Performance of a RAD51-based functional HRD test on paraffin-embedded breast cancer tissue.

PURPOSE: BRCA-deficient breast cancers (BC) are highly sensitive to platinum-based chemotherapy and PARP inhibitors due to their deficiency in the homologous recombination (HR) pathway. However, HR deficiency (HRD) extends beyond BRCA-associated BC, highlighting the need for a sensitive method to enrich for HRD tumors in an alternative way. A promising approach is the use of functional HRD tests which evaluate the HR capability of tumor cells by measuring RAD51 protein accumulation at DNA damage sites. This study aims to evaluate the performance of a functional RAD51-based HRD test for the identification of HRD BC. METHODS: The functional HR status of 63 diagnostic formalin-fixed paraffin-embedded (FFPE) BC samples was determined by applying the RAD51-FFPE test. Samples were screened for the presence of (epi)genetic defects in HR and matching tumor samples were analyzed with the RECAP test, which requires ex vivo irradiated fresh tumor tissue on the premise that the HRD status as determined by the RECAP test faithfully represented the functional HR status. RESULTS: The RAD51-FFPE test identified 23 (37%) of the tumors as HRD, including three tumors with pathogenic variants in BRCA1/2. The RAD51-FFPE test showed a sensitivity of 88% and a specificity of 76% in determining the HR-class as defined by the RECAP test. CONCLUSION: Given its high sensitivity and compatibility with FFPE samples, the RAD51-FFPE test holds great potential to enrich for HRD tumors, including those associated with BRCA-deficiency. This potential extends to situations where DNA-based testing may be challenging or not easily accessible in routine clinical practice. This is particularly important considering the potential implications for treatment decisions and patient stratification.

RAD51 as a biomarker for homologous recombination deficiency in high-grade serous ovarian carcinoma: robustness and interobserver variability of the RAD51 test.

The RAD51 test is emerging as a promising biomarker for the assessment of functional homologous recombination deficiency (HRD). Yet, the robustness and reproducibility of the immunofluorescence-based RAD51 test, in different academic laboratories, have not been systematically investigated. Therefore, we tested the performance of the RAD51 assay in formalin-fixed paraffin-embedded (FFPE) high-grade serous ovarian carcinoma (HGSOC) samples in four European laboratories. Here, we confirm that subtle differences in staining procedures result in low variability of RAD51 and γH2AX scores. However, substantial variability in RAD51 scoring was observed in some samples, likely due to complicating technical and biological features, such as high RAD51 signal-to-noise ratio and RAD51 heterogeneity. These results support the need to identify and perform additional quality control steps and/or automating image analysis. Altogether, resolving technical issues should be a priority, as identifying tumours with functional HRD is urgently needed to guide the individual treatment of HGSOC patients. Follow-up studies are needed to define the key tissue quality requirements to assess HRD by RAD51 in FFPE tumour samples, as this test could help in guiding the individual treatment of HGSOC patients.

High-Resolution Multiparameter DNA Flow Cytometry for Accurate Ploidy Assessment and the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin-Embedded Tissues.

This article contains detailed protocols for the simultaneous flow cytometric identification of tumor cells and stromal cells and measurement of DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for accurate DNA content assessments of FFPE carcinoma tissues. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 (near-haploidy) and of keratin-positive tumor cells with a DNA index close to 1.0 in overall DNA aneuploid samples, thus improving DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiparameter DNA content analysis of FFPE carcinomas Alternate Protocol 1: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue and red excitation Alternate Protocol 2: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue excitation Support Protocol: Sorting cell population from FFPE carcinomas.

QPOLE: A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction.

PURPOSE: Detection of 11 pathogenic variants in the POLE gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, POLE status is determined by DNA sequencing, which can be expensive, relatively time-consuming, and unavailable in hospitals without specialized equipment and personnel. This may hamper the implementation of POLE-testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost POLE hotspot test by a quantitative polymerase chain reaction (qPCR) assay, QPOLE. MATERIALS AND METHODS: Primer and fluorescence-labeled 5'-nuclease probe sequences of the 11 established pathogenic POLE mutations were designed. Three assays, QPOLE-frequent for the most common mutations and QPOLE-rare-1 and QPOLE-rare-2 for the rare variants, were developed and optimized using DNA extracted from formalin-fixed paraffin-embedded tumor tissues. The simplicity of the design enables POLE status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay. RESULTS: Cutoffs for POLE wild-type, POLE-mutant, equivocal, and failed results were predefined on the basis of a subset of POLE mutants and POLE wild-types for the internal and external validation. For equivocal cases, additional DNA sequencing is recommended. Performance in 282 EC cases, of which 99 were POLE-mutated, demonstrated an overall accuracy of 98.6% (95% CI, 97.2 to 99.9), a sensitivity of 95.2% (95% CI, 90.7 to 99.8), and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% (95% CI, 92.1 to 99.8) and 100%. External validation confirmed feasibility and accuracy. CONCLUSION: QPOLE is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. QPOLE detects all pathogenic variants in the exonuclease domain of the POLE gene. QPOLE will make low-cost POLE-testing available for all women with EC around the globe.

