Whole-exome sequencing of vulvar squamous cell carcinomas reveals an impaired prognosis in patients with TP53 mutations and concurrent CCND1 gains.

Very little information is available on the mutational landscape of vulvar squamous cell carcinoma (VSCC), a disease that mainly affects older women. Studies focusing on the mutational patterns of the currently recognized etiopathogenic types of this tumor [human papillomavirus (HPV)-associated (HPV-A), HPV-independent (HPV-I) with TP53 mutation (HPV-I/TP53mut), and HPV-I with wild-type TP53 (HPV-I/TP53wt]) are particularly rare, and there is almost no information on the prognostic implications of these abnormalities. Whole-exome DNA sequencing of 60 VSCC and matched normal tissues from each patient was performed. HPV detection, immunohistochemistry (IHC) for p16, p53, and mismatch repair proteins were also performed. Ten tumors (16.7%) were classified as HPV-A, 37 (61.7%) as HPV-I/TP53mut, and 13 (21.6%) as HPV-I/TP53wt. TP53 was the most frequently mutated gene (66.7%), followed by FAT1 (28.3%), CDKN2A (25.0%), RNF213 (23.3%), NFE2L2 (20%) and PIK3CA (20%). All the 60 tumors (100%) were DNA mismatch repair proficient. Seventeen tumors (28.3%) showed CCND1 gain. Bivariate analysis, adjusted for FIGO stage, revealed that TP53 mutation, CCND1 gain, and the combination of the two alterations were strongly associated with impaired recurrence-free survival (hazard ratio=4.4, p<0.001) and disease-specific survival (hazard ratio=6.1, p=0.002). Similar results were obtained when p53 IHC status was used instead of TP53 status and when considering only HPV-I VSCC. However, in the latter category, p53 IHC maintained its prognostic impact only in combination with CCND1 gains. All tumors carried at least one potentially actionable genomic alteration. In conclusion, VSCCs with CCND1 gain represent a prognostically adverse category among HPV-I/TP53mut tumors. All patients with VSCCs are potential candidates for targeted therapy.

Differential etiopathogenic features of vulvar squamous cell carcinomas in sub-Saharan Africa and Europe.

Two pathways have been described for vulvar squamous cell carcinomas (VSCC), one associated with human papillomavirus (HPV), and the other HPV-independent. We compared the etiopathogenic features of a series of VSCC from Mozambique, a sub-Saharan country with high prevalence of HPV and HIV, with those of Spain, a European country with low prevalence of HPV and HIV. All VSCC diagnosed at the two institutions from January 2018 to December 2020 were included (n = 35 and n = 41, respectively). HPV DNA detection and genotyping, and immunohistochemistry for p16 and p53 were performed. Tumors showing p16 positive staining and/or HPV DNA positivity were considered HPV-associated. 34/35 tumors (97%) from Mozambique and 8/41 (19%) from Spain were HPV-associated (P < .001). Mean age of the patients from Mozambique and Spain was 45 ± 12 and 72 ± 14, respectively (P < .001). No differences were found in terms of HPV genotypes or multiple HPV infection rates. 1/35 tumors (3%) from Mozambique and 29/41 (70%) from Spain showed abnormal p53 immunostaining (P < .001). In contrast with the predominance of HPV-independent VSCC affecting old women in Europe, most VSCC in sub-Saharan Africa are HPV-associated and arise in young women. This data may have important consequences for primary prevention of VSCC worldwide.

Vulvar squamous cell carcinoma arising on human papillomavirus-independent precursors mimicking high-grade squamous intra-epithelial lesion: a distinct and highly recurrent subtype of vulvar cancer.

AIMS: Each category of vulvar squamous cell carcinoma (VSCC), human papillomavirus (HPV)-associated and HPV-independent, arises on a specific intra-epithelial precursor: high-grade squamous intra-epithelial lesions (HSIL) and differentiated vulvar intra-epithelial neoplasia (dVIN), respectively. However, a subset of HPV-independent VSCC arises on an intra-epithelial precursor closely mimicking HSIL. We aimed to explore the clinicopathological features of the HPV-independent tumours with HSIL-like lesions and compare them with HPV-independent VSCC with dVIN and HPV-associated tumours with HSIL. METHODS AND RESULTS: We retrospectively identified 105 cases of surgically treated VSCC with adjacent intra-epithelial precursors. The cases were classified into three groups based on the HPV status and the adjacent precursor identified: (i) HPV-associated VSCC with HSIL (n = 26), (ii) HPV-independent VSCC with dVIN lesions (n = 54) and (iii) HPV-independent VSCC with HSIL-like lesions (n = 25). We analysed the histological and clinical features including the recurrence-free survival and disease-specific survival in the three groups. Patients with HPV-independent VSCC with HSIL-like lesions and with dVIN were older than patients with HPV-associated VSCC (76 and 77 versus 66 years, respectively, P < 0.001). HPV-independent VSCC with HSIL-like lesions recurred more frequently [hazard ratio (HR) = 3.87; P < 0.001] than HPV-independent VSCC with dVIN (HR = 2.27; P = 0.1) and HPV-associated VSCC (HR = 1). In the multivariate analysis, HPV-independent VSCC with HSIL-like lesions remained significant for recurrence. No differences in disease-specific survival were observed between the three groups. CONCLUSIONS: Even though VSCC with HSIL-like lesions are not associated with higher mortality, they are more likely to recur and might benefit from more intensive treatment strategies and closer surveillance after treatment.

Human papillomavirus and p53 status define three types of vulvar squamous cell carcinomas with distinct clinical, pathological, and prognostic features.

INTRODUCTION: Based on their etiological relationship with human papillomavirus (HPV), the 2020 WHO classification has divided vulvar squamous cell carcinomas (VSCC) into two distinct types, HPV-associated and HPV-independent, and HPV-independent tumours have recently been divided according to p53 status. Nevertheless, the clinical and prognostic significance of this classification has not been clearly established. We analysed the differential clinical, pathological, and behavioural characteristics of these three types of VSCC in a large series of patients. METHODS AND RESULTS: VSCC samples from patients who underwent primary surgery at the Hospital Clinic of Barcelona, Spain, during a 47-year period (January 1975 to January 2022) were analysed (n = 190). HPV detection, p16, and p53 immunohistochemical staining were evaluated. We also analysed recurrence-free survival (RFS) and disease-specific survival (DSS). Thirty-three tumours (17.4%) were HPV-associated and 157 (82.6%) HPV-independent. Of these, 20 showed normal and 137 abnormal p53 expression. The two types of HPV-independent tumours showed worse RFS in the multivariate analysis (hazard ratio [HR] = 3.63; P = 0.023 for the HPV-independent p53 normal VSCC and HR = 2.78; P = 0.028 for the HPV-independent p53 abnormal VSCC). Although the differences were not significant, HPV-independent VSCC had worse DSS than HPV-associated VSCC. Although patients with HPV-independent p53 normal tumours had worse RFS than patients with HPV-independent p53 abnormal tumours, the DSS was better for the former group. Only advanced FIGO stage was associated with worse DSS in multivariate analysis (HR = 2.83; P = 0.010). CONCLUSION: The association of HPV and p53 status have prognostic implications, reinforcing a three-tier molecular classification of VSCC (HPV-associated VSCC, HPV-independent VSCC with normal p53, HPV-independent VSCC with abnormal p53).

Minimally Invasive Tissue Sampling as an Alternative to Complete Diagnostic Autopsies in the Context of Epidemic Outbreaks and Pandemics: The Example of Coronavirus Disease 2019 (COVID-19).

BACKGROUND: Infectious diseases' outbreak investigation requires, by definition, conducting a thorough epidemiological assessment while simultaneously obtaining biological samples for an adequate screening of potential responsible pathogens. Complete autopsies remain the gold-standard approach for cause-of-death evaluation and characterization of emerging diseases. However, for highly transmissible infections with a significant associated lethality, such as COVID-19, complete autopsies are seldom performed due to biosafety challenges, especially in low-resource settings. Minimally invasive tissue sampling (MITS) is a validated new approach based on obtaining postmortem samples from key organs and body fluids, a procedure that does not require advanced biosafety measures or a special autopsy room. METHODS: We aimed to review the use of MITS or similar procedures for outbreak investigation up to 27 March 2021 and their performance for evaluating COVID-19 deaths. RESULTS: After a literature review, we analyzed in detail the results of 20 studies conducted at international sites, whereby 216 COVID-19-related deaths were investigated. MITS provided a general and more granular understanding of the pathophysiological changes secondary to the infection and high-quality samples where the extent and degree of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related damage could be evaluated. CONCLUSIONS: MITS is a useful addition in the investigation and surveillance of infections occurring in outbreaks or epidemics. Its less invasive nature makes the tool more acceptable and feasible and reduces the risk of procedure-associated contagion, using basic biosafety measures. Standardized approaches protocolizing which samples should be collected-and under which exact biosafety measures-are necessary to facilitate and expand its use globally.

