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Purpose: Recent studies have shown that 7% to 12% of endometrial cancers are ultramutated due to somatic mutation in the proofreading exonuclease domain of the DNA replicase POLE. Interestingly, these tumors have an excellent prognosis. In view of the emerging data linking mutation burden, immune response, and clinical outcome in cancer, we investigated whether POLE-mutant endometrial cancers showed evidence of increased immunogenicity.Experimental Design: We examined immune infiltration and activation according to tumor POLE proofreading mutation in a molecularly defined endometrial cancer cohort including 47 POLE-mutant tumors. We sought to confirm our results by analysis of RNAseq data from the TCGA endometrial cancer series and used the same series to examine whether differences in immune infiltration could be explained by an enrichment of immunogenic neoepitopes in POLE-mutant endometrial cancers.Results: Compared with other endometrial cancers, POLE mutants displayed an enhanced cytotoxic T-cell response, evidenced by increased numbers of CD8+ tumor-infiltrating lymphocytes and CD8A expression, enrichment for a tumor-infiltrating T-cell gene signature, and strong upregulation of the T-cell cytotoxic differentiation and effector markers T-bet, Eomes, IFNG, PRF, and granzyme B. This was accompanied by upregulation of T-cell exhaustion markers, consistent with chronic antigen exposure. In silico analysis confirmed that POLE-mutant cancers are predicted to display more antigenic neoepitopes than other endometrial cancers, providing a potential explanation for our findings.Conclusions: Ultramutated POLE proofreading-mutant endometrial cancers are characterized by a robust intratumoral T-cell response, which correlates with, and may be caused by an enrichment of antigenic neopeptides. Our study provides a plausible mechanism for the excellent prognosis of these cancers. Clin Cancer Res; 21(14); 3347–55. ©2015 AACR.

This file describes additional methodology, Supplementary Tables and Supplementary Figure legends.

POLE mutations identified in endometrial cancers in the observation arm of the PORTEC-1 trial

Frequency of amplifications, GISTIC q-value and Cox regression P value for all regions in 1q locus for cluster 2, 3 and 4 ECs (n= 268).

Purpose: Molecular classification of endometrial cancer identified distinct molecular subgroups. However, the largest subset of endometrial cancers remains poorly characterized and is referred to as the “nonspecific molecular profile” (NSMP) subgroup. Here, we aimed at refining the classification of this subgroup by profiling somatic copy-number aberrations (SCNAs).Experimental Design: SCNAs were analyzed in 141 endometrial cancers using whole-genome SNP arrays and pooled with 361 endometrial cancers from The Cancer Genome Atlas. Genomic Identification of Significant Targets in Cancer (GISTIC) identified statistically enriched SCNAs and penalized Cox regression assessed survival effects. The prognostic significance of relevant SCNAs was validated using multiplex ligation-dependent probe amplification in 840 endometrial cancers from the PORTEC-1/2 trials. Copy-number status of genes was correlated with gene expression to identify potential cancer drivers. One plausible oncogene was validated in vitro using antisense oligonucleotide-based strategy.Results: SCNAs affecting chromosome 1q32.1 significantly correlated with worse relapse-free survival (RFS) in the NSMP subgroup (HR, 2.12; 95% CI, 1.26–3.59; P = 0.005). This effect was replicated in NSMP endometrial cancers from PORTEC-1/2 (HR, 2.34; 95% CI, 1.17–4.70; P = 0.017). A new molecular classification including the 1q32.1 amplification improved risk prediction of recurrence. MDM4 gene expression strongly correlated with 1q32.1 amplification. Silencing MDM4 inhibited cell growth in cell lines carrying 1q32.1 amplification, but not in those without MDM4 amplification. Vice versa, increasing MDM4 expression in nonamplified cell lines stimulated cell proliferation.Conclusions: 1q32.1 amplification was identified as a prognostic marker for poorly characterized NSMP endometrial cancers, refining the molecular classification of this subgroup. We functionally validated MDM4 as a potential oncogenic driver in the 1q32.1 region. Clin Cancer Res; 23(23); 7232–41. ©2017 AACR.

Fluorescent In Situ Hybridization of Pole on metaphase spreads of Pole-mutant cell line

Multivariable analysis on the prognostic role of the clinicopathological characteristics, molecular subgroups, and potential other classifiers in all cases of early-stage endometrial cancer (n=834) and in the subset of EC without substantial LVSI, >10% L1CAM, p53 and POLE mutation (n=620).

Purpose: Recommendations for adjuvant treatment for women with early-stage endometrial carcinoma are based on clinicopathologic features. Comprehensive genomic characterization defined four subgroups: p53-mutant, microsatellite instability (MSI), POLE-mutant, and no specific molecular profile (NSMP). We aimed to confirm the prognostic capacity of these subgroups in large randomized trial populations, investigate potential other prognostic classifiers, and integrate these into an integrated molecular risk assessment guiding adjuvant therapy.Experimental Design: Analysis of MSI, hotspot mutations in 14 genes including POLE, protein expression of p53, ARID1a, β-catenin, L1CAM, PTEN, ER, and PR was undertaken on 947 available early-stage endometrioid endometrial carcinomas from the PORTEC-1 and -2 trials, mostly high-intermediate risk (n = 614). Prognostic value was determined using univariable and multivariable Cox proportional hazard models. AUCs of different risk stratification models were compared.Results: Molecular analyses were feasible in >96% of the patients and confirmed the four molecular subgroups: p53-mutant (9%), MSI (26%), POLE-mutant (6%), and NSMP (59%). Integration of prognostic molecular alterations with established clinicopathologic factors resulted in a stronger model with improved risk prognostication. Approximately 15% of high-intermediate risk patients had unfavorable features (substantial lymphovascular space invasion, p53-mutant, and/or >10% L1CAM), 50% favorable features (POLE-mutant, NSMP being microsatellite stable, and CTNNB1 wild-type), and 35% intermediate features (MSI or CTNNB1-mutant).Conclusions: Integrating clinicopathologic and molecular factors improves the risk assessment of patients with early-stage endometrial carcinoma. Assessment of this integrated risk profile is feasible in daily practice, and holds promise to reduce both overtreatment and undertreatment. Clin Cancer Res; 22(16); 4215–24. ©2016 AACR.

Supplementary table 1. Demographic and clinicopathological characteristics of cases by EC molecular subtype

Supplementary Methods

Purpose: Pathogenic POLE proofreading domain mutations are found in many malignancies where they are associated with ultramutation and favorable prognosis. The extent to which this prognosis depends on their sensitivity to adjuvant treatment is unknown, as is the optimal therapy for advanced-staged or recurrent POLE-mutant cancers.Experimental Design: We examined the recurrence-free survival of women with POLE-mutant and POLE–wild-type endometrial cancers (EC) in the observation arm of the randomized PORTEC-1 endometrial cancer trial (N = 245 patients with stage I endometrial cancer for analysis). Sensitivity to radiotherapy and selected chemotherapeutics was compared between Pole-mutant mouse-derived embryonic stem (mES) cells, generated using CRISPR-Cas9 (Pole mutations D275A/E275A, and cancer-associated P286R, S297F, V411L) and isogenic wild-type cell lines.Results: In the observation arm of the PORTEC-1 trial (N = 245), women with POLE-mutant endometrial cancers (N = 16) had an improved recurrence-free survival (10-year recurrence-free survival 100% vs. 80.1% for POLE–wild-type; HR, 0.143; 95% confidence interval, 0.001–0.996; P = 0.049). Pole mutations did not increase sensitivity to radiotherapy nor to chemotherapeutics in mES cells. In contrast, Pole-mutant cells displayed significantly increased sensitivity to cytarabine and fludarabine (IC50 Pole P286R–mutant vs. wild-type: 0.05 vs. 0.17 μmol/L for cytarabine, 4.62 vs. 11.1 μmol/L for fludarabine; P < 0.001 for both comparisons).Conclusions: The favorable prognosis of POLE-mutant cancers cannot be explained by increased sensitivity to currently used adjuvant treatments. These results support studies exploring minimization of adjuvant therapy for early-stage POLE-mutant cancers, including endometrial and colorectal cancers. Conversely, POLE mutations result in hypersensitivity to nucleoside analogues, suggesting the use of these compounds as a potentially effective targeted treatment for advanced-stage POLE-mutant cancers. Clin Cancer Res; 24(13); 3197–203. ©2018 AACR.