Prognostic relevance of the molecular classification in high-grade endometrial cancer for patients staged by lymphadenectomy and without adjuvant treatment.

INTRODUCTION: The clinical role of the molecular endometrial cancer (EC) classification has not been fully explored in patients staged with lymphadenectomy or without adjuvant treatment, conditions that could potentially moderate the prognostic value of the classification. We aimed to evaluate the clinical outcome of the molecular subgroups in patients with high-grade EC staged by lymphadenectomy and those without adjuvant treatment. METHODS: DNA-sequencing for the detection of pathogenic POLE-exonuclease domain mutations and immunohistochemistry for mismatch repair (MMR) proteins and p53 expression were performed on 412 high-grade EC from the Danish Gynaecological Cancer Database (2005-2012) to classify them as POLE-ultramutated (POLEmut), MMR-deficient (MMRd), p53-mutant (p53abn), or no specific molecular profile (NSMP). Patients with stage IV or residual disease after surgery were excluded. Kaplan-Meier method, log-rank test and Cox proportional hazard models were used for analysis. RESULTS: Molecular analysis was successful in 367 EC; 251 patients had undergone lymphadenectomy. Five-year recurrence rates in this subgroup of patients was 36.7% for women with p53abn EC, 0.0% for POLEmut EC, 13.4% for MMRd EC and 42.9% for NSMP EC (p < 0.001). Similar results were observed among stage IA-IB patients. Among patients without adjuvant treatment (n = 264), none with POLEmut EC (n = 26) had a recurrence. CONCLUSION: The molecular EC classification has strong prognostic value, independent of clinicopathological factors, also among high-grade EC patients staged by lymphadenectomy and those without adjuvant treatment. The unfavourable prognosis of early-stage p53abn EC is not due to undetected lymph node metastasis, and the indolent behaviour of POLEmut EC is independent of adjuvant treatment.

Histological and Somatic Mutational Profiles of Mismatch Repair Deficient Endometrial Tumours of Different Aetiologies.

BACKGROUND: Mismatch repair deficient (MMRd) tumours may arise from somatic events acquired during carcinogenesis or in the context of Lynch syndrome (LS), an inherited cancer predisposition condition caused by germline MMR pathogenic variants. Our aim was to explore whether sporadic and hereditary MMRd endometrial cancers (EC) display distinctive tumour biology. METHODS: Clinically annotated LS-EC were collected. Histological slide review was performed centrally by two specialist gynaecological pathologists. Mutational analysis was by a bespoke 75- gene next-generation sequencing panel. Comparisons were made with sporadic MMRd EC. Multiple correspondence analysis was used to explore similarities and differences between the cohorts. RESULTS: After exclusions, 135 LS-EC underwent independent histological review, and 64 underwent mutational analysis. Comparisons were made with 59 sporadic MMRd EC. Most tumours were of endometrioid histological subtype (92% LS-EC and 100% sporadic MMRd EC, respectively, p = NS). Sporadic MMRd tumours had significantly fewer tumour infiltrating lymphocytes (p ≤ 0.0001) and showed more squamous/mucinous differentiation than LS-EC (p = 0.04/p = 0.05). PTEN mutations were found in 88% sporadic MMRd and 61% LS-EC, respectively (p < 0.001). Sporadic MMRd tumours had significantly more mutations in PDGFRA, ALK, IDH1, CARD11, CIC, MED12, CCND1, PTPN11, RB1 and KRAS, while LS-EC showed more mutations affecting SMAD4 and ARAF. LS-EC showed a propensity for TGF-ß signalling disruption. Cluster analysis found that wild type PTEN associates predominantly with LS-EC, whilst co-occurring mutations in PTEN, PIK3CA and KRAS predict sporadic MMRd EC. CONCLUSIONS: Whilst MMRd EC of hereditary and sporadic aetiology may be difficult to distinguish by histology alone, differences in infiltrating immune cell counts and mutational profile may predict heterogenous responses to novel targeted therapies and warrant further study.

The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks.