Building Global Capacity to Conduct Pathology-Based Postmortem Examination: Establishing a New Training Hub for Minimally Invasive Tissue Sampling.

BACKGROUND: Minimally invasive tissue sampling (MITS), an alternative to complete diagnostic autopsy, is a pathology-based postmortem examination that has been validated in low- and middle-income countries (LMICs) and can provide accurate cause of death information when used with other data. The MITS Surveillance Alliance was established in 2017 with the goal to expand MITS globally by increasing training capacity, accessibility, and availability in LMICs. Between January 2019 and May 2020, the MITS Surveillance Alliance convened a multidisciplinary team of technical advisors to attain this goal. METHODS: This article describes the process used to develop criteria and identify an optimal location for a MITS training hub, establish a cadre of LMIC-based trainers, refine standardized MITS sample collection protocols, develop a training program, and release a telepathology platform for quality assessment of MITS histological samples. RESULTS: Results include the creation of a training hub and curriculum, with a total of 9 pathologists and technicians trained as part of the training of the trainers. Those trainers trained 15 participants from seven MITS projects representing 6 LMICs trained in MITS sample collection. The 15 participants have gone on to train more than 50 project-level staff in MITS sample collection. CONCLUSIONS: Lessons learned include an appreciation for using an iterative process for establishing standardized procedures, creating opportunities for all stakeholders to deliver critical feedback, and highlighting the importance of complementing in-person trainings with ongoing technical assistance.

Minimally Invasive Tissue Sampling: A Tool to Guide Efforts to Reduce AIDS-Related Mortality in Resource-Limited Settings.

BACKGROUND: Available information on the causes of death among people living with human immunodeficiency virus (PLHIV) in low- and middle-income countries (LMICs) remains scarce. We aimed to provide data on causes of death in PLHIV from two LMICs, Brazil and Mozambique, to assess the impact of clinical misdiagnosis on mortality rates and to evaluate the accuracy of minimally invasive tissue sampling (MITS) in determining the cause of death in PLHIV. METHODS: We performed coupled MITS and complete autopsy on 164 deceased PLHIV (18 children, 36 maternal deaths, and 110 adults). HIV antibody levels and HIV RNA viral loads were determined from postmortem serum samples. RESULTS: Tuberculosis (22.7%), toxoplasmosis (13.9%), bacterial infections (13.9%), and cryptococcosis (10.9%) were the leading causes of death in adults. In maternal deaths, tuberculosis (13.9%), bacterial infections (13.9%), cryptococcosis (11.1%), and cerebral malaria (8.3%) were the most frequent infections, whereas viral infections, particularly cytomegalovirus (38.9%), bacterial infections (27.8%), pneumocystosis (11.1%), and HIV-associated malignant neoplasms (11.1%) were the leading cause among children. Agreement between the MITS and the complete autopsy was 100% in children, 91% in adults, and 78% in maternal deaths. The MITS correctly identified the microorganism causing death in 89% of cases. CONCLUSIONS: Postmortem studies provide highly granular data on the causes of death in PLHIV. The inaccuracy of clinical diagnosis may play a significant role in the high mortality rates observed among PLHIV in LMICs. MITS might be helpful in monitoring the causes of death in PLHIV and in highlighting the gaps in the management of the infections.

Minimally Invasive Tissue Sampling Findings in 12 Patients With Coronavirus Disease 2019.