Supplementary table 2. Antigen presentation pathway mutations in TCGA POLE proofreading-mutant ECs

Supplementary figure 1. Tumor infiltrating lymphocytes and Crohn's like reaction in POLE- mutant endometrioid endometrial cancers

Supplementary figure 4. CD20+ and FOXP3+ tumor infiltrate according to EC molecular subtype

Legends to supplementary figures and tables

Supplementary figure 2. CD3+ tumor infiltrate according to EC molecular subtype

Supplementary figure 3. Number of TIA-1+ cytolytic T cells according to molecular subtype

Purpose: Molecular classification of endometrial cancer identified distinct molecular subgroups. However, the largest subset of endometrial cancers remains poorly characterized and is referred to as the “nonspecific molecular profile” (NSMP) subgroup. Here, we aimed at refining the classification of this subgroup by profiling somatic copy-number aberrations (SCNAs). Experimental Design: SCNAs were analyzed in 141 endometrial cancers using whole-genome SNP arrays and pooled with 361 endometrial cancers from The Cancer Genome Atlas. Genomic Identification of Significant Targets in Cancer (GISTIC) identified statistically enriched SCNAs and penalized Cox regression assessed survival effects. The prognostic significance of relevant SCNAs was validated using multiplex ligation-dependent probe amplification in 840 endometrial cancers from the PORTEC-1/2 trials. Copy-number status of genes was correlated with gene expression to identify potential cancer drivers. One plausible oncogene was validated in vitro using antisense oligonucleotide-based strategy. Results: SCNAs affecting chromosome 1q32.1 significantly correlated with worse relapse-free survival (RFS) in the NSMP subgroup (HR, 2.12; 95% CI, 1.26–3.59; P = 0.005). This effect was replicated in NSMP endometrial cancers from PORTEC-1/2 (HR, 2.34; 95% CI, 1.17–4.70; P = 0.017). A new molecular classification including the 1q32.1 amplification improved risk prediction of recurrence. MDM4 gene expression strongly correlated with 1q32.1 amplification. Silencing MDM4 inhibited cell growth in cell lines carrying 1q32.1 amplification, but not in those without MDM4 amplification. Vice versa, increasing MDM4 expression in nonamplified cell lines stimulated cell proliferation. Conclusions: 1q32.1 amplification was identified as a prognostic marker for poorly characterized NSMP endometrial cancers, refining the molecular classification of this subgroup. We functionally validated MDM4 as a potential oncogenic driver in the 1q32.1 region. Clin Cancer Res; 23(23); 7232–41. ©2017 AACR.

Purpose: Pathogenic POLE proofreading domain mutations are found in many malignancies where they are associated with ultramutation and favorable prognosis. The extent to which this prognosis depends on their sensitivity to adjuvant treatment is unknown, as is the optimal therapy for advanced-staged or recurrent POLE-mutant cancers. Experimental Design: We examined the recurrence-free survival of women with POLE-mutant and POLE–wild-type endometrial cancers (EC) in the observation arm of the randomized PORTEC-1 endometrial cancer trial (N = 245 patients with stage I endometrial cancer for analysis). Sensitivity to radiotherapy and selected chemotherapeutics was compared between Pole-mutant mouse-derived embryonic stem (mES) cells, generated using CRISPR-Cas9 (Pole mutations D275A/E275A, and cancer-associated P286R, S297F, V411L) and isogenic wild-type cell lines. Results: In the observation arm of the PORTEC-1 trial (N = 245), women with POLE-mutant endometrial cancers (N = 16) had an improved recurrence-free survival (10-year recurrence-free survival 100% vs. 80.1% for POLE–wild-type; HR, 0.143; 95% confidence interval, 0.001–0.996; P = 0.049). Pole mutations did not increase sensitivity to radiotherapy nor to chemotherapeutics in mES cells. In contrast, Pole-mutant cells displayed significantly increased sensitivity to cytarabine and fludarabine (IC50 Pole P286R–mutant vs. wild-type: 0.05 vs. 0.17 μmol/L for cytarabine, 4.62 vs. 11.1 μmol/L for fludarabine; P < 0.001 for both comparisons). Conclusions: The favorable prognosis of POLE-mutant cancers cannot be explained by increased sensitivity to currently used adjuvant treatments. These results support studies exploring minimization of adjuvant therapy for early-stage POLE-mutant cancers, including endometrial and colorectal cancers. Conversely, POLE mutations result in hypersensitivity to nucleoside analogues, suggesting the use of these compounds as a potentially effective targeted treatment for advanced-stage POLE-mutant cancers. Clin Cancer Res; 24(13); 3197–203. ©2018 AACR.

Purpose: Recent studies have shown that 7% to 12% of endometrial cancers are ultramutated due to somatic mutation in the proofreading exonuclease domain of the DNA replicase POLE. Interestingly, these tumors have an excellent prognosis. In view of the emerging data linking mutation burden, immune response, and clinical outcome in cancer, we investigated whether POLE-mutant endometrial cancers showed evidence of increased immunogenicity. Experimental Design: We examined immune infiltration and activation according to tumor POLE proofreading mutation in a molecularly defined endometrial cancer cohort including 47 POLE-mutant tumors. We sought to confirm our results by analysis of RNAseq data from the TCGA endometrial cancer series and used the same series to examine whether differences in immune infiltration could be explained by an enrichment of immunogenic neoepitopes in POLE-mutant endometrial cancers. Results: Compared with other endometrial cancers, POLE mutants displayed an enhanced cytotoxic T-cell response, evidenced by increased numbers of CD8+ tumor-infiltrating lymphocytes and CD8A expression, enrichment for a tumor-infiltrating T-cell gene signature, and strong upregulation of the T-cell cytotoxic differentiation and effector markers T-bet, Eomes, IFNG, PRF, and granzyme B. This was accompanied by upregulation of T-cell exhaustion markers, consistent with chronic antigen exposure. In silico analysis confirmed that POLE-mutant cancers are predicted to display more antigenic neoepitopes than other endometrial cancers, providing a potential explanation for our findings. Conclusions: Ultramutated POLE proofreading-mutant endometrial cancers are characterized by a robust intratumoral T-cell response, which correlates with, and may be caused by an enrichment of antigenic neopeptides. Our study provides a plausible mechanism for the excellent prognosis of these cancers. Clin Cancer Res; 21(14); 3347–55. ©2015 AACR.

Immunotherapy of usual vulvar intraepithelial neoplasia (uVIN) is promising; however, many patients still fail to show clinical responses, which could be explained by an immune escape through alterations in human leukocyte antigen (HLA) expression. Therefore, we analyzed a cohort of patients with a primary (n = 43) and subsequent recurrent uVIN lesion (n = 20), vaccine-treated uVIN patients (n = 12), patients with human papillomavirus (HPV)-induced vulvar carcinoma (n = 21) and healthy controls (n = 26) for the expression of classical HLA-class I/II and nonclassical HLA-E/-G and MHC class I chain-related molecule A (MICA). HLA-class I was downregulated in 70% of uVIN patients, including patients with a clinical response to immunotherapy. Downregulation of HLA-class I is probably reversible, as only 15% of the uVIN cases displayed loss of heterozygosity (LOH) and HLA-class I could be upregulated in uVIN keratinocyte cultures by interferon γ. HLA-class I downregulation is more frequently associated with LOH in vulvar carcinomas (25-55.5%). HLA-class II was found to be focally expressed in 65% of uVIN patients. Of the nonclassical molecules, MICA was downregulated in 80% of uVIN whereas HLA-E and -G were expressed in a minority of cases. Their expression was more prominent in vulvar carcinoma. No differences were found between the alterations observed in paired primary and recurrent uVIN. Importantly, downregulation of HLA-B/C in primary uVIN lesions was associated with the development of recurrences and progression to cancer. We conclude that downregulation of HLA is frequently observed in premalignant HPV-induced lesions, including clinical responders to immunotherapy, and is associated with worse clinical outcome. However, in the majority of cases downregulation may still be reversible.

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.

