as good as the current method and for HPV the quality remains at least as good as in research setting " HPV testing procedure has to be monitored to continuous and rigorous quality assurance to avoid sub-optimal, potentially harmful practices: " Quality control program also for Triage Test (PAP-Test) and for each phase of the protocol F. Carozzi Workshop F. Carozzi Pre-congruessuale 2015 GISCi 2015

24 Quality Assurance, Internal Quality Control and External Quality Assurance - Quality Assurance (QA): is essential to guarantee precise and accurate analysis to support optimal patient care. - ensures the right result for the right test, in the right time, the right sample for the right patient and interpreted with the correct reference data using detailed check lists - Internal Quality Control (IQC): - verifies the precision of the investigations in the single lab, - system precision and stability - shall be repeated in every analytical session - External Quality Assessment (EQA): data accuracy, comparison with external labs - can detect differences between centers that measure the same analyte - allows to verify single lab accuracy (Bias) compared to the medium value obteined by all participant labs Since the determination is periodic and retrospective, is commonly used the term "assessment" rather than "control".

25 Implementation of appropriate quality control procedures for HPV test in a screening setting The laboratory must prepare a SOP (Standard Operating Procedure) with the definition of alarm and rejection of analytic series based on the value of the control samples All results of Internal QC should be recorded, daily archived and registered in the form like control charts. "out of control" results must be registered Definition of corrective action to be taken in the event of nonobservance of quality parameters.

26 Registrazione giornaliera dei dati Controllo Interno RLU/CO RCS 1 DS 2 DS 1,8 3DS 1,7 RLU/CO 1,6 1,5 1,4 1,3 1,2 1,1 1 0,9 0,8 0,7 1. Standard Operating Procedure with corrective actions and management of situations that deviate from the expected value Serie1 0,6 0,5 0,4 0,3 0,2 0,

27 14% NORD CENTRO SUD e ISOLE HPV screening % di donne Program POSITIVE in al Italy Test HPV women aged 35-64yo % HPV test positive women 12% 10% HPV Test validated for screening Quality Controls for HPV test within screening programs are necessary to be sure that difference in HPV positivity depends by prevalence of infection in the different areas/cities 9,1% 12,4% 8% 6,3% 6,6% 7,1% 7,8% 6% 4,6% 4,7% 4,8% 4,8% 5,0% 5,1% 5,6% 5,8% 6,0% 6,0% 4% 4,0% 2% 0% ROMA G SAVONESE MOLISE TRENTO ALTA PADOVANA PADOVA ROVIGO VENEZIANA ADRIA VALLECAMONICA ESTE AVEZZANO TORINO LATINA REGGIO EMILIA FIRENZE TERAMO LANCIANO PESCARA

28 External Quality Assurance Programs for HR- HPV in a screening setting HPV is a virus and normally in clinical virology, it is important to apply methods with highest sensitivity for the micro-organism that is responsible for an infection " The HPV test applied to the screening program doesnt follow this rule BUT it must to be validated and optimized for clinical sensitivity and specificity in a screening context the cut-off used in this contest does not correspond with the minimum detectable level of the method HPV EQA specific for HPV screening Test " So EQA panel applied to HPV test in cervical cancer MUST : Be optimised for screening test Evaluate all 12 HR HPV types in a cycle of EQA panel

29 EQA, examples of existing schemes " WHO HPV LabNet (Proficiency Panel) Plasmid material. Annual proficiency panel of 46 samples (43 extracted DNA + 3 cell samples to be extracted). Designed to test genotyping assays " UK NEQAS VEQ Pooled material of clinical origin, 4 specimens 3 times a year scores on qualitative performance (presence/absence of HR HPV types) In ThinPrep " " QCMD VEQ Cell line material and clinical samples. Annual panel (8-10 samples). 2 kind of samples: CORE and EDUCATIONAL. Score assigned on the basis of CORE samples result. In Thin Prep " w.qcmd.com DicoCare VEQ " (Italian EQA) Clinical samples. 8 specimens 4 times a year. Designed to test clinical performance of the methodology used and genotyping assays- lyophilized sample to re-suspend in used medium " Online.dicocare.org

30 HPV"primario"e"qualità"dell intero"processo:"dalla" fase"prexanali:ca"a"quella"postxanali:ca" Prevenire"gli"errori" Il"percorso"dello""screening"è"un"sistema"complesso"in"cui" interagiscono"molteplici"famori"" come"in"altri"sistemi"complessi,"possono"verificarsi"inciden:" ed"errori,"la"centralizzazione"genera"efficienza"ma"dobbiamo" valutare"le"fasi"di"nuova"cri:cità,"non"solo"da"parte"del" laboratorio"." Vanno"pertanto"progeMa:"specifici"modelli"di"controllo"del" rischio"clinico,"con"l obie<vo"di"prevenire"il"verificarsi"di"un" errore"e,"qualora"questo"accada,"contenerne"le"conseguenze." Solo"aMraverso"opportune"analisi"è"possibile"iden:ficare"le" cause"di"un"errore"e"ridisegnare"i"processi"al"fine"di"ridurre"la" probabilità"che"esso"si"ripeta."

31 La!prevenzione!degli!errori!nel!processo!di!implementazione!del!test!HPV!come! test!di!screening!primario." XISPO,"Firenze"""X"Centro"per"la"Ges:one"del"Rischio"Clinico"e"la"Sicurezza"del" Paziente,"Regione"Toscana" Val"Camonica,""Roma"G,""IOV"Veneto,"AO"Reggio"Emilia" " Individuare"le"insufficienze"nel"sistema"e"progeMare"le"idonee"barriere"prote<ve.""" ""due"approcci:"" proa<vo,"in"cui"l analisi"parte"dalla"revisione"dei"processi"e"delle"procedure"esisten:," iden:ficando,"nelle"varie"fasi,"i"pun:"di"cri:cità;"per"validare"delle"procedure"o"per" monitorare"la"sicurezza"delle"pra:che"di"lavoro" "rea<vo:"l analisi"parte"da"un"evento"avverso"e"ricostruisce"a"ritroso"la"sequenza"degli" avvenimen:"con"lo"scopo"di"iden:ficare"i"famori"che"hanno"causato"o"contribuito"il" verificarsi"dell evento" Vedi Sessione Poster: G.P. Pompeo L approccio"proa<vo"è"quello"che"può"garan:re"la"realizzazione"di"un"progemo" sanitario"più"sicuro"e"per"la"sua"realizzazione"lo"strumento"da"preferire"è"la" cosiddema"fmea"(failure(mode(and(effect(cri2caly(analysis)" Applicazione"di""questo"metodo"nell ambito"della"implementazione"del" Programma"HPV"della"Regione"Toscana"e"di"altri"programmi"che"sono"par::"o"in" fase"di"implementazione""

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33 !Il#Test#HPV+HR#nei#programmi#di#screening! 5.1"CaraMeris:che"del""test#hr+HPV# 5.2""Indicazioni"sulle"modalità"di"risposta"del" test"hrxhpv"nello"screening" 5.3"Introduzione"di"idonee"procedure"di" controllo"di"qualità"dei"test"molecolari""

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35 Grazie"per"l amenzione"!!" "