BACKGROUND: Minimally invasive tissue sampling (MITS), a postmortem procedure that uses core needle biopsy samples and does not require opening the body, may be a valid alternative to complete autopsy (CA) in highly infectious diseases such as coronavirus disease-19 (COVID-19). This study aimed to (1) compare the performance of MITS and CA in a series of COVID-19 deaths and (2) evaluate the safety of the procedure. METHODS: From October 2020 to February 2021, MITS was conducted in 12 adults who tested positive before death for COVID-19, in a standard, well-ventilated autopsy room, where personnel used reinforced personal protective equipment. In 9 cases, a CA was performed after MITS. A thorough histological evaluation was conducted, and the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was evaluated by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: The diagnoses provided by MITS and CA matched almost perfectly. In 9 patients, COVID-19 was in the chain of events leading to death, being responsible for diffuse alveolar damage and mononuclear T-cell inflammatory response in the lungs. No specific COVID-19 features were identified. Three deaths were not related to COVID-19. All personnel involved in MITS repeatedly tested negative for COVID-19. SARS-CoV-2 was identified by RT-PCR and immunohistochemistry in the MITS samples, particularly in the lungs. CONCLUSIONS: MITS is useful for evaluating COVID-19-related deaths in settings where a CA is not feasible. The results of this simplified and safer technique are comparable to those of CA.

Molecular Landscape of Vulvar Squamous Cell Carcinoma.

Vulvar squamous cell carcinoma (VSCC) is a rare malignancy with dual pathogenesis, Human papillomavirus (HPV)-associated and HPV-independent, with a poorly explored molecular landscape. We aimed to summarize the findings of the series analyzing molecular hallmarks of this neoplasm. In January 2021, we conducted a comprehensive literature search using Pubmed Medline and Scopus to identify publications focused on genomic profiling of VSCC. Observational studies, including both prospective and retrospective designs, evaluating molecular alterations in VSCC were deemed eligible. A total of 14 studies analyzing 749 VSCC were identified. The study series were heterogeneous in HPV testing and sequencing strategies, included small sets of tumors and cancer genes, and commonly lacked survival analysis. Only one extensive targeted next-generation sequencing-based study comprised a large cohort of 280 VSCC. The mutated genes, their number, and frequencies were highly variable between the series. Overall, TP53 and CDKN2A, followed by PIK3CA, HRAS, and PTEN, were the most frequently studied and mutated genes. Mutations involved in the PI3K/AKT/mTOR pathway, including TP53, HRAS, KRAS, and PIK3CA, have been consistently reported across the studies. However, the role of individual mutations or pathways in the development of VSCC remains unclear. In conclusion, heterogeneity and the small sample size of available molecular series contribute to a limited view of the molecular landscape of VSCC. Large-scale genome- or exome-wide studies with robust HPV testing are necessary to improve the molecular characterization of VSCC.

Pathogenesis of Penile Squamous Cell Carcinoma: Molecular Update and Systematic Review.

Penile squamous cell carcinoma (PSCC) is a rare but aggressive neoplasm with dual pathogenesis (human papillomavirus (HPV)-associated and HPV-independent). The development of targeted treatment is hindered by poor knowledge of the molecular landscape of PSCC. We performed a thorough review of genetic alterations of PSCC focused on somatic mutations and/or copy number alterations. A total of seven articles have been identified which, overall, include 268 PSCC. However, the series are heterogeneous regarding methodologies employed for DNA sequencing and HPV detection together with HPV prevalence, and include, in general, a limited number of cases, which results in markedly different findings. Reported top-ranked mutations involve TP53, CDKN2A, FAT1, NOTCH-1 and PIK3CA. Numerical alterations involve gains in MYC and EGFR, as well as amplifications in HPV integration loci. A few genes including TP53, CDKN2A, PIK3CA and CCND1 harbor both somatic mutations and copy number alterations. Notch, RTK-RAS and Hippo pathways are frequently deregulated. Nevertheless, the relevance of the identified alterations, their role in signaling pathways or their association with HPV status remain elusive. Combined targeting of different pathways might represent a valid therapeutic approach in PSCC. This work calls for large-scale sequencing studies with robust HPV testing to improve the genomic understanding of PSCC.

Prognostic implications of genotyping and p16 immunostaining in HPV-positive tumors of the uterine cervix.