Background: Mismatch repair (MMR)-deficiency analysis is increasingly recommended for all endometrial cancers, as it identifies Lynch syndrome-patients, and is emerging as a prognostic classifier to guide adjuvant treatment. The aim of this study was to define the optimal approach for MMR-deficiency testing and to clarify discrepancies between microsatellite instability (MSI) analysis and immunohistochemical (IHC) analysis of MMR protein expression. Patients and Methods: 696 endometrial cancers were analyzed for MSI (pentaplex panel) and MMR protein expression (IHC). Agreement between methodologies was calculated using Cohen’s Kappa. MLH1 promoter hypermethylation, dinucleotide microsatellite markers and somatic MMR and POLE exonuclease domain (EDM) gene variants (using next-generation/Sanger sequencing) were analyzed in discordant cases. Results: MSI was found in 180 patients. Complete loss of expression of one or more MMR proteins was observed in 196 cases. A PMS2- and MSH6-antibody panel detected all cases with loss of MMR protein expression. The results of MSI and MMR protein expression were concordant in 655/696 cases (kappa=0.854, PConclusion: MSI and IHC analysis are highly concordant in endometrial cancer. This holds true for cases with subclonal loss of MMR protein expression. Discordant MMR-proficient/MSI-high cases (

Purpose: Recommendations for adjuvant treatment for women with early-stage endometrial carcinoma are based on clinicopathologic features. Comprehensive genomic characterization defined four subgroups: p53-mutant, microsatellite instability (MSI), POLE-mutant, and no specific molecular profile (NSMP). We aimed to confirm the prognostic capacity of these subgroups in large randomized trial populations, investigate potential other prognostic classifiers, and integrate these into an integrated molecular risk assessment guiding adjuvant therapy. Experimental Design: Analysis of MSI, hotspot mutations in 14 genes including POLE, protein expression of p53, ARID1a, β-catenin, L1CAM, PTEN, ER, and PR was undertaken on 947 available early-stage endometrioid endometrial carcinomas from the PORTEC-1 and -2 trials, mostly high-intermediate risk (n = 614). Prognostic value was determined using univariable and multivariable Cox proportional hazard models. AUCs of different risk stratification models were compared. Results: Molecular analyses were feasible in >96% of the patients and confirmed the four molecular subgroups: p53-mutant (9%), MSI (26%), POLE-mutant (6%), and NSMP (59%). Integration of prognostic molecular alterations with established clinicopathologic factors resulted in a stronger model with improved risk prognostication. Approximately 15% of high-intermediate risk patients had unfavorable features (substantial lymphovascular space invasion, p53-mutant, and/or >10% L1CAM), 50% favorable features (POLE-mutant, NSMP being microsatellite stable, and CTNNB1 wild-type), and 35% intermediate features (MSI or CTNNB1-mutant). Conclusions: Integrating clinicopathologic and molecular factors improves the risk assessment of patients with early-stage endometrial carcinoma. Assessment of this integrated risk profile is feasible in daily practice, and holds promise to reduce both overtreatment and undertreatment. Clin Cancer Res; 22(16); 4215–24. ©2016 AACR.

JAK1 frameshift mutations may promote cancer cell immune evasion by impeding upregulation of the antigen presentation pathway in microsatellite unstable endometrial cancers (ECs). This study investigated the JAK1 mutation frequency, its functional implication in immune evasion and its prognostic significance in microsatellite unstable EC. Microsatellite instability and three microsatellite repeats within JAK1 were analyzed in 181 ECs. Sixty-two (34%) ECs showed microsatellite instability, of which 22 (35%) had a JAK1 mutation. LMP7, TAP1 and HLA class I protein expression and the presence of CD8-positive T-cells were analyzed in the microsatellite unstable ECs. JAK1 mutant microsatellite unstable ECs showed impaired upregulation of LMP7 (P=0.074) and HLA class I (P

