F. Carozzi Workshop Pre-congruessuale GISCi 2015 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
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Lo"Screening"Cervicale"in"Italia""
Età Screening Intervallo
Da 25y a 30-35 y Pap-test con HPV di triage per ASC-US
3 anni
Da > 30-35y a 64 HPV con Pap di Traige 5 anni
25-64 HPV test nel the follow-up di donne : - Trattate per lesioni CIN2 - Con alterazioni citologiche e negative alla colposcopia
F. Carozzi Workshop Pre-congruessuale GISCi 2015
• Trento, new tender
Piemonte TP HC2
• Latina , surepath
Pap conv +stm
Hc2+?
Abruzzo TP, Hc2
• Primary Screening with HPV test in Italy
• Hc2
Cobas Roche
Basilicata Pap Conv+STM, Hc2
Toscana: Pap conv+HC2
TP+ HC2
Liguria pap conv + STM, HC2
• HC2 • Emilia Romagna: TP, Roche
• HPV Locals Programs
• HPV Regional Programs
• HPV Programs in the activation phase
HPV test and citology Triage centralized in 1-2 Labs for Regions
HPV test used in HPV screening programs in Italy
• Roma G pap conv, STMAbbott
Veneto TP,Roche
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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?)"
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F. Carozzi Workshop Pre-congruessuale GISCi 2015
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,"""
………….."
F. Carozzi Workshop Pre-congruessuale GISCi 2015
.""
ACCESSO AL PROGRAMMA PERCORSO DIAGNOSTICOREALIZZAZIONE TEST FOLLOW UP
ConvocazioneAccettazioneaccoglienza
Esecuzionetest
Follow up
Intervento chirurgico/
trattamento terapeuticoUlteriore
accertamentodiagnostico
Ulteriore accertamentodiagnostico
Valutazionediagnostica
Valutazionediagnostica
Positivi al testPotenziale alto rischio Esito positivo
Esito negativo
Negativi al testRischio basso
Richiamo precoce
Diagramma di flusso screening
localizzata
Laboratorio centrale
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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"80.000"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"
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F. Carozzi Workshop Pre-congruessuale GISCi 2015
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
F. Carozzi Workshop Pre-congruessuale GISCi 2015
CDC , Atlanta USA
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Variations in Cervical Samples
" Collection Device - Brush, broom, spatula, swab, tampon
" Collection Media - STM, PreservCyt, SurePath, alcohol, paper
" Collection Method - Clinician collected
- Self-collected
F. Carozzi Workshop Pre-congruessuale GISCi 2015 12"
" 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.
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 2015 21 April 2015 23 April 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
• 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
Four HPV assays – four different technologies
QiaSymphony"RCS"HC2®"work"flow"
Roche cobas®4800 workflow
Gen-Probe PANTHER® APTIMA workflow
Genomica!CLART®HPV2"
The"HORIZON"Study"
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
38 CIN 3+ in 2,869 women screened at age 30-65 years*
Screening test CIN 3+ Detection rate per
100 screened women
Sensitivity
Cytology 31 1.1% -- 1 (ref) 82%
Hybrid Capture 2 34 1.2% -- +10% 89%
cobas 36 1.3% -- +16% 95%
CLART 36 1.3% -- +16% 95%
APTIMA 33 1.2% -- +6% 87%
Sensi:vity"for"CIN"3+"
[Rebolj et al., in preparation]
* Histological follow-up will be complete by end of 2013
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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
HPV e rischio cancerogeno: 2011
F. Carozzi Workshop Pre-congruessuale GISCi 2015
• Come mantenerlo aggiornato? • Chi lo fa? • Con quale frequenza? • Come renderlo disponibile? • Come valutare i dati che saranno pubblicati?
Elenco test hrHPV clinicamente validati
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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""
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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.
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Quality control
""""""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) 135–144
endogenous, within-sample controls
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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 2015 21 April 2015 23 April 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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".
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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 non-observance of quality parameters.
F. Carozzi Workshop Pre-congruessuale GISCi 2015
Registrazione giornaliera dei dati
Controllo Interno RLU/CO RCS
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
1,1
1,2
1,3
1,4
1,5
1,6
1,7
1,8
0 50 100 150 200
RLU
/CO
Serie1
1 DS
2 DS
3DS
1. Standard Operating Procedure with corrective actions and management of situations that deviate from the expected value
F. Carozzi Workshop Pre-congruessuale GISCi 2015
% di donne 35-64 POSITIVE al Test HPV
4,0%4,6% 4,7% 4,8% 4,8% 5,0% 5,1%
5,6% 5,8% 6,0% 6,0%6,6%
7,1%
7,8%
9,1%
12,4%
0%
2%
4%
6%
8%
10%
12%
14%
ROMA G
SAVONESE
MOLISE
TRENTO
ALTA PADOVANA
PADOVA
ROVIGO
VENEZIANA
ADRIA
VALLECAMONICA
ESTE
AVEZZANO
TORINO
LATINA
REGGIO EMILIA
FIRENZE
TERAMO
LANCIANO
PESCARA
6,3%
NORDCENTROSUD e ISOLE
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
HPV screening Program in Italy women aged 35-64yo % HPV test positive women
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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 " www.ukneqas.org.uk
" 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
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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."
F. Carozzi Workshop Pre-congruessuale GISCi 2015
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"
• 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""
Vedi Sessione Poster: G.P. Pompeo
F. Carozzi Workshop Pre-congruessuale GISCi 2015
F. Carozzi Workshop Pre-congruessuale GISCi 2015
!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""
F. Carozzi Workshop Pre-congruessuale GISCi 2015