Human papillomaviruses (HPVs) are the causative agents of carcinoma of the uterine cervix. A number of HPV genotypes have been associated with cervical cancer and almost all tumors associated with HPV show strong p16 expression. However, there is little information on the possible impact of the HPV genotype and p16 immunostaining on the clinicopathological features or their prognostic value in cervical carcinoma. We evaluated a series of 194 patients with HPV-positive cervical cancers treated at our institution, focusing on the clinicopathological features and the relationship of the HPV genotypes and p16 immunostaining with the prognosis. A single HPV type was identified in 149 (77%) tumors, multiple HPV infection was detected in 30 cases (15%), and undetermined HPV type/s were identified in 15 (8%) carcinomas. HPV 16 and/or 18 were detected in 156 (80%) tumors. p16 was positive in 186 (96%) carcinomas, but eight tumors (4%) were negative for p16 (seven squamous cell carcinomas, one adenocarcinoma); 5/8 caused by HPV 16 and/or 18. Patients with HPV 16 and/or 18 were younger (49 ± 15 vs. 57 ± 17 years, p < 0.01) and more frequently had nonsquamous tumors than patients with other HPV types (24% [37/156] vs. 0% [0/38]; p = 0.01). Neither the HPV type nor multiple infection showed any prognostic impact. Patients with p16-negative tumors showed a significantly worse overall survival than women with p16-positive carcinomas (45 vs. 156 months, p = 0.03), although no significant differences in disease-free survival were observed. In the multivariate analysis, negative p16 immunostaining was associated with a worse overall survival together with advanced FIGO stage and lymph node metastases. In conclusion, the HPV genotype has limited clinical utility and does not seem to have prognostic value in cervical cancer. In contrast, a negative p16 result in patients with HPV-positive tumors is a prognostic marker associated with a poor overall survival.

p53 Immunohistochemical Patterns in HPV-Independent Squamous Cell Carcinomas of the Vulva and the Associated Skin Lesions: A Study of 779 Cases.

Human papillomavirus (HPV)-independent vulvar squamous cell carcinomas (VSCC) and its precursors frequently harbour TP53 mutations. Recently, six p53 immunohistochemical (IHC) patterns have been defined, which have shown strong correlation with TP53 mutation status. However, few studies have applied this new six-pattern framework and none of them exhaustively compared p53 IHC positivity and patterns between invasive VSCC and adjacent skin lesion. We performed p53 IHC in a series of 779 HPV-independent VSCC with adjacent skin and evaluated the IHC slides following the newly described classification. Some 74.1% invasive VSCC showed abnormal p53 IHC staining. A skin lesion was identified in 450 cases (57.8%), including 254 intraepithelial precursors and 196 inflammatory/reactive lesions. Two hundred and ten of 450 (47%) VSCC with associated skin lesions showed an abnormal p53 IHC stain, with an identical staining pattern between the VSCC and the adjacent skin lesion in 80% of the cases. A total of 144/450 (32%) VSCC showed wild-type p53 IHC both in the invasive VSCC and adjacent skin lesion. Finally, 96/450 (21%) VSCC showed p53 IHC abnormal staining in the invasive VSCC but a wild-type p53 staining in the skin lesion. Most of the discordant cases (70/96; 73%) showed adjacent inflammatory lesions. In conclusion, the p53 IHC staining and pattern are usually identical in the VSCC and the intraepithelial precursor.

Standardization of Minimally Invasive Tissue Sampling Specimen Collection and Pathology Training for the Child Health and Mortality Prevention Surveillance Network.

BACKGROUND: Minimally invasive tissue sampling (MITS) is a simplified postmortem examination technique that has shown to be an adequate approach for cause of death investigation in low-resource settings. It requires relatively low level of infrastructures and can be performed by health professionals with no background in pathology. A training program has been developed for the Child Health and Mortality Prevention Surveillance (CHAMPS) network to guarantee standardization of specimen collection techniques, procedures, and laboratory methods. METHODS: The training program has included assessment of the site capacities and training on a standardized protocol of MITS sampling and histological processing. The project has also introduced a program of training for trainers for the personnel from Mozambique. To guarantee the adequacy of the procedure in each site, a trainer accompanied the local teams when the activities started. Training outcomes were assessed by evaluating the quality of the samples obtained and the quality of the slides produced locally. RESULTS: Between June 2016 and October 2018, the laboratories of 7 sites (Bangladesh, Ethiopia, Kenya, Mali, Mozambique, Sierra Leone, and South Africa) have been evaluated and upgraded. Training has been delivered to 63 staff members from all sites. More than 600 MITS procedures have been performed. The quantity of tissue obtained in the MITS by the local teams was sufficient or abundant in 73%, and 87% of the slides were considered as technically acceptable or excellent. CONCLUSIONS: Satisfactory standardization of MITS and histology procedures has been achieved across all CHAMPS sites through organized capacity-building plans.

Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology-screened referral population.