Objective The standard treatment of early-stage (FIGO [International Federation of Gynecology and Obstetrics] I) endometrioid endometrial cancer (EEC) is hysterectomy with bilateral salpingo-oophorectomy. An alternative approach for younger women with low-grade EEC who wish to preserve fertility may be hormonal treatment. Previous studies have suggested that progesterone may elicit its antitumor effect in EEC by interacting with the Wingless (Wnt) and/or phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Therefore, we explored whether common activating genetic alterations in Wnt and PI3K/Akt signaling correlated with nonresponsiveness to progesterone therapy for low-grade EEC. In addition, we investigated whether benign morphology under progesterone treatment is accompanied by the absence of genetic changes. Methods We analyzed molecular alterations in the Wnt and PI3K/Akt signaling in 84 serial endometrial samples from 11 premenopausal patients with progesterone receptor–positive low-grade EEC conservatively treated with progesterone and correlated these with histological and clinical follow-up. Results There were 6 responders and 5 nonresponders to progesterone treatment. The response rate to progesterone treatment was 55%, and the relapse rate was 83%. All responders had alterations in both the Wnt and PI3K/Akt pathway before treatment. In the nonresponder group, tumors inconsistently showed alterations in none, 1, or both pathways. Normalization of the endometrium morphology under progesterone treatment is accompanied by the absence of the genetic changes found in the specimen before treatment. Conclusions We found that activating molecular alterations in either Wnt or PI3K/Akt signaling pathways did not predict resistance to progesterone treatment. It seems that morphological response goes along with disappearance of the established mutations. This exploratory study suggests that Wnt or PI3K/Akt status is unable to predict response to progesterone treatment in patients with EEC.

Genetic variation of antigen-processing machinery (APM) components has been shown to be associated with cervical carcinoma risk and outcome in a genetically homogeneous Dutch population. However, the role of APM component single nucleotide polymorphisms (SNPs) in genetically heterogeneous populations with different distributions of human papillomavirus (HPV) subtypes remains unclear. Eleven non-synonymous, coding SNPs in the TAP1, TAP2, LMP2, LMP7 and ERAP1 genes were genotyped in cervical carcinoma patients and healthy controls from two distinct Indonesian populations (Balinese and Javanese). Individual genotype and allele distributions were investigated using single-marker analysis, and combined SNP effects were assessed by haplotype construction and haplotype interaction analysis. Allele distribution patterns in Bali and Java differed in relation to cervical carcinoma risk, with four ERAP1 SNPs and one TAP2 SNP in the Javanese population showing significant association with cervical carcinoma risk, while in the Balinese population, only one TAP2 SNP showed this association. Multimarker analysis demonstrated that in the Javanese patients, one specific haplotype, consisting of the ERAP1-575 locus on chromosome 5 and the TAP2-379 and TAP2-651 loci on chromosome 6, was significantly associated with cervical carcinoma risk (global P = 0.008); no significant haplotype associations were found in the Balinese population. These data indicate not only that genetic variation in APM component genes is associated with cervical carcinoma risk in Indonesia but also that the patterns of association differ depending on background genetic composition and possibly on differences in HPV type distribution.

Background: The tumour microenvironment comprises a network of immune response and vascularization factors. From this network, we identified immunological and vascularization gene expression clusters and the correlations between the clusters. We subsequently determined which factors were correlated with patient survival in cervical carcinoma. Methods: The expression of 42 genes was investigated in 52 fresh frozen squamous cervical cancer samples by qRT-PCR. Weighted gene co-expression network analysis and mixed-model analyses were performed to identify gene expression clusters. Correlations and survival analyses were further studied at expression cluster and single gene level. Results: We identified four immune response clusters: 'T cells' (CD3E/CD8A/TBX21/IFNG/FOXP3/IDO1), 'Macrophages' (CD4/CD14/CD163), 'Th2' (IL4/IL5/IL13/IL12) and 'Inflammation' (IL6/IL1B/IL8/IL23/IL10/ARG1) and two vascularization clusters: 'Angiogenesis' (VEGFA/FLT1/ANGPT2/ PGF/ICAM1) and 'Vessel maturation' (PECAM1/VCAM1/ANGPT1/SELE/KDR/LGALS9). The 'T cells' module was correlated with all modules except for 'Inflammation', while 'Inflammation' was most significantly correlated with 'Angiogenesis' (p < 0.001). High expression of the 'T cells' cluster was correlated with earlier TNM stage (p = 0.007). High CD3E expression was correlated with improved disease-specific survival (p = 0.022), while high VEGFA expression was correlated with poor disease-specific survival (p = 0.032). Independent predictors of poor disease-specific survival were IL6 (hazard ratio = 2.3, p = 0.011) and a high IL6/IL17 ratio combined with low IL5 expression (hazard ratio = 4.2, p = 0.010). Conclusions: 'Inflammation' marker IL6, especially in combination with low levels of IL5 and IL17, was correlated with poor survival. This suggests that IL6 promotes tumour growth, which may be suppressed by a Th17 and Th2 response. Measuring IL6, IL5 and IL17 expression may improve the accuracy of predicting prognosis in cervical cancer.

