High-grade vulvar intraepithelial neoplasia: comprehensive characterization and long-term vulvar carcinoma risk.

AIMS: Adequate diagnosis of human papillomavirus (HPV)-associated high-grade squamous intraepithelial lesion (HSIL) and HPV-independent vulvar intraepithelial neoplasia (VIN) is essential but can be challenging. We comprehensively characterized a large population-based series of vulvar lesions, originally reported as high-grade VIN, and assessed the cancer risk. METHODS AND RESULTS: Baseline high-grade VIN of 751 patients were categorized by histopathological reassessment, integrating the results of immunohistochemistry (p16INK4a , p53, Ki-67) and HPV DNA testing. Integrated analyses resulted in 88.4% HPV-associated lesions (77.0% HSIL, 10.9% low-grade SIL [LSIL], and 0.4% vulvar squamous cell carcinoma [VSCC]), 10.9% HPV-independent lesions (6.1% HPV-independent VIN, 4.7% nondysplastic lesions, and 0.1% VSCC) and 1.1% inconclusive lesions. HSIL demonstrated p16INK4a block-positivity in 99.0%, increased Ki-67 in ≥2/3rd of the epithelium in 93.6%, and HPV positivity in 99.6%. In HSIL, a p53 wildtype mid-epithelial staining pattern was common (51.6%) while this was not observed in HPV-independent lesions. HPV-independent VIN harboured mutant p53 patterns in 65.2% and showed a wide morphological spectrum, ranging from differentiated to nondifferentiated ('HPV-associated-like', in 41.3%). Kaplan-Meier analyses showed a 10-year cancer risk of 8.0% in HPV-associated HSIL, 67.4% in HPV-independent VIN/p53mutant, and 27.8% in HPV-independent VIN/p53wildtype. Strikingly, the 10-year cancer risk was 73.3% in HPV-independent VIN with nondifferentiated ('HPV-associated-like') morphology. CONCLUSION: Immunohistochemistry by p16INK4a and p53 is highly recommended for optimal categorization into HPV-associated and HPV-independent VIN, which is of utmost importance given the different cancer risk. The high cancer risk of HPV-independent VIN underscores the need for surgical treatment and close follow-up, especially in case of a p53 mutant pattern and/or nondifferentiated morphology.

Clinical validation of methylation biomarkers for optimal detection of high-grade vulvar intraepithelial neoplasia.

The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)-associated and HPV-independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high-grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high-grade VIN, were reassessed and categorized into HPV-associated or HPV-independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation-specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high-grade VIN was determined by logistic regression analysis. SST was the best-performing individual marker (AUC 0.90), detecting 80% of high-grade VIN cases, with excellent detection of HPV-independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST. Selection of a marker panel, including ZNF582, SST and miR124-2, resulted in a comparably high accuracy for detection of high-grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high-grade VIN. SST, as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high-grade VIN in need of treatment, particularly HPV-independent VIN, from low-grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.

Prognostic value of proliferation, PD-L1 and nuclear size in patients with superior sulcus tumours treated with chemoradiotherapy and surgery.

AIMS: The aim of this study was to determine the relationship between proliferative activity, PD-L1 status and nuclear size changes after preoperative chemoradiotherapy (CRT) and the clinical outcome in patients with superior sulcus tumours. METHODS: Proliferative activity (MIB-1) and PD-L1 status were estimated by immunohistochemistry in the tumour cells of resection specimen in a series of 33 patients with residual tumour after trimodality therapy for a sulcus superior tumour between 2005 and 2014. A morphometric analysis of both pretreatment and post-treatment tumour materials was also performed. Results were related to disease-free survival and overall survival. RESULTS: Low proliferative activity (<20% MIB-1) was associated with better overall survival: 2-year overall survival of 73% compared with 43% and 25%, respectively, for moderate (MIB-1 20%-50%) and high (MIB-1 >50%) proliferative activity (p=0.016). A negative PD-L1 status (<1% positive tumour cells) was also associated with better overall survival (p=0.021). The mean nuclear size of normal lung tissue pneumocytes was significantly smaller compared with the mean nuclear size of tumour cells of the resection specimens (median difference -38.1; range -115.2 to 16.0; p<0.001). The mean nuclear size of tumour cells did not differ between pretreatment biopsies and resection specimens (median difference -4.6; range -75.2 to 86.7; p=0.14). Nuclear size was not associated with survival (p=0.82). CONCLUSIONS: Low proliferative activity determined by MIB-1 as well as a negative PD-L1 expression are significantly associated with better overall survival in patients with residual tumour after CRT for superior sulcus tumour.