PARP inhibitor (PARPi) sensitivity is related to tumor-specific defects in homologous recombination (HR). Therefore, there is great clinical interest in tests that can rapidly and reliably identify HR deficiency (HRD). Functional HRD tests determine the actual HR status by using the (dis)ability to accumulate RAD51 protein at sites of DNA damage as read-out. In this study, we further improved and calibrated a previously described RAD51-based functional HRD test on 74 diagnostic formalin-fixed paraffin-embedded (FFPE) specimens (RAD51-FFPE test) from endometrial cancer (EC n = 25) and epithelial ovarian cancer (OC n = 49) patients. We established optimal parameters with regard to RAD51 foci cut-off (≥2) and HRD threshold (15%) using matched endometrial and ovarian carcinoma specimens for which HR status had been established using a RAD51-based test that required ex vivo irradiation of fresh tissue (RECAP test). The RAD51-FFPE test detected BRCA deficient tumors with 90% sensitivity and RECAP-HRD tumors with 87% sensitivity, indicating that it is an attractive alternative to DNA-based tests with the potential to be applied in routine diagnostic pathology.

The RECAP Test Rapidly and Reliably Identifies Homologous Recombination-Deficient Ovarian Carcinomas.

Recent studies have shown that the efficacy of PARP inhibitors in epithelial ovarian carcinoma (EOC) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2 deficient EOC. A robust method with which to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we investigated the proficiency of a functional HR assay based on the detection of RAD51 foci, the REcombination CAPacity (RECAP) test, in identifying HRD tumors in a cohort of prospectively collected epithelial ovarian carcinomas (EOCs). Of the 39 high-grade serous ovarian carcinomas (HGSOC), the RECAP test detected 26% (10/39) to be HRD, whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Of the HRD tumors that could be sequenced, 8/9 showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation, indicating that the RECAP test reliably identifies HRD, including but not limited to tumors related to BRCA1/2 deficiency. Furthermore, we found a trend towards better overall survival (OS) of HGSOC patients with RECAP-identified HRD tumors compared to patients with HRP tumors. This study shows that the RECAP test is an attractive alternative to DNA-based HRD tests, and further development of a clinical grade RECAP test is clearly warranted.

Frequent Homologous Recombination Deficiency in High-grade Endometrial Carcinomas.

PURPOSE: The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics. EXPERIMENTAL DESIGN: Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort. RESULTS: Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001). CONCLUSIONS: HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.

Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue.

BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1 promoter hypermethylation and pedigree analysis were also performed. In the training cohort, 45 of 46 germline BRCA1/2 variants were detected (sensitivity, 98%). In the prospective cohort (n = 62), all six germline variants were identified (sensitivity, 100%), together with five somatic BRCA1/2 variants and eight cases with BRCA1 promoter hypermethylation. In four BRCA1/2 variant-negative patients, surveillance or prophylactic management options were offered on the basis of positive family histories. We conclude that BRCA1/2 formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2 variants. When taking family history of BRCA1/2 variant-negative patients into account, tumor BRCA1/2 variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counseling and simultaneously selects patients who benefit most from targeted treatment.

The landscape of somatic mutations in Indonesian cervical cancer is predominated by the PI3K pathway.

OBJECTIVE: To investigate the prevalence of somatic mutations in Indonesian cervical carcinoma patients in the context of histology and human papillomavirus (HPV) type. METHODS: In total 174 somatic hot-spot mutations in 13 genes were analyzed by mass spectrometry in 137 Indonesian cervical carcinomas. RESULTS: In 66/137 tumors (48%) 95 mutations were identified. PIK3CA was most frequently mutated (24%), followed by FBXW7 (7%), CTNNB1 (6%), and PTEN (6%). In squamous cell carcinomas more often multiple mutations per sample (p=0.040), and more PIK3CA (p=0.039) and CTNNB1 (p=0.038) mutations were detected compared to adenocarcinomas. PIK3CA mutations were associated with HPV 16 positivity, CDKN2A mutations with HPV 52 positivity, and, interestingly, PTEN mutations with HPV negativity. Balinese tumor samples more often carried multiple mutations (p=0.019), and more CTNNB1, CDKN2A, and NRAS mutations compared to Javanese tumor samples. CONCLUSIONS: Potentially targetable somatic mutations occurred in 48% of Indonesian cervical carcinomas. The landscape of mutations is predominated by mutations concerning the PI3K pathway, and we prompt for more research on developing therapies targeting this pathway, explicitly for the more advanced stage cervical carcinoma patients.

L1 cell adhesion molecule (L1CAM) is a strong predictor for locoregional recurrences in cervical cancer.

BACKGROUND: L1 cell adhesion molecule (L1CAM) has been shown to be a prognostic marker in various cancer types, and has been suggested to play a role in epