Test de VPH en estrategia de screening primario para detección de cáncer cervicouterino / HPV test in primary screening strategy for cervical cancer detection

INTRODUCCIÓN: El cáncer del cuello uterino es una enfermedad prevenible, e incluso puede curarse si se detecta en una fase temprana y se trata debidamente. A pesar de ello, sigue siendo uno de los tipos de cáncer más comunes y una de las causas de muerte por cáncer entre mujeres de todo el mundo. Más del 85% de las mujeres afectadas son mujeres jóvenes y con escasa instrucción que viven en los países más pobres. Muchas de ellas también tienen hijos de corta edad cuya supervivencia se ve truncada por la muerte prematura de sus madres1 . Pocas enfermedades reflejan las desigualdades a nivel mundial como el cáncer del cuello uterino. En los países de ingresos bajos y medianos, su incidencia es de casi el doble y su tasa de mortalidad el triple que las de los países de ingresos altos. En palabras del Director General de la OMS: «Mediante intervenciones costo-eficaces y basadas en pruebas científicas, en particular la vacunación de las niñas contra el virus del papiloma humano, la detección y el tratamiento de lesiones precancerosas y la mejora del acceso al diagnóstico y el tratamiento de cánceres invasivos, podemos eliminar el cáncer del cuello uterino como problema de salud pública y lograr que se convierta en una enfermedad del pasado¼2 . Un reciente comunicado a todos los países en mayo de 2018 aboga por el fin al sufrimiento provocado por el CCU y una renovada voluntad política para hacer realidad su eliminación, instando a todas las partes interesadas a que se unieran en pos de ese objetivo común3 . Este esfuerzo mundial está en consonancia con los instrumentos que consagran la salud como derecho humano, 4 así como con la Agenda 2030 para el Desarrollo Sostenible y su principio general de no dejar a nadie atrás. Respalda el logro de varios Objetivos de Desarrollo Sostenible y metas conexas 5 y es uno de los componentes de la Estrategia Mundial del Secretario General de las Naciones Unidas para la Salud de la Mujer, el Niño y el Adolescente (2016-2030). OBJETIVO: El objetivo del presente informe será evaluar entre sí las distintas tecnologías para test de VPH disponibles en nuestro país, utilizadas en estrategias de detección precoz de CCU. Se analizarán diferencias en eficacia, seguridad, conveniencia y costos para el sistema de salud de Argentina. METODOLOGÍA: Se realizó la búsqueda bibliográfica de manera independiente por distintos investigadores. Se revisaron bases de datos y buscadores: MEDLINE, LILACS, Cochrane, Universidad de York, HTAi, OMS, Tripdatabase, Google académico, agencias de ETS, agencias reguladoras de alimentos y medicamentos, repositorios de guías de práctica clínica y productores de guías como OMS-OPS, Ministerio de Salud de la República Argentina, Royal College of Obstetricians and Gynaecologist,Faculty of Sexual and Reproductive Health Care, American College of Obstetricians and Gynecologist y Society of Obstetricians and Gynaecologist of Canada. Se realizó una búsqueda de informes en el repositorio BRISA de la OPS-RedETSA y en el repositorio de RedArets, así como otras agencias de ETS de nuestro país y del mundo. Se solicitó a través de las autoridades de CONETEC a todos los productores y vendedores de marcas y modelos de test de VPH disponibles en Argentina que acerquen evidencias sobre eficacia, seguridad y conveniencia de sus marcas y modelos. RESULTADOS: La prueba de detección del VPH ofrece máxima especificidad y tiene un sólido valor de predicción negativa, lo que significa que la mujer que obtiene un resultado negativo no necesita ser examinada de nuevo en un mínimo de cinco años. A su vez, dar a las mujeres la posibilidad de realizar por sí mismas la prueba (auto-toma) mejora la aceptabilidad y facilita el acceso a los servicios, aunque se requiere una compleja y dinámica articulación entre las capacidades de detección y atención del sistema de salud para captar la gran cantidad de casos positivos en la primera prueba, y continuar los estudios confirmatorios con PAP y las instancias terapéuticas derivadas. A diferencia de otros países, Argentina no logra reducir la mortalidad por CCU en forma significativa, pero existe desde 2011 una decisión de política sanitaria para implementar el test de VPH como prueba primaria. Como otros países, existe en Argentina una elevada inequidad en el acceso a rastreo y mortalidad por CCU, relacionándose las mismas con los determinantes sociales de la salud. CONCLUSIÓN: La detección y el tratamiento del CCU constituyen una prioridad en políticas sanitárias. Existe en Argentina una decisión de política sanitaria para implementar el test de VPH como prueba primaria en el screening de CCU (iniciada progresivamente desde 2011), que debe enfrentar de manera primordial la alta inequidad en el acceso a rastreo y mortalidad por CCU, relacionándose las mismas con los determinantes sociales de la salud. El test de VPH respecto al test de PAP: Es más sensible aunque levemente menos específico. Se recomienda en Argentina como rastreo en serie (HPV seguido de PAP). Permite la auto-toma que incrementa la tasa de realización del rastreo. Comparado con PAP, el test HPV demostró salvar vidas y ser costo-ahorrativo. En la revisión de la evidencia realizada no se identificaron diferencias en performance diagnóstica entre las diferentes marcas y modelos de tests de VPH disponibles en Argentina; algunas marcas comerciales aún no tienen validada la auto-toma y otras requieren adquirir por separado (con otro proveedor) este dispositivo. Las guías clínicas propuesta por OMS y varios países en el mundo apoyan el uso de estas metodologías como estrategia diagnóstica; su cobertura es explícita en países desarrollados y se encuentra en implementación en nuestra región Las evaluaciones económicas evaluadas de organismos de referencia avalan su utilización, demostrando ahorros de costos y mejor perfil de eficacia global en la patología, aunque consideran mandatorio un gran cambio operativo y cultural para los médicos, pacientes y laboratorios para que la implementación sea exitosa (requiriendo adecuada planificación, financiación y coordinación).

Clinical validation of the HPVIR high-risk HPV test on cervical samples according to the international guidelines for human papillomavirus DNA test requirements for cervical cancer screening.

BACKGROUND: The indicating FTA card is a dry medium used for collection of cervical samples. HPVIR is a multiplex real-time PCR test that detects 12 high-risk human papillomavirus types (hrHPV) and provides single genotype information for HPV16, - 31, - 35, - 39, - 51, - 56, and - 59 and pooled type information for HPV18/45 and HPV33/52/58. The aim of this study was to evaluate whether a strategy with cervical samples collected on the FTA card and subsequently analysed with the HPVIR test complies with the criteria of the international guidelines for a clinically validated method for cervical screening. METHODS: We performed a non-inferiority test comparing the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a clinically validated reference test (Cobas® HPV test) based on liquid-based cytology (LBC) samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. For evaluation of the specificity we used 799 women without ≥ CIN2, and for clinical sensitivity we used 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples. RESULTS: The clinical sensitivity and specificity for samples collected on the FTA card and analysed using the HPVIR test were non-inferior to samples analysed with the Cobas® HPV test based on LBC samples (non-inferiority test score, p = 1.0 × 10- 2 and p = 1.89 × 10- 9, respectively). Adequate agreement of > 87% was seen in both the intra- and inter-laboratory comparisons. CONCLUSIONS: Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.

[Relationship between cervical lesions and the type-specific viral load of high risk HPV reflected by the Ct value of Cobas 4800 HPV system].

Objective: To explore the relationship between cervical lesions and high risk HPV (HR-HPV) viral load reflected by the cycle threshold (Ct) values of Cobas 4800 HPV (Cobas 4800) system. Methods: From August 2016 to September 2017, 7 000 women from Shenzhen, were recruited for cervical cancer screening with Cobas 4800 system and cytology co-test. Colposcope biopsies were performed on women who were positive of HPV 16, 18, and positive of HPV types other than 16,18 with cytology [≥ atypical squamous cell of undetermined signification (ASCUS)], or HPV negative but abnormal of cytology [≥ low grade squamous intraepithelial lesion (LSIL)]. The Ct values of HPV 16, 18 and all combined other types coming from Cobas 4800 system were used as an indicator of viral load to analyze the relationship between type-specific HPV load and the cervical lesions. Results: (1) Among the 7 000 screening women, 370 cases were positive for cervical cancer screening, 325 of them underwent colposcope biopsies, and coloposcopy referred rate was 87.8% (325/370). Among 325 women undergoing cervical biopsy, pathological diagnosis was 119 cases of normal cervical cervix, 151 cases of LSIL, and 55 cases of high-grade squamous intraepithelial lesion (HSIL) and above (HSIL(+); including 53 cases of HSIL, 1 case of cervical adenocarcinoma, and 1 case of cervical squamous cell carcinoma). (2) The Ct value of HPV 16 was inversely correlated with the upgrading of the lesions (r=-0.617, P=0.000), and significant different among normal cervix,LSIL and HSIL(+) (35.4±4.5 vs 31.0±6.0 vs 26.5±4.0; F=25.537, P=0.000). There was no correlation between Ct value of HPV 18 and cervical lesions (r=-0.021, P=0.902). The Ct value of other 12 HPV types was statistically difference among normal normal cervix, HSIL(+) and cervicitis (33.0±5.3 vs 29.9±7.2 vs 29.8±5.8; F=5.087, P=0.007). Among them, LSIL and HSIL(+) were significantly lower than normal cervix (P<0.05), but there was no significant difference between LSIL and HSIL(+) (P>0.05). Conclusion: The Ct value of HPV 16 detecting in Cobas 4800 system as an indicator of virus load obviously correlates with different grades of cervical lesions, therefore could be a reference of cervical lesion existence and an indicator of lesion prognosis.

Performance of Aptima and Cobas HPV testing platforms in detecting high-grade cervical dysplasia and cancer.

BACKGROUND: Human papillomavirus (HPV) tests and genotyping have been used in clinical risk assessment. The purpose of this study was to analyze the performance of 2 common HPV testing platforms in detecting high-grade cervical lesions (high-grade squamous intraepithelial lesion [HSIL] or worse [≥HSIL]). METHODS: Between January 1 and December 31, 2015, 2041 Papanicolaou (Pap) tests with biopsy confirmation were analyzed along with HPV tests performed on Cobas or Aptima platforms. A biopsy diagnosis of grade 2 cervical intraepithelial neoplasia was confirmed with p16/Ki-67 immunohistochemistry. RESULTS: In total, 1866 and 175 Pap cases were tested on Cobas and Aptima platforms, respectively. Both platforms were highly sensitive (97% for both) for biopsy-confirmed ≥HSIL. Cobas HPV testing had higher positive rates for the diagnosis of benign lesions (84% vs 51%) and low-grade squamous intraepithelial lesions (89% vs 63%) on biopsy compared with Aptima. Aptima testing had significantly higher specificity for ≥HSIL than Cobas (41% vs 13%; P < .0001). Overall, performance of the Aptima platform was superior to that of the Cobas platform in detecting biopsy-confirmed ≥HSIL, resulting from its significantly higher positive predictive value (25% vs 16%; P < .03) and overall accuracy (50% vs 26%; P < .0001). CONCLUSIONS: Although both the Cobas and Aptima platforms offer highly sensitive tests for high-grade cervical lesions, Aptima HPV testing demonstrated significantly higher specificity and positive predictive value than Cobas testing for biopsy-confirmed ≥HSIL. The considerable difference may be related to the significant increase in E6/E7 expression after HPV DNA integration. The significantly higher specificity and overall accuracy of Aptima testing for ≥HSIL, resulting in the identification of high-risk populations that require immediate treatment and close follow-up, may prove useful in clinical risk stratification. Cancer Cytopathol 2017;125:652-7. © 2017 American Cancer Society.

Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma.

Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting.

Low prevalence of human papillomavirus in oral cavity squamous cell carcinoma in Queensland, Australia.

BACKGROUND: While human papillomavirus (HPV) is an accepted risk factor for oropharyngeal squamous cell carcinoma (SCC), its aetiological role in oral cavity SCC remains unclear. This study aimed to determine the HPV prevalence in an Australian population. METHODS: DNA was extracted from 63 formalin-fixed paraffin-embedded tumour specimens histologically confirmed as SCC of the oral cavity, diagnosed during 2006-2012. Clinical data were extracted from medical records. HPV presence was determined by polymerase chain reaction. Positive samples were typed by sequencing. Immunohistochemistry was used to assess p16INK4A , p53, pRB, Ki67, Cyclin D1 and p21WAF1 expression. RESULTS: Five of the 63 tumours (8%) were positive for HPV DNA (three HPV-16 positive and two HPV-18 positive). Two tumours overexpressed p16INK4A (3%) and one of these was also HPV positive. Overexpression of Cyclin D1 correlated significantly with tumour recurrence (P = 0.029) and death (P = 0.002). CONCLUSIONS: This study has identified a low prevalence of high-risk HPV in Queensland, Australia.

Maximizing the adequacy of Hologic(®) Cervista(®) HPV HR results on ThinPrep(®) Pap samples treated with glacial acetic acid.