Meeting abstracts Cervical cancer is the second leading cause of cancer death in young women worldwide. The aim of the current study was to identify which combination of immunological and vascular factors in the tumor microenvironment of cervical carcinoma is most significant for survival. Using

Somatic mutations play a major role in tumour initiation and progression. The mutation status of a tumour may predict prognosis and guide targeted therapies. The majority of techniques to study oncogenic mutations require high quality and quantity DNA or are analytically challenging. Mass-spectrometry based mutation analysis however is a relatively simple and high-throughput method suitable for formalin-fixed, paraffin-embedded (FFPE) tumour material. Targeted gene panels using this technique have been developed for several types of cancer. These current cancer hotspot panels are not focussed on the genes that are most relevant in gynaecological cancers. In this study, we report the design and validation of a novel, mass-spectrometry based panel specifically for gynaecological malignancies and present the frequencies of detected mutations. Using frequency data from the online Catalogue of Somatic Mutations in Cancer, we selected 171 somatic hotspot mutations in the 13 most important genes for gynaecological cancers, being BRAF, CDKN2A, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A and PTEN. A total of 546 tumours (205 cervical, 227 endometrial, 89 ovarian, and 25 vulvar carcinomas) were used to test and validate our panel, and to study the prevalence and spectrum of somatic mutations in these types of cancer. The results were validated by testing duplicate samples and by allele-specific qPCR. The panel presented here using mass-spectrometry shows to be reproducible and high-throughput, and is usefull in FFPE material of low quality and quantity. It provides new possibilities for studying large numbers of gynaecological tumour samples in daily practice, and could be useful in guided therapy selection.

Background: Vulvar cancer is the fourth most common gynaecological malignancy, with an annual incidence of 2 out of 100 000 women. Although most cases of early stage vulvar cancer have a good prognosis, recurrence and rapid tumour progression can occur. We investigated the prevalence of spindle cell morphology in vulvar cancer and its association with survival. Methods: This retrospective cohort study included 108 patients with primary vulvar squamous cell carcinoma who were treated at the Leiden University Medical Center during 2000–2009. Paraffin-embedded tissue was examined for the presence of spindle cell morphology. Survival and histology data were compared between cases with spindle and without spindle cell morphology. Results: Twenty-two (20%) tumours showed spindle cells infiltrating the stromal tissue. All spindle cell tumours were human papillomavirus (HPV) negative. Spindle cell morphology was strongly associated with poor prognosis and with a high risk of lymph node involvement at the time of diagnosis (relative risk 2.26 (95% CI 1.47–3.47)). Five-year disease-specific survival was lower in patients with vs without spindle cell morphology (45.2% vs 79.7%, respectively; P=0.00057). Conclusion: Vulvar spindle cell morphology occurs frequently and seems to develop through the non-HPV pathway. It is associated with a worse prognosis than conventional vulvar squamous cell carcinoma.

Cervical cancer is typically well infiltrated by immune cells. Because of the intricate relationship between cancer cells and immune cells, we aimed to identify both cancer cell and immune cell expressed biomarkers. Using a novel approach, we isolated RNA from flow-sorted viable EpCAM+ tumor epithelial cells and CD45+ tumor-infiltrating immune cells obtained from squamous cell cervical cancer samples (n = 24). Total RNA was sequenced and differential gene expression analysis of the CD45+ immune cell fractions identified TCL1A as a novel marker for predicting improved survival (p = 0.007). This finding was validated using qRT-PCR (p = 0.005) and partially validated using immunohistochemistry (p = 0.083). Importantly, TCL1A was found to be expressed in a subpopulation of B cells (CD3-/CD19+/CD10+/CD34-) using multicolor immunofluorescence. A high TCL1A/CD20 (B cell) ratio, determined in total tumor samples from a separate patient cohort using qRT-PCR (n = 52), was also correlated with improved survival (p = 0.027). This is the first study demonstrating the prognostic value of separating tumor epithelial cells from tumor-infiltrating immune cells and determining their RNA expression profile for identifying putative cancer biomarkers. Our results suggest that intratumoral TCL1A+ B cells are important for controlling cervical cancer development.