Validation of ASCL1 and LHX8 Methylation Analysis as Primary Cervical Cancer Screening Strategy in South African Women with Human Immunodeficiency Virus.

BACKGROUND: Compared with women who are human immunodeficiency virus (HIV) negative, women with human immunodeficiency virus (WWH) have a higher human papillomavirus (HPV) prevalence and increased cervical cancer risk, emphasizing the need for effective cervical cancer screening in this population. The present study aimed to validate methylation markers ASCL1 and LHX8 for primary screening in a South African cohort of WWH. METHODS: In this post hoc analysis within the DIAgnosis in Vaccine And Cervical Cancer Screen (DiaVACCS) study, a South African observational multicenter cohort study, cervical scrape samples from 411 HIV-positive women were analyzed for hypermethylation of ASCL1 and LHX8 genes, HPV DNA, and cytology. Sensitivities, specificities, and positive and negative predictive values of primary methylation-based, HPV-based and cytology-based screening were calculated for the detection of cervical intraepithelial neoplasia of grade 3 or higher. RESULTS: Single markers ASCL1 and LHX8 resulted in a good performance for the detection of cervical intraepithelial neoplasia of grade 3 or higher, with sensitivities of 85.9% (95% confidence interval [CI], 78.2%-93.6%) and 89.7% (83.0%-96.5%), respectively, and specificities of 72.9% (67.3%-78.5%) and 75.0% (69.5%-80.5%). Combining markers ASCL1 and LHX8 resulted in a lower sensitivity compared with HPV testing (84.6% vs 93.6%, respectively; ratio, 0.90 [95% CI, .82-.99]) and a higher specificity (86.7% vs 78.3%; ratio 1.11 [1.02-1.20]) and reduced the referral rate from 46.8% to 33.4%. ASCL1/LHX8 methylation had a significantly higher sensitivity than cytology (threshold, high-grade intraepithelial squamous lesion or worse), (84.6% vs 74.0%, respectively; ratio, 1.16 [95% CI, 1.01-1.32]) and similar specificity (86.7% vs 91.0%; ratio, 0.95 [.90-1.003]). CONCLUSIONS: Our results validate the accuracy of ASCL1/LHX8 methylation analysis for primary screening in WWH, which offers a full-molecular alternative to cytology- or HPV-based screening, without the need for additional triage testing.

DNA methylation markers for cancer risk prediction of vulvar intraepithelial neoplasia.

Current clinical and histological classifications are unable to determine the risk of vulvar squamous cell carcinoma (VSCC) in high-grade vulvar intraepithelial neoplasia (VIN), making prognostic biomarkers highly needed. We studied host-cell DNA methylation markers in high-grade squamous intraepithelial lesion (HSIL) and differentiated VIN (dVIN) without VSCC, in HSIL and dVIN adjacent to VSCC and in human papillomavirus (HPV) positive and negative VSCC, relative to control vulvar tissues. A series of 192 formalin-fixed paraffin-embedded vulvar samples, including VSCC (n = 58), VIN adjacent to VSCC (n = 30), VIN without VSCC during follow-up (n = 41) and normal vulvar tissues (n = 63), were tested for 12 DNA methylation markers with quantitative multiplex methylation-specific PCR (qMSP). HPV status was determined by p16INK4A immunohistochemistry and high-risk HPV PCR analysis. Logistic regression analyses were used to determine methylation patterns and methylation marker performance for VIN and VSCC detection. Methylation markers showed significantly higher methylation levels with increasing severity of disease. VIN adjacent to VSCC showed a similar methylation-high pattern as VSCC, while VIN without VSCC displayed a heterogeneous methylation pattern. Vulvar carcinogenesis is associated with increased DNA methylation. Higher DNA methylation levels in VIN seem to reflect higher cancer risk, emphasizing the high potential of DNA methylation biomarkers in the diagnostic workup of VIN. As a next step, longitudinal studies are needed to verify the prognostic value of methylation biomarkers as a clinical tool for stratification of cancer risk in women with VIN.

