G. Bea A. Wisman

B cells critical for outcome in high grade serous ovarian carcinoma

Abstract Recent work has shown evidence for the prognostic significance of tumor infiltrating B cells (B‐TIL) in high grade serous ovarian carcinoma (HGSOC), the predominant histological subtype of ovarian cancer. However, it remains unknown how the favorable prognosis associated with B‐TIL relates to the current standard treatments of primary debulking surgery (PDS) followed by chemotherapy or (neo‐)adjuvant chemotherapy (NACT) combined with interval debulking surgery. To address this, we analyzed the prognostic impact of B‐TIL in relationship to primary treatment and tumor infiltrating T cell status in a highly homogenous cohort of HGSOC patients. This analysis involved a combined approach utilizing histological data and high‐dimensional flow cytometry analysis. Our findings indicate that while HGSOC tumors pre‐treated with NACT are infiltrated with tumor‐reactive CD8 + and CD4 + TIL subsets, only B‐TIL and IgA plasma blasts confer prognostic benefit in terms of overall survival. Importantly, the prognostic value of B‐TIL and IgA plasma blasts was not restricted to patients treated with NACT, but was also evident in patients treated with PDS. Together, our data point to a critical prognostic role for B‐TIL in HGSOC patients independent of T cell status, suggesting that alternative treatment approaches focused on the activation of B cells should be explored for HGSOC.

Genome-wide DNA methylation in relation to ARID1A deficiency in ovarian clear cell carcinoma

Abstract Background The poor chemo-response and high DNA methylation of ovarian clear cell carcinoma (OCCC) have attracted extensive attentions. Recently, we revealed the mutational landscape of the human kinome and additional cancer-related genes and found deleterious mutations in ARID1A , a component of the SWI/SNF chromatin-remodeling complex, in 46% of OCCC patients. The present study aims to comprehensively investigate whether ARID1A loss and genome-wide DNA methylation are co-regulated in OCCC and identify putative therapeutic targets epigenetically regulated by ARID1A. Methods DNA methylation of ARID1A mt/ko and ARID1A wt OCCC tumors and cell lines were analyzed by Infinium MethylationEPIC BeadChip. The clustering of OCCC tumors in relation to clinical and mutational status of tumors were analyzed by hierarchical clustering analysis of genome-wide methylation. GEO expression profiles were used to identify differentially methylated (DM) genes and their expression level in ARID1A mt/ko vs ARID1A wt OCCCs. Combining three pre-ranked GSEAs, pathways and leading-edge genes epigenetically regulated by ARID1A were revealed. The leading-edge genes that passed the in-silico validation and showed consistent ARID1A -related methylation change in tumors and cell lines were regarded as candidate genes and finally verified by bisulfite sequencing and RT-qPCR. Results Hierarchical clustering analysis of genome-wide methylation showed two clusters of OCCC tumors. Tumor stage, ARID1A/PIK3CA mutations and TP53 mutations were significantly different between the two clusters. ARID1A mutations in OCCC did not cause global DNA methylation changes but were related to DM promoter or gene-body CpG islands of 2004 genes. Three pre-ranked GSEAs collectively revealed the significant enrichment of EZH2- and H3K27me3-related gene-sets by the ARID1A -related DM genes. 13 Leading-edge DM genes extracted from the enriched gene-sets passed the expression-based in-silico validation and showed consistent ARID1A -related methylation change in tumors and cell lines. Bisulfite sequencing and RT-qPCR analysis showed promoter hypermethylation and lower expression of IRX1 , TMEM101 and TRIP6 in ARID1A mt compared to ARID1A wt OCCC cells, which was reversed by 5-aza-2′-deoxycytidine treatment. Conclusions Our study shows that ARID1A loss is related to the differential methylation of a number of genes in OCCC. ARID1A -dependent DM genes have been identified as key genes of many cancer-related pathways that may provide new candidates for OCCC targeted treatment.

