Robert Jach

Worldwide human papillomavirus genotype attribution in over 2000 cases of intraepithelial and invasive lesions of the vulva

BACKGROUND Human papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions. METHODS Histologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16(INK4a) by immunohistochemistry (CINtec histology kit, ROCHE). An IVC was considered HPV driven if both HPV-DNA and p16(INK4a) overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI). RESULTS Of 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16(INK4a) positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC_WB) (N=326) were more likely to be HPV and p16(INK4a) positive (AP=69.5%, CI=63.6-74.8) versus KSCC (AP=11.5%, CI=9.7-13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold). CONCLUSION Combined data from HPV-DNA and p16(INK4a) testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide.

Cancer cell detection in tissue sections using AFM.

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Youngs modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.

Human papillomavirus DNA prevalence and type distribution in anal carcinomas worldwide

Knowledge about human papillomaviruses (HPV) types involved in anal cancers in some world regions is scanty. Here, we describe the HPV DNA prevalence and type distribution in a series of invasive anal cancers and anal intraepithelial neoplasias (AIN) grades 2/3 from 24 countries. We analyzed 43 AIN 2/3 cases and 496 anal cancers diagnosed from 1986 to 2011. After histopathological evaluation of formalin‐fixed paraffin‐embedded samples, HPV DNA detection and genotyping was performed using SPF‐10/DEIA/LiPA25 system (version 1). A subset of 116 cancers was further tested for p16INK4a expression, a cellular surrogate marker for HPV‐associated transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance in the anal cancer data set. HPV DNA was detected in 88.3% of anal cancers (95% confidence interval [CI]: 85.1–91.0%) and in 95.3% of AIN 2/3 (95% CI: 84.2–99.4%). Among cancers, the highest prevalence was observed in warty–basaloid subtype of squamous cell carcinomas, in younger patients and in North American geographical region. There were no statistically significant differences in prevalence by gender. HPV16 was the most frequent HPV type detected in both cancers (80.7%) and AIN 2/3 lesions (75.4%). HPV18 was the second most common type in invasive cancers (3.6%). p16INK4a overexpression was found in 95% of HPV DNA‐positive anal cancers. In view of the results of HPV DNA and high proportion of p16INK4a overexpression, infection by HPV is most likely to be a necessary cause for anal cancers in both men and women. The large contribution of HPV16 reinforces the potential impact of HPV vaccines in the prevention of these lesions.

Large contribution of human papillomavirus in vaginal neoplastic lesions: A worldwide study in 597 samples

AIM This work describes the human papillomavirus (HPV) prevalence and the HPV type distribution in a large series of vaginal intraepithelial neoplasia (VAIN) grades 2/3 and vaginal cancer worldwide. METHODS We analysed 189 VAIN 2/3 and 408 invasive vaginal cancer cases collected from 31 countries from 1986 to 2011. After histopathological evaluation of sectioned formalin-fixed paraffin-embedded samples, HPV DNA detection and typing was performed using the SPF-10/DNA enzyme immunoassay (DEIA)/LiPA25 system (version 1). A subset of 146 vaginal cancers was tested for p16(INK4a) expression, a cellular surrogate marker for HPV transformation. Prevalence ratios were estimated using multivariate Poisson regression with robust variance. RESULTS HPV DNA was detected in 74% (95% confidence interval (CI): 70-78%) of invasive cancers and in 96% (95% CI: 92-98%) of VAIN 2/3. Among cancers, the highest detection rates were observed in warty-basaloid subtype of squamous cell carcinomas, and in younger ages. Concerning the type-specific distribution, HPV16 was the most frequently type detected in both precancerous and cancerous lesions (59%). p16(INK4a) overexpression was found in 87% of HPV DNA positive vaginal cancer cases. CONCLUSIONS HPV was identified in a large proportion of invasive vaginal cancers and in almost all VAIN 2/3. HPV16 was the most common type detected. A large impact in the reduction of the burden of vaginal neoplastic lesions is expected among vaccinated cohorts.