Host Cell Deoxyribonucleic Acid Methylation Markers for the Detection of High-grade Anal Intraepithelial Neoplasia and Anal Cancer.

BACKGROUND: High-grade anal intraepithelial neoplasia (AIN2/3; HGAIN) is highly prevalent in human immunodeficiency virus positive (HIV+) men who have sex with men (MSM), but only a minority will eventually progress to cancer. Currently, the cancer risk cannot be established, and therefore all HGAIN is treated, resulting in overtreatment. We assessed host cell deoxyribonucleic acid (DNA) methylation markers for detecting HGAIN and anal cancer. METHODS: Tissue samples of HIV+ men with anal cancer (n = 26), AIN3 (n = 24), AIN2 (n = 42), AIN1 (n = 22) and HIV+ male controls (n = 34) were analyzed for methylation of 9 genes using quantitative methylation-specific polymerase chain reaction. Univariable and least absolute shrinkage and selection operator logistic regression, followed by leave-one-out cross-validation, were used to determine the performance for AIN3 and cancer detection. RESULTS: Methylation of all genes increased significantly with increasing severity of disease (P < 2 × 10-6). HGAIN samples revealed heterogeneous methylation patterns, with a subset resembling cancer. Four genes (ASCL1, SST, ZIC1,ZNF582) showed remarkable performance for AIN3 and anal cancer detection (area under the curve [AUC] > 0.85). ZNF582 (AUC = 0.89), detected all cancers and 54% of AIN3 at 93% specificity. Slightly better performance (AUC = 0.90) was obtained using a 5-marker panel. CONCLUSIONS: DNA methylation is associated with anal carcinogenesis. A marker panel that includes ZNF582 identifies anal cancer and HGAIN with a cancer-like methylation pattern, warrantingvalidation studies to verify its potential for screening and management of HIV+ MSM at risk for anal cancer.

DNA hypermethylation analysis in sputum of asymptomatic subjects at risk for lung cancer participating in the NELSON trial: argument for maximum screening interval of 2†years.

AIMS: Lung cancer is the major contributor to cancer mortality due to metastasised disease at time of presentation. The current study investigated DNA hypermethylation of biomarkers RASSF1A, APC, cytoglobin, 3OST2, FAM19A4, PHACTR3 and PRDM14 in sputum of asymptomatic high-risk individuals from the NELSON lung cancer low-dose spiral CT screening trial to detect lung cancer at preclinical stage. METHODS: Subjects were selected with (i) lung cancer in follow-up (cases; n=65), (ii) minor cytological aberrations (controls; n=120) and (iii) a random selection of subjects without cytological aberrations (controls; n=99). Median follow-up time for controls was 80 months. Cut-off values were based on high specificity to assess diagnostic value of the biomarkers. RESULTS: RASSF1A may denote presence of invasive cancer because of its high specificity (93% (95% CI 89% to 96%); sensitivity 17% (95% CI 4% to 31%), with best performance in a screening interval of 2 years. The panel of RASSF1A, 3OST2 and PRDM14 detected 28% (95% CI 11% to 44%) of lung cancer cases within 2 years, with specificity of 90% (95% CI 86% to 94%). Sputum cytology did not detect any lung cancers. CONCLUSIONS: In a lung cancer screening setting with maximum screening interval of 2 years, DNA hypermethylation analysis in sputum may play a role in the detection of preclinical disease, but complementary diagnostic markers are needed to improve sensitivity.

Combined sputum hypermethylation and eNose analysis for lung cancer diagnosis.

AIMS: The aim of this study is to explore DNA hypermethylation analysis in sputum and exhaled breath analysis for their complementary, non-invasive diagnostic capacity in lung cancer. METHODS: Sputum samples and exhaled breath were prospectively collected from 20 lung cancer patients and 31 COPD controls (Set 1). An additional 18 lung cancer patients and 8 controls only collected exhaled breath as validation set (Set 2). DNA hypermethylation of biomarkers RASSF1A, cytoglobin, APC, FAM19A4, PHACTR3, 3OST2 and PRDM14 was considered, and breathprints from exhaled breath samples were created using an electronic nose (eNose). RESULTS: Both DNA hypermethylation markers in sputum and eNose were independently able to distinguish lung cancer patients from controls. The combination of RASSF1A and 3OST2 hypermethylation had a sensitivity of 85% with a specificity of 74%. eNose had a sensitivity of 80% with a specificity of 48%. Sensitivity for lung cancer diagnosis increased to 100%, when RASSF1A hypermethylation was combined with eNose, with specificity of 42%. Both methods showed to be complementary to each other (p≤0.011). eNose results were reproducible in Set 2. CONCLUSIONS: When used in concert, RASSF1A hypermethylation in sputum and exhaled breath analysis are complementary for lung cancer diagnosis, with 100% sensitivity in this series. This finding should be further validated.