Trends in cervical cancer incidence in the Netherlands: A join‐point and age–period–cohort analysis (1989–2023)

Abstract Cervical cancer remains a significant public health issue, ranking as the fourth most common cancer in women globally. In the Netherlands, cervical cancer incidence declined steadily from 1989 to 2001 but increased between 2001 and 2007. This study updates trends in cervical cancer incidence from 1989 to 2023 in the Netherlands and evaluates the impact of screening practices and participation rates in the national population‐based screening program. This ecological study uses group‐level data from the Netherlands Cancer Registry (1989–2023) to analyze trends across three temporal dimensions—age, period, and cohort—using age–period–cohort analysis and join‐point regression to identify significant changes in trends over time. Cervical cancer incidence declined steadily from 1989 to 2003 but increased from 2003 to 2023, particularly among younger birth cohorts (1976–1995). This increase aligns with the decline in screening participation, which dropped from over 77% before 2003 to below 60% in subsequent years, as well as the introduction of primary high‐risk human papillomavirus testing in 2017, which has higher sensitivity than cytology, leading to more cases being detected and, consequently, an increase in incidence. Trends in cervical cancer incidence in the Netherlands indicate a positive impact of the screening program, with a steady decline over the years. However, efforts should focus on increasing participation in both screening and vaccination, as this is crucial for achieving the goal of reducing incidence to below 4 per 100,000 women, in line with the global strategy to eliminate cervical cancer.

Methylation Analysis to Detect CIN3+ in High-Risk Human Papillomavirus-Positive Self-Samples From the Population-Based Cervical Cancer Screening Program

Since 2017, a self-sampling device has been introduced to the Dutch population-based screening program to enable higher participation rates. However, routine triage cytology cannot be performed on self-sampling material. Methylation is an alternative triage method that can be performed directly on DNA extracted from self-samples. Recently, we tested a set of 15 published cervical intraepithelial neoplasia grade 3 or worse (CIN3+)-specific methylation markers and found a panel of 3 markers with a sensitivity of 82% and a specificity of 74%. In this study, we determined the sensitivity and specificity of 2 commercial assays using quantitative methylation-specific PCR. DNA from the same cohort of high-risk human papillomavirus-positive self-sampled material obtained through the population-based screening program in the North of the Netherlands from women with CIN2 or less (<CIN3, n = 208) and women with CIN3+ (n = 96) was used. The QIAsure methylation test (consisting of 2 methylation markers) showed a sensitivity of 65% and a specificity of 72%, whereas the Gyntect assay (consisting of 6 methylation markers) showed a sensitivity of 59% and a specificity of 91% for CIN3+. When we compared all individual 23 methylation markers, receiver operating characteristic analysis showed an area under the curve of ≥0.7 for 11 of 23 markers (P < .001). By model-based recursive partitioning and robustness analysis, we found a panel with a better sensitivity compared with QIAsure and Gyntect (P < .001). This new panel, consisting of ITGA4, ASCL1, and FAM19A4, has a sensitivity of 84% and a specificity of 70%, similar to our previously identified panel (ANKRD18CP, LHX8, and EPB41L3). Thus, in addition to our previously identified panel, the combination of ITGA4, ASCL1, and FAM19A4 showed good diagnostic performance and potentially can replace cytology, thereby avoiding additional doctor visits for many women worldwide and reducing the time for decision making for referral to the gynecologist.

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients

CD103-positive tissue resident memory-like CD8

NOTCH Signaling Limits the Response of Low-Grade Serous Ovarian Cancers to MEK Inhibition