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3-95.2) and 31.8% (95%CI 27.7-36.1) for hrHPV, 77.8% (95%CI 66.9-85.8) and 69.3% (95%CI 65.0-73.3) for methylation biomarkers and 93.1% (95%CI 84.8-97.0) and 49.4% (95%CI 44.8-53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6-95.8), methylation positivity in 75.8% (95%CI 64.2-84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5-96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6-99.7) with a specificity of 46.3% (95%CI 40.8-51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions.

HPV-negative tumors of the uterine cervix.

Human papillomaviruses (HPV) are the causative agents of virtually all cervical carcinomas. Nevertheless, a small proportion of cervical cancer are negative for HPV, although the significance of this finding remains unclear. We aimed to provide insight into the differential clinico-pathological characteristics of this unusual subset of HPV-negative cervical cancer. We performed HPV-DNA detection using a highly sensitive PCR test (SPF10) and p16 immunostaining in 214 cervical carcinomas specimens from women treated at the Gynecological Oncology Unit of the Hospital Clinic (Barcelona, Spain) from 2012 to 2015. The clinical and pathological characteristics, including disease-free survival and overall survival, of HPV-negative and -positive cervical carcinomas were compared. Twenty-one out of 214 tumors (10%) were negative for HPV DNA. HPV-negative tumors were more frequently of the non-squamous type (9/21, 43% vs. 37/193, 19%; p < 0.01) and showed negative p16 staining (9/21; 43% vs. 7/193; 4%; p < 0.01). HPV-negative tumors were more frequently diagnosed at advanced FIGO stage (19/21, 91% vs. 110/193, 57%; p < 0.01) and more frequently had lymph node metastases (14/21, 67% vs. 69/193, 36%; p < 0.01). Patients with HPV-negative cervical cancer had a significantly worse disease-free survival (59.8 months, 95% confidence interval 32.0-87.6 vs. 132.2 months, 95% confidence interval 118.6-145.8; p < 0.01) and overall survival (77.0 months, 95% confidence interval 47.2-106.8 vs. 153.8 months, 95% confidence interval 142.0-165.6; p = 0.01) than women with HPV-positive tumors. However, only advanced FIGO stage and lymph node metastases remained associated with a poor disease-free survival and overall survival on multivariate analysis. In conclusion, our results suggest that a low percentage of cervical cancer arise via an HPV-independent pathway. These HPV-negative tumors are diagnosed at advanced stages, show higher prevalence of lymph nodes metastases and have an impaired prognosis.

CADM1, MAL, and miR124 Promoter Methylation as Biomarkers of Transforming Cervical Intrapithelial Lesions.

BACKGROUND: Squamous intraepithelial lesions/cervical intraepithelial neoplasias (SIL/CIN) are high-risk human papilloma virus (hrHPV)-related lesions which are considered as high grade (HSIL/CIN2-3) or low grade (LSIL/CIN1) lesions according to their risk of progression to cervical cancer (CC). Most HSIL/CIN2-3 are considered as transforming hrHPV infections, so truly CC precursors, although some clear spontaneously. hrHPV testing has a high sensitivity for the detection of HSIL/CIN2-3 but a relatively low specificity for identifying transforming lesions. We aimed to determine whether the combination of CADM1, MAL and miR124 promoter methylation status assessed in histological samples can be used as a biomarker in the identification of transforming HSIL/CIN lesions. DESIGN: 131 cervical biopsies, including 8 cases with no lesion and a negative hrHPV test result (control group), 19 low-grade (L)SIL/CIN1, 30 HSIL/CIN2, 60 HSIL/CIN3, and 14 CC were prospectively collected. hrHPV was detected and genotyped using the polymerase chain reaction (PCR)-based technique SPF10 HPV LIPA. A multiplex quantitative methylation-specific PCR (qMSP) was used to identify the methylation status of the CADM1, MAL, and miR124 promoter genes. RESULTS: Significantly higher methylation levels of CADM1, MAL and miR-124 were found in HSIL/CIN2-3 and CC compared with normal and LSIL lesions. DNA methylation of at least one gene was detected in 12.5% (1/8) of normal samples, 31.5% (6/19) of LSIL/CIN1, 83.3% (25/30) of HSIL/CIN2, 81.6% (49/60) of HSIL/CIN3 and 100% (14/14) of CC (p < 0.001). The sensitivity and specificity for HSIL/CIN2-3 and CC of having at least one methylated gene were 84.6% and 74.0%, respectively. The sensitivity and specificity of the combination of at least one methylated gene and a positive hrHPV test were 80.7% and 85.1% for HSIL/CIN2-3 and CC, respectively. CONCLUSIONS: The methylation rate of CADM1, MAL and miR124 increases with the severity of the lesion. Further research is warranted to evaluate the usefulness of these biomarkers for the identification of transforming HSIL/CIN.

p16 staining has limited value in predicting the outcome of histological low-grade squamous intraepithelial lesions of the cervix.

In order to evaluate the usefulness of p16 staining in predicting the outcome of histological low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 (LSIL/CIN1) we prospectively recruited all the patients referred to colposcopy from 2003 to 2011 due to abnormal screening test results and diagnosed with LSIL/CIN1 at biopsy (n=507). All biopsies were stained for p16 and re-evaluated after three years by the same gynecological pathologist using the LAST criteria. Follow-up was conducted every 6 months and included a Pap test (liquid-based cytology), high-risk human papillomavirus testing (Hybrid Capture 2 test), and colposcopy. The mean follow-up was 28 months. An outcome diagnosis of HSIL was defined as a histological diagnosis of high-grade SIL/CIN (HSIL/CIN2-3). The diagnosis of LSIL/CIN1 was confirmed in 416 out of 507 biopsies (82%), whereas 58 (11%) were reclassified as negative and 33 (6%) as HSIL/CIN2-3. During follow-up, 86/507 women initially diagnosed with LSIL/CIN1 (17%) showed an outcome diagnosis of HSIL/CIN2-3, with the rate of HSIL final diagnosis of 3% (2/58) in the women with biopsies reclassified as negative, 17% (70/416) in the group with confirmed LSIL and 42% (14/33) in the women with biopsies reclassified as HSIL (P<0.001). p16 was positive in 245/507 patients (48%) and in 210/416 patients (50%) with confirmed LSIL/CIN1 at re-evaluation. Although positive p16 immunostaining was associated with risk of HSIL/CIN2-3 outcome in the multivariate analysis (Hazard ratio (HR) 1.9; 95% confidence interval (CI): 1.2-3.1; P=0.009) in the overall group of patients with LSIL/CIN1, this association was not verified in the subset of patients with confirmed LSIL/CIN1 after re-evaluation (HR: 1.6; 95% CI: 0.9-2.6; P=0.095). In conclusion, in LSIL/CIN1 lesions p16 should be limited to equivocal cases in which HSIL/CIN2 is included in the differential diagnosis since it has low value in clinical practice as a marker of progression of LSIL/CIN1.

mRNA biomarker detection in liquid-based cytology: a new approach in the prevention of cervical cancer.

Several high-risk human papillomavirus (HPV)-induced cell biomarkers have been proposed as possible candidates to identify patients harboring high-grade squamous intraepithelial lesions (HSILs) of the uterine cervix. We aimed to determine the feasibility of the detection of the mRNA of six biomarkers in cervical smear specimens obtained by liquid-based cytology and to evaluate whether this approach might be useful in the identification of patients with HSIL. One-hundred and twenty three women referred to colposcopy in the Hospital Clinic of Barcelona were included in the study. After a thorough study, including Pap test, high-risk HPV testing (Hybrid Capture 2 test), and colposcopy with directed biopsy and/or endocervical curettage, 48 patients were diagnosed with HSIL, whereas 75 were classified as negative (n=28), or harboring low-grade SIL (n=47). CDKN2A/p16, BIRC5, MMP9, TOP2A, MCM5, and MKI67 mRNA expression was analyzed by reverse transcription quantitative polymerase chain reaction in liquid-based cytology after the Pap test and Hybrid Capture 2 performance. The tissue expression of these biomarkers was analyzed by immunohistochemistry in the biopsy material. One-hundred and thirteen out of 123 (92%) liquid-based cytology yielded adequate material for mRNA analysis. TOP2A was the most sensitive (97%) biomarker for the detection of HSIL and CDKN2A/p16 the most specific (78%). The combination of TOP2A and CDKN2A/p16 showed a sensitivity of 96% (95% confidence interval (CI): 88-99) and a specificity of 71% (95% CI: 55-82). In the immunohistochemistry analysis, all biomarkers showed a high sensitivity but low specificity for HSIL, except CDKN2A/p16 which had a sensitivity of 100% and a specificity of 63%. The combination of TOP2A and CDKN2A/p16 showed a sensitivity of 100% (95% CI: 91-100) and a specificity of 43% (95% CI: 32-55). The detection of mRNA of cell biomarkers in liquid-based cytology material is feasible. The combination TOP2A and CDKN2A/p16 has a good balance between sensitivity and specificity for the detection of women with HSIL.