Prolonged sampling of spontaneous sputum improves sensitivity of hypermethylation analysis for lung cancer.

AIMS: The adequacy of lung cancer diagnosis with sputum cytology depends on duration of sputum sampling. The aim of this methodological study was to determine whether the hypermethylation detection rate of RASSF1A, adenomatous polyposis coli (APC) and cytoglobin (CYGB) is influenced by the duration of sputum collection. METHODS: Prospective sputum samples were collected from 53 lung cancer patients and 47 chronic obstructive pulmonary disease patients as controls. Subjects collected spontaneous sputum at home during nine consecutive days in three canisters I, II and III (ie, days 1-3, days 4-6, days 7-9, respectively). Quantitative methylation-specific PCR was performed to assess gene promoter methylation status of RASSF1A, APC and CYGB. RESULTS: Analysis of each canister separately showed hypermethylation of RASSF1A, APC and/or CYGB in samples I, II and III, in 43%, 40% and 47% of cases, respectively. In control samples, these numbers were 4%, 2% and 4%, respectively. Cumulative analysis for days 1-6 and days 1-9 revealed an increase in sensitivity to 53% and 64%, and specificity of 94% and 91%, respectively. CONCLUSION: Sputum collected over multiple successive days results in a gain in sensitivity for the detection of lung cancer, at the expense of a small loss in specificity. Condensed abstract Assessment of hypermethylation sensitivity of biomarkers in sputum collected over a prolonged period for the detection of lung cancer resulted in a promising gain in sensitivity, at the expense of a small loss in specificity.

DNA copy number alterations in endobronchial squamous metaplastic lesions predict lung cancer.

RATIONALE: Autofluorescence bronchoscopy (AFB) is a valid strategy for detecting premalignant endobronchial lesions. However, no biomarker can reliably predict lung cancer risk of subjects with AFB-visualized premalignant lesions. OBJECTIVES: The present study set out to identify AFB-visualized squamous metaplastic (SqM) lesions with malignant potential by DNA copy number profiling. METHODS: Regular AFB examinations in 474 subjects at risk of lung cancer identified six subjects with SqM lesions at baseline, and carcinoma in situ or carcinoma (carcinoma in situ or greater) at the initial SqM site at follow-up bronchoscopy. These progressive SqM lesions were compared for immunostaining pattern and array comparative genomic hybridization-based chromosomal profiles with 23 SqM lesions of subjects who remained cancer-free. Specific DNA copy number alterations (CNAs) linked to cancer risk were identified and accuracy of CNAs to predict endobronchial cancer in this series was determined. MEASUREMENTS AND MAIN RESULTS: At baseline, p53, p63, and Ki-67 immunostaining were not predictive for a differential clinical outcome of SqM lesions. The mean number of CNAs in baseline SqM of cases was significantly higher compared with control subjects (P < 0.01). Chromosomal regions significantly more frequently altered in SqM of cases were 3p26.3-p11.1, 3q26.2-q29, 9p13.3-p13.2, and 17p13.3-p11.2 (family-wise error rate <0.10). CNAs were specifically detected at the site of future cancer. In cases, baseline-detected CNAs persisted in subsequent biopsies taken from the initial site, and levels increased toward cancer progression. In this series, a model based on CNAs at 3p26.3-p11.1, 3q26.2-29, and 6p25.3-24.3 predicted cancer with 97% accuracy. CONCLUSIONS: The data suggest that the presence of specific CNAs in SqM lesions predict endobronchial cancer.

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population.

BACKGROUND: Undifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection. METHODS: Nasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied. RESULTS: NPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%. CONCLUSION: Multiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.