Abstract Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian cancer with high fatality rates in advanced stages due to its chemoresistant properties. LGSOC is characterized by activation of MAPK signaling, and recent clinical trials indicate that the MEK inhibitor (MEKi) trametinib may be a good treatment option for a subset of patients. Understanding MEKi-resistance mechanisms and subsequent identification of rational drug combinations to suppress resistance may greatly improve LGSOC treatment strategies. Both gain-of-function and loss-of-function CRISPR-Cas9 genome-wide libraries were used to screen LGSOC cell lines to identify genes that modulate the response to MEKi. Overexpression of MAML2 and loss of MAP3K1 were identified, both leading to overexpression of the NOTCH target HES1, which has a causal role in this process as its knockdown reversed MEKi resistance. Interestingly, increased HES1 expression was also observed in selected spontaneous trametinib-resistant clones, next to activating MAP2K1 (MEK1) mutations. Subsequent trametinib synthetic lethality screens identified SHOC2 downregulation as being synthetic lethal with MEKis. Targeting SHOC2 with pan-RAF inhibitors (pan-RAFis) in combination with MEKi was effective in parental LGSOC cell lines, in MEKi-resistant derivatives, in primary ascites cultures from patients with LGSOC, and in LGSOC (cell line–derived and patient-derived) xenograft mouse models. We found that the combination of pan-RAFi with MEKi downregulated HES1 levels in trametinib-resistant cells, providing an explanation for the synergy that was observed. Combining MEKis with pan-RAFis may provide a promising treatment strategy for patients with LGSOC, which warrants further clinical validation.

DNA methylation markers as triage test for the early identification of cervical lesions in a Chinese population

AbstractObjective strategies are required in cervical cancer screening. We have identified several DNA methylation markers with high sensitivity and specificity to detect cervical intraepithelial neoplasia 2 or worse (CIN2+) in Dutch women. Our study aims to analyze the diagnostic characteristics of these markers in a Chinese cohort. A total of 246 liquid‐based cytology samples were included, of which 205 women underwent colposcopy due to an abnormal cytology result (atypical squamous cells of undetermined significance [ASCUS] or worse), while 227 were tested high‐risk human papillomavirus (hrHPV) positive. All six individual markers (ANKRD18CP, C13ORF18, EPB41L3, JAM3, SOX1 and ZSCAN1) showed enhanced methylation levels and frequency with increasing severity of the underlying lesion (P ≤ .001). In cytological abnormal women, sensitivity to detect CIN2+ was 79%, 76% and 72% for the three panels (C13ORF18/EBP41L3/JAM3, C13ORF18/ANKRD18CP/JAM3 and ZSCAN1/SOX1, respectively), with a specificity of 57%, 65% and 68%. For the first two panels, these diagnostic characteristics were similar to the Dutch cohort, while for ZSCAN1/SOX1 the sensitivity was higher in the Chinese cohort, but with a lower specificity (both P &lt; .05). In hrHPV‐positive samples, similar sensitivity and specificity for the detection of CIN2+ were found as for the abnormal cytology cohort, which were now all similar between both cohorts and non‐inferior to HPV16/18 genotyping. Our analysis reveals that the diagnostic performances are highly comparable for C13ORF18/EBP41L3/JAM3 and C13ORF18/ANKRD18CP/JAM3 methylation marker panels in both Chinese and Dutch cohorts. In conclusion, methylation panels identified in a Dutch population are also applicable for triage testing in cervical cancer screening in China.

Evaluation of Six Methylation Markers Derived from Genome-Wide Screens for Detection of Cervical Precancer and Cancer

Combined STING levels and CD103+ T cell infiltration have significant prognostic implications for patients with cervical cancer

Activation of STimulator of INterferon Genes (STING) is important for induction of anti-tumor immunity. A dysfunctional STING pathway is observed in multiple cancer types and associates with poor prognosis and inferior response to immunotherapy. However, the association between STING and prognosis in virally induced cancers such as HPV-positive cervical cancer remains unknown. Here, we investigated the prognostic value of STING protein levels in cervical cancer using tumor tissue microarrays of two patient groups, primarily treated with surgery (n = 251) or radio(chemo)therapy (n = 255). We also studied CD103, an integrin that marks tumor-reactive cytotoxic T cells that reside in tumor epithelium and that is reported to associate with improved prognosis. Notably, we found that a high level of STING protein was an independent prognostic factor for improved survival in both the surgery and radio(chemo)therapy group. High infiltration of CD103+ T cells was associated with improved survival in the radio(chemo)therapy group. The combination of STING levels and CD103+ T cell infiltration is strongly associated with improved prognosis. We conclude that combining the prognostic values of STING and CD103 may improve the risk stratification of cervical cancer patients, independent from established clinical prognostic parameters.