BACKGROUND: Although glacial acetic acid (GAA) treatment of bloody cervical samples has reduced the rate of unsatisfactory Pap Tests, recent studies suggest that it may negatively impact high-risk (hr)-HPV test results. The objectives of this study were to compare the levels of genomic DNA between GAA treated and nontreated ThinPrep(®) samples using the Hologic(®) Cervista(®) HPV-HR assay and to compare the adequacy of the ThinPrep(®) Pap Test between aliquoted and nonaliquoted samples. METHODS: Prior to GAA treatment, 2.5 ml of the cervical sample was prealiquoted from 102 bloody ThinPrep(®) vials. Both GAA treated and nontreated samples were analyzed for hr-HPV using the Cervista(®) HPV HR assay. The levels of genomic DNA were measured and compared between these samples. In addition, ThinPrep(®) Pap Test adequacy rates were calculated and compared on aliquoted and nonaliquoted cervical samples. RESULTS: Of the 102 cervical samples, 95 (93%) nontreated aliquots contained satisfactory levels of genomic DNA as compared to 36 (35%) GAA-treated samples (p < 0.00001). Ninety-nine (97%) aliquoted cervical samples were satisfactory for cytologic evaluation as compared to 1,326 (96%) GAA treated samples from 2013 (nonaliquoted); not statistically significant (p = 0.7968). CONCLUSION: The levels of genomic DNA were significantly decreased in GAA treated vs non-treated TP samples. Aliquoting from the TP sample prior to treatment with GAA enables accurate measurement of DNA without affecting the adequacy of the TP cytology slide.

A new system for the amplification of biological signals: RecA and complimentary single strand DNA probes on a leaky surface acoustic wave biosensor.

This research describes a new amplification signals system of the leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor for the simple, sensitive and rapid detection of the target double-stranded DNA (dsDNA). The system consists of a RecA protein-coated complementary single-stranded DNA (cssDNA) probe complex that amplifies the biological signal to improve the sensitivity of the biosensor. The bis-PNA probe for detecting HPV was first immobilized on a gold surface membrane of the detection channel. After the probe was completely hybridized with the corresponding target DNA, different concentrations of the "RecA protein-complementary single strand DNA probe" were added to react with the bis-PNA/dsDNA complex. The phase shift of the LSAW biosensors, which was measured and found to be most significant when the RecA protein was 45 µg/mL and the ATPγS was 2.5 mmol/L. Compared with other concentrations (P<0.01) of RecA and ATPγS, the value of the phase shift was (11.74 ± 1.03) degrees and the ratio of the phase shift and hybridization time clearly outperformed that of the other concentrations. Compared to the direct hybridization of the bis-PNA probe and the target DNA sequence, the sensitivity was effectively improved and the detection time was significantly shortened. PNA binding adjacent to the area of the target sequence homologous to the probe significantly increased the yield of the hybridization reaction between the PNA/dsDNA complex and the RecA protein-coated cssDNA probe. In this condition, the phase shift was significantly obvious and the detection time was significantly shortened. In conclusion, the combination of the RecA protein-coated cssDNA probe and the LSAW bis-PNA biosensor provides sensitivity and simple and rapid detection of clinical trace pathogenic microorganisms.

A second generation cervico-vaginal lavage device shows similar performance as its preceding version with respect to DNA yield and HPV DNA results.

BACKGROUND: Attendance rates of cervical screening programs can be increased by offering HPV self-sampling to non-attendees. Acceptability, DNA yield, lavage volumes and choice of hrHPV test can influence effectiveness of the self-sampling procedures and could therefore play a role in recruiting non-attendees. To increase user-friendliness, a frequently used lavage sampler was modified. In this study, we compared this second generation lavage device with the first generation device within similar birth cohorts. METHODS: Within a large self-sampling cohort-study among non-responders of the Dutch cervical screening program, a subset of 2,644 women received a second generation self-sampling lavage device, while 11,977 women, matched for age and ZIP-code, received the first generation model. The second generation device was different in shape, color, lavage volume, and packaging, in comparison to its first generation model. The Cochran's test was used to compare both devices for hrHPV positivity rate and response rate. To correct for possible heterogeneity between age and ZIP codes in both groups the Breslow-Day test of homogeneity was used. A T-test was utilized to compare DNA yields of the obtained material in both groups. RESULTS: Median DNA yields were 90.4 µg/ml (95% CI 83.2-97.5) and 91.1 µg/ml (95% CI 77.8-104.4, p= 0.726) and hrHPV positivity rates were 8.2% and 6.9% (p= 0.419) per sample self-collected by the second - and the first generation of the device (p= 0.726), respectively. In addition, response rates were comparable for the two models (35.4% versus 34.4%, p= 0.654). CONCLUSIONS: Replacing the first generation self-sampling device by an ergonomically improved, second generation device resulted in equal DNA yields, comparable hrHPV positivity rates and similar response rates. Therefore, it can be concluded that the clinical performance of the first and second generation models are similar. Moreover, participation of non-attendees in cervical cancer screening is probably not predominantly determined by the type of self-collection device.