Autologous muscle-derived cells for the treatment of female stress urinary incontinence: A 2-year follow-up of a polish investigation

We evaluated the safety, feasibility and initial effects of therapy with muscle‐derived cells (MDCs) for women with stress urinary incontinence (SUI).

Diagnostic accuracy of three-dimensional sonohysterography compared with office hysteroscopy and its interrater/intrarater agreement in uterine cavity assessment after hysteroscopic metroplasty.

OBJECTIVE To compare the diagnostic accuracy of three-dimensional sonohysterography (3D-SIS) and office hysteroscopy in uterine cavity assessment after hysteroscopic metroplasty (HM) and determine the interrater/intrarater agreement for 3D-SIS. DESIGN Prospective observational study. SETTING University hospital, private hospital, and clinic. PATIENT(S) One hundred forty-one women undergoing HM for septate uterus with a history of miscarriage and/or infertility. INTERVENTION(S) 3D-SIS and office hysteroscopy at 6-8 weeks after HM. MAIN OUTCOME MEASURE(S) Shape of the uterine cavity, length of the fundal notch (≥1 or <1 cm), and the presence of intrauterine adhesions were assessed, and the interrater/intrarater agreement of 3D-SIS was evaluated in 30 randomly selected patients. RESULT(S) Uterine abnormalities were detected with the use of hysteroscopy in 18 (12.8%) of 141 women. 3D-SIS was highly accurate (97.2%), sensitive (97%), and specific (100%), with a positive predictive value of 100% and a negative predictive value of 85%. The diagnostic values of hysteroscopy and 3D-SIS were not significantly different (McNemar test). 3D-SIS showed substantial interrater/intrarater agreement regarding overall uterine cavity evaluation (κ = 0.79 and 0.78, respectively). CONCLUSION(S) 3D-SIS demonstrated substantial interrater/intrarater agreement for the postoperative evaluation of the uterine cavity, being as diagnostically accurate as hysteroscopy. The use of second-look hysteroscopy may be limited to cases that require reoperation.

Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM

High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16INK4a and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16INK4a and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16INK4a was present. Extensive p16INK4a expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM/E4 or, more promisingly, p16INK4a/E4 as an adjunct to conventional pathology.

Reply: Are the ESHRE/ESGE criteria of female genital anomalies for diagnosis of septate uterus appropriate?

Sir, We are following with interest the European Society of Human Reproduction and Embryology/European Society for Gynaecological Endoscopy (ESHRE/ESGE) initiative to create a new classification of female genital tract congenital anomalies (Grimbizis et al. 2013). We applied the European criteria routinely using three-dimensional (3D) ultrasonography, while simultaneously classifying our patients based on the American Society for Reproductive Medicine (ASRM) criteria (Buttram et al. 1988), complemented by additional common morphometric criteria used to differentiate septate, bicornuate and arcuate uteri (Bermejo et al. 2010, Ludwin et al. 2013). We encountered three problems: (i) practical adaptation of the ESHRE–ESGE classification criteria for septate uterus diagnosis, (ii) an increased frequency of septate uterus diagnoses and all uterine congenital anomalies according to the ESHRE–ESGE compared with the ASRM classification (because a septate uterus was diagnosed in cases where uteri fulfilled previous morphometric criteria for arcuate and in some cases normal uteri) and (iii) potentially important clinical implications of the new classification. We believe that application of ESHRE–ESGE criteria may cause difficulties because (i) the thickness of the uterine wall as the reference value for the septate uterus diagnosis may vary in different regions of the uterus. Thus, it is important to determine where and how it should be measured and (ii) the original definition of septate uterus is an abnormally shaped uterine cavity. The ratio of myometrial thickness to the size of the internal fundal indentation may not reflect the actual anatomical relationships in the uterus and lead to false diagnoses (Fig. 1). Another drawback of the relative criteria is that they are not compatible with endoscopic techniques since myometrial thickness cannot be assessed. The application of the ESHRE/ESGE criteria may have serious clinical implications. The diagnosis of uterine septum on the basis of relative ESHRE/ESGE criteria is not supported by retrospective results and prospective studies of corrective surgery. This applies to eligibility for hysteroscopic metroplasty of patients with recurrent miscarriages who have a small internal fundal indentation; the proposed treatment may not improve obstetric results. A more serious concern is that prophylactic metroplasty may be performed in these cases prior to assist reproductive technology procedures. Additionally, the new classification may affect the assessment of anatomical results of corrective surgery, which could result in frequent repeat procedures, possibly without any significant effect on reproductive performance. This might affect the decision to re-operate in cases whereprevious retrospective studies suggested no indication for complementary metroplasty (residual septum

Transrectal Ultrasound-Guided Hysteroscopic Myomectomy of Submucosal Myomas With a Varying Degree of Myometrial Penetration

STUDY OBJECTIVE To predict the 1-step complete resection rate after transrectal ultrasound-guided hysteroscopic myomectomy and to determine the usefulness of intraoperative transrectal ultrasonography (TRUS) in monitoring hysteroscopic electroresection of submucosal myomas. DESIGN Prospective cohort study (Canadian Task Force classification II-1). SETTING University hospital. PATIENTS One hundred twenty women with symptomatic (abnormal uterine bleeding or reproductive disorder), single, submucosal myomas underwent hysteroscopic electroresection. Groups 1 and 2 were monitored, respectively, with or without TRUS. Anatomical inclusion criteria were myoma ≤5 cm and myometrial free margin ≥3 mm above the myoma. INTERVENTIONS Myomas were evaluated preoperatively via sonohysterograpy and were graded according to the guidelines outlined by the European Society of Hysteroscopy (ESH), including size and myometrial free margin, and according to the STEPW (size, topography, extension, penetration, and lateral wall) classification. On the basis of sonographic findings, patients with myomas >3 cm received gonadotropin-releasing hormone therapy for 1 to 3 months. Hysteroscopic myomectomy was performed with or without TRUS guidance. At 4 to 8 weeks after the initial procedure, postoperative transvaginal ultrasonography, sonohysterography, or second-look hysteroscopy was performed. MEASUREMENTS AND MAIN RESULTS In the TRUS group, a significantly higher percentage of 1-step complete resections was observed than in the group without TRUS (91% vs 73%) (p = .02). This was associated with a statistically significant difference in the subgroups of myomas that were deeply penetrating into the myometrium (89% vs 55%) (p < .01). One-way logistic analysis of data for all treated patients indicated the use of TRUS, as well as the ESH and STEPW classifications, as significant factors influencing the 1-step complete resection. At multivariable logistic regression analysis, use of TRUS (odds ratio [OR], 2.74; p < .001), myomas graded 0 or 1 according to ESH (OR, 3.55; p < .001), and size <3 cm (OR, 2.35; p < .05) were significantly associated with 1-step complete resection (area under the curve, 0.80; p < .001). In the TRUS group there were two significant predictors: size <3 cm (OR = 5.21; p < .05) and myometrial free margin <5 mm (OR, 0.18; p < .05). CONCLUSION Intraoperative use of TRUS during hysteroscopic myomectomy increases the chance of complete 1-step removal of submucosal myomas that deeply penetrate the myometrium.

Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a

Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (&kgr;=0.43 for CIN1/negative, 0.54 for CIN2/⩽CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: &kgr;=0.896; 95% confidence interval [CI]: 0.763-0.969; p16INK4a: &kgr;=0.798; 95% CI: 0.712-0.884; MCM: &kgr;=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative, although 9 were MCM-positive. There were variable extents of p16INK4a positivity in 11/11 CIN1 and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a, and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a, indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life-cycle completion (and which might ultimately regress) from purely transforming CIN2/3 needing treatment warrants further research.