High-risk human papillomavirus is transcriptionally active in a subset of sinonasal squamous cell carcinomas.

It has been reported that high-risk human papillomavirus (HPV) is a causative agent of a subgroup of oropharyngeal carcinomas. In these tumors, the presence of the transcriptionally active HPV has been proved through the identification of HPV E6 or E7 messenger RNA (mRNA) transcripts. The aim of the study was to assess the HPV-active transcription in a series of sinonasal carcinomas, in correlation with the HPV DNA identification and the p16 immunohistochemistry. Seventy patients with squamous cell carcinomas of the sinonasal tract were included in the survey. The main clinicopathological characteristics were recorded. All tumors were investigated for HPV through the HPV DNA detection by PCR, using the SPF10 primers and by in situ hybridization, using the high-risk GenPoint probe (Dako, Glostrup, Denmark). HPV16 E7 mRNA transcripts detection was performed by RT-PCR in 27 cases. The immunostaining for p16 was performed in all cases. Fourteen carcinomas (20%) were positive for high-risk HPV by PCR: 13 HPV16 and one HPV35. In situ hybridization showed a dotted nuclear positivity in all these cases. HPV16 E7 mRNA was detected in seven tumors harboring HPV16; in the remaining HPV-positive cases, RNA did not reach the quality for analysis. Strong, diffuse positivity for p16 was observed only in the HPV-positive cases. The 14 HPV-positive squamous cell carcinomas were non-keratinizing or scarcely keratinizing tumors. No significant differences were found in terms of gender, age, or staging at diagnosis between HPV-positive and HPV-negative tumors. However, differences in disease-free survival and overall survival between both groups of patients were significant (P=0.004 and P=0.028, respectively). In conclusion, we have shown that HPV is the etiological agent of a subset of sinonasal carcinomas demonstrating the transcriptionally active HPV in these tumors. Immunostaining for p16 can be used as a surrogate marker to identify these tumors.

p53 Immunohistochemistry Defines a Subset of Human Papillomavirus-Independent Penile Squamous Cell Carcinomas With Adverse Prognosis.

Penile squamous cell carcinoma (PSCC) is classified into 2 prognostically distinct types: human papillomavirus (HPV)-associated and HPV-independent. However, the impact of p53 status on prognosis remains controversial. We correlated HPV and p53 status with the prognosis of a large series of patients with PSCC. p53 was analyzed according to a recently described immunohistochemical (IHC) pattern-based framework that includes 2 normal and 4 abnormal patterns and closely correlates with TP53 mutational status. A total of 122 patients with surgically treated PSCC in 3 hospitals were included. Based on HPV in situ hybridization and p16 and p53 IHC, the tumors were classified into 3 subtypes: HPV-associated, HPV-independent/p53 normal, and HPV-independent/p53 abnormal. All patients were followed up for at least 22 months (median: 56.9 months). Thirty-six tumors (29%) were HPV-associated, 35 (29%) were HPV-independent/p53 normal, and 51 (42%) were HPV-independent/p53 abnormal. Disease-related deaths were observed in 3/36 (8%), 0/35 (0%) and 14/51 (27%) of the patients, respectively (P< 0.001). A total of 7/14 deaths in the latter group were patients with tumors showing p53 abnormal patterns not recognized in the classic p53 IHC interpretation (basal, null, and cytoplasmic). According to our multivariate analysis, HPV-independent/p53 abnormal tumors and advanced stage were associated with impaired disease-specific survival (hazard ratio = 23.4, 95% CI = 2.7-3095.3; P= 0.001 and 16.3, 95% CI = 1.8-2151.5; P= 0.008, respectively). In conclusion, compared with patients with HPV-associated and HPV-independent/p53-normal PSCC, patients with HPV-independent/p53 abnormal PSCC have worse clinical outcomes. p53 IHC results define 2 prognostic categories in HPV-independent PSCC: HPV-independent/p53-normal tumors as low-risk tumors, whereas HPV-independent/p53-abnormal tumors as aggressive neoplasms.