Deborah French

Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test

ABSTRACT There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases.

Human papillomavirus and colorectal cancer: evidences and pitfalls of published literature

PurposeThe aim of this study is to review published literature regarding a possible role of human papillomavirus (HPV) infection in colorectal cancer in order to understand if HPV infection plays an active role in colorectal carcinogenesis and to highlight evidences and pitfalls of published studies.MethodsWe reviewed literature by searching PubMed, Ovid, and the Cochrane databases for published series investigating HPV and colorectal cancer from 1988 to date.ResultsTwenty-one studies investigating a possible correlation between HPV infection and colon cancer have been published. We reviewed 15 case–control studies and six studies investigating a possible role for HPV virus in colorectal carcinogenesis. HPV was detected in the majority of reported series with a significant difference in HPV infection between tumors and disease-free controls or tumor-adjacent tissue; the HPV mean detection rate within carcinomas was 41.7%, comparing to a mean detection rate of 32.8% in adjacent colic mucosae, and 5.8% in disease-free controls (Chi-square test, p = 0.001). The correlation between HPV infection and c-myc amplification, k-ras mutation, and p53 polymorphism or mutations has been investigated; however, the possible role of HPV in colorectal carcinogenesis was not defined.ConclusionsHPV has been detected in the majority of reported series, but published literature lacks in definitive data regarding standard methods of investigation and stratification of groups and population. These data encourage further studies with the aim to investigate the presence of the virus in larger series, its possible role in oncogenesis, the integration in host genome, the expression of viral oncoproteins, the mutations in HPV positive cancers and routes of colon infection (hematologic/lymphatic spreading or perineal diffusion).

Expression of HPV16 E5 down-modulates the TGFbeta signaling pathway

BackgroundInfection with high-risk human papillomavirus (HR-HPV) genotypes, mainly HPV16 and HPV18, is a major risk factor for cervical cancer and responsible for its progression. While the transforming role of the HPV E6 and E7 proteins is more characterized, the molecular mechanisms of the oncogenic activity of the E5 product are still only partially understood, but appear to involve deregulation of growth factor receptor expression. Since the signaling of the transforming growth factor beta (TGFbeta) is known to play crucial roles in the epithelial carcinogenesis, aim of this study was to investigate if HPV16 E5 would modulate the TGF-BRII expression and TGFbeta/Smad signaling.FindingsThe HPV16 E5 mRNA expression pattern was variable in low-grade squamous intraepithelial lesions (LSIL), while homogeneously reduced in high-grade lesions (HSIL). Parallel analysis of TGFBRII mRNA showed that the receptor transcript levels were also variable in LSILs and inversely related to those of the viral protein. In vitro quantitation of the TGFBRII mRNA and protein in human keratinocytes expressing 16E5 in a dose-dependent and time-dependent manner showed a progressive down-modulation of the receptor. Phosphorylation of Smad2 and nuclear translocation of Smad4 were also decreased in E5-expressing cells stimulated with TGFbeta1.ConclusionsTaken together our results indicate that HPV16 E5 expression is able to attenuate the TGFbeta1/Smad signaling and propose that this loss of signal transduction, leading to destabilization of the epithelial homeostasis at very early stages of viral infection, may represent a crucial mechanism of promotion of the HPV-mediated cervical carcinogenesis.

Prognostic Value of HPV E6/E7 mRNA Assay in Women with Negative Colposcopy or CIN1 Histology Result: A Follow-Up Study

Pap test, and especially HPV DNA test, identify a large group of women who do not have any clinically relevant lesions, i.e., CIN2+ (Cervical Intraepithelial Neoplasia grade 2 or worse), but who are at greater risk of getting lesions in the future. The follow up of these women needs new biomarkers with prognostic value. The objective of this study is to evaluate the prognostic value of E6/E7 mRNA over-expression assay (PreTect HPV-Proofer, Norchip) for 5 HR-HPV types (16, 18, 31, 33, and 45) for progression to CIN2+ after a negative colposcopy. This prospective study, conducted at four Italian centres, enrolled 673 women with either a negative colposcopy or a negative or CIN1 histology. The clinical end-point was histological confirmation of CIN2+. Women were classified at baseline according to mRNA results and managed according to local colposcopy protocols. At least one conclusive follow-up test was obtained for 347 women (25 months average lapse since recruitment, range 5–74). Only seven CIN2+ were detected during follow up, three among the 82 women positive for mRNA at baseline, two among the 250 negative (Fisher exact test, p = 0.02), and two among the 12 with an invalid test. Absolute CIN2+ risk was 6.7/1,000 person/years in the whole cohort. The absolute CIN2+ risk was 18.4/1,000 person/years and 3.6/1,000 person/years in mRNA-positive and mRNA-negative women, respectively. In conclusion, E6/E7 mRNA over-expression appears to be a good candidate as a prognostic biomarker to manage HR-HPV DNA-positive women with negative colposcopy or histology, particularly in order to decrease follow-up intensity in those who are negative.

In vivo HPV 16 E5 mRNA: Expression pattern in patients with squamous intra-epithelial lesions of the cervix

BACKGROUND Human Papillomavirus (HPV) type 16 E5 is a small protein, which is reported to display transforming activity in vitro and in animal studies. The E5 transcriptional activity, however, has been rarely reported in vivo in literature. OBJECTIVES (a) To detect the E5 transcripts in vivo in a population of HPV 16 positive patients with abnormal cytology and (b) to correlate the level of expression to the degree of the cytological lesion. STUDY DESIGN AND METHODS 250 cytological samples of HPV positive women were obtained and tested for the E6/E7 mRNA expression. Patients were selected if HPV 16 only mRNA positive and with a cytology consistent with low-grade/high-grade squamous intra-epithelial (LSIL/HSIL) lesions. Selected patients were tested for the E5 transcripts by reverse RT PCR, comparing the expression level in vivo with a transfected HPV 16 E5 HaCaT cell line. RESULTS 27 HPV 16 E6/E7 mRNA positive LSIL/HSIL patients were selected. 13 out of 17 LSIL patients were tested positive for the E5 mRNA, showing an ample range of positivity. In the HSIL group 7 out of 10 patients were tested positive, displaying lower and more homogeneous levels of expression if compared with the transfected cells. CONCLUSION The HPV 16 E5 transcripts levels showed a broad distribution in vivo; the discrepancy was wider in LSIL patients, with HSIL patients displaying a more homogeneous profile. However, because of the limited number of patients, we could not draw a firm conclusion about the correlation between the E5 expression and the disease progression.

Impact of human papillomavirus infection on the neovaginal and vulval tissues of women who underwent surgical treatment for Mayer-Rokitansky-Kuster-Hauser syndrome

OBJECTIVE To evaluate the impact of human papillomavirus (HPV) infections on the neovaginal and vulval tissues of women who underwent surgical treatment for Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. DESIGN Multicenter observational study. SETTING University and community hospitals. PATIENT(S) Thirty-three women who had previously undergone neovagina reconstruction due to MRKH and who were referred to our centers for the evaluation and treatment of HPV neovaginal/vulval-related lesions. INTERVENTION(S) HPV infections were confirmed by polymerase chain reaction analysis or hybrid capture 2 tests; the patients underwent vaginoscopy, pap smear, and biopsy of the lesion and were treated by laser vaporization. Follow-up was conducted for 5 years. MAIN OUTCOME MEASURE(S) HPV-related neovaginal/vulval lesions, HPV testing, follow-up, recurrence rate. RESULT(S) Seventeen patients showed vulval lesions, and 16 patients neovaginal lesions. HPV testing results were positive for low-risk HPVs in 27 patients and high-risk HPVs in six patients. All the vulval lesions were condylomata, whereas 10 neovaginal lesions were condylomata, three were vaginal intraepithelial neoplasia (VAIN) degree 1, two were VAIN degree 2, and one was an adenocarcinoma. Eight patients were lost to follow-up. Twenty patients tested positive for an HPV infection, and seven patients (28%) had a recurrence of the lesion in the follow-up time. CONCLUSION(S) Patients who underwent neovagina reconstruction have sexual relationships and are HPV exposed. These patients should be evaluated after surgery for HPV infections to prevent HPV-related diseases and cancers.

Assessment of HPV-mRNA test to predict recurrent disease in patients previously treated for CIN 2/3

BACKGROUND The use of HPV-mRNA test in the follow-up after LEEP is still matter of debate, with regard to its capacity of prediction relapse. OBJECTIVE The aim of the present study is to evaluate the reliability of HPV-mRNA test to predict the residual and recurrent disease, and its accuracy in the follow-up of patients treated for CIN 2/3. STUDY DESIGN Multicenter prospective cohort study. Patients who underwent LEEP after a biopsy diagnosing CIN 2/3 were followed at 3, 6, 12, 24 and 36 months. Each check up included cytology, colposcopy, HPV-DNA test (LiPA) and HPV-mRNA test (PreTect HPV Proofer Kit NorChip). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), of HPV-DNA test and HPV-mRNA test to predict relapse, recurrent and residual disease. Using multiple logistic regression, the statistical significant variables as assessed in univariate analysis were entered and investigated as predictors of relapse disease. RESULTS The mRNA-test in predicting a residual disease had a sensitivity of 52% and a NPV of 91%, whereas DNA-test had 100% and 100%, respectively. On the contrary in the prediction of recurrent disease mRNA-test had a sensitivity and a NPV of 73.5% and 97%, whereas DNA-test had 44% and 93%. On the multivariate analysis, age, cytology, HPV DNA and mRNA test achieved the role of independent predictors of relapse. CONCLUSION HPV-mRNA test has a higher sensitivity and a higher NPV in predicting recurrent disease, for this reason it should be used in the follow-up of patients treated with LEEP for CIN 2/3 in order to individualize the timing of check up.

Prognostic implication of high risk human papillomavirus E6 and E7 mRNA in patients with intraepithelial lesions of the cervix in relationship to age.

Since the introduction of the cytological screening programs, a significant reduction in the incidence of cervical cancer has been achieved. Almost all of these cancers are related to high-risk (HR) Human Papillomavirus (HPV) cervical infections. However, the natural history of HPV infection seems to be different in younger patients, resulting in a higher rate of regression. There is, therefore, the need to identify HPV-related biomarkers in order to enhance the effectiveness of screening of high-risk cytological lesions, in particular in women over 35 years of age. This study aims to evaluate the prognostic value of the HR HPV E6 and E7 mRNA expression in women with intraepithelial lesions of the cervix, older or younger than 35 years of age. One hundred and eighty-four HR HPV DNA positive patients with a low squamous intraepithelial lesion (LSIL) were tested for mRNA expressions, included in an observational study, and evaluated at follow-up with standard cytology up to 24 months from the mRNA test. The frequency of HSIL/LSIL cytology in the older cohort of mRNA positive patients was significantly higher compared to mRNA-negative patients, both at 1 and 2 years of follow-up (Chi-square: p 0.007 and p 0.009), but this difference was not found in the younger cohort. According to our results, the E6/E7 mRNA test could be a biomarker for viral activity, useful in identifying patients at higher risk of abnormal cytology, and in implementing the management of HR HPV DNA-positive women over 35 years of age.

Human papillomavirus does not have a causal role in colorectal carcinogenesis

AIM To investigate the presence of human papillomavirus (HPV) DNA along with the integration, the quantification and the expression of the HPV16 in colorectal cancers. METHODS A prospective series of colorectal tumors were genotyped for HPV DNA. The clinical and pathological variables of the HPV-positive tumors were compared to those of HPV-negative samples. The integration status of HPV16 was evaluated by calculating E2/E6 ng ratios. HPV16-positive tumors were also evaluated for (1) E2, E4, E5, E6 and E7 viral gene ng quantification; (2) relative quantification compared to W12 cells; and (3) viral E2, E4, E5, E6 and E7 mRNA transcripts by real-time polymerase chain reaction. RESULTS HPV infection was detected in 16.9% of all tumors examined, and HPV16 was the most frequent type detected (63.6% of positive tissues). Notably, the clinical and pathological features of HPV-positive colorectal cancers were not significantly different than those of HPV-negative cancers (χ (2) and t-test for all clinical and pathological features of HPV-positive vs HPV-negative colorectal cancers: p ns). HPV16 DNA was present exclusively in episomal form, and the HPV16 E2, E4, E5, E6 and E7 genes were detected in trace nanogram quantities. Furthermore, the HPV16 genes ranged from 10(-3) to 10(-9) compared to W12 cells at an episomal stage. Although the extractions were validated by housekeeping gene expression, all the HPV16 positive tissues were transcriptionally inactive for the E2, E4, E5, E6 and E7 mRNAs. CONCLUSION Based on our results, HPV is unlikely involved in colorectal carcinogenesis.

The re-infection rate of high-risk HPV and the recurrence rate of vulvar intraepithelial neoplasia (VIN) usual type after surgical treatment.

Summary Background VIN usual type appears to be related to the HPV’s oncogenic types. The aim of this prospective multicenter study was to evaluate the re-infection rate of high-risk HPV and the recurrence rate of VIN usual type after surgical treatment. Material/Methods The study enrolled 103 women affected by VIN usual type. They underwent wide local excision by CO2 laser. The patients were investigated by clinical evaluation and HPV DNA test 6 months after surgical treatment, and then were followed-up at 12, 18, 24, and 36 months. The recurrences were treated with re-excision. Results The rate of HPV infection after surgical treatment was 34% at 6 months, 36.9% at 12 months, 40% at 18 months, 41.7% at 24 months and 44.7% at 36 months. The mean time from HPV infection to the development of VIN was 18.8 months. Conclusions HPV testing in the follow-up of VIN usual type patients might be useful for identifying those patients with a higher risk of recurrence after surgical treatment, although more studies are needed. These preliminary data suggest that the test, in addition to clinical examination, can improve the efficacy of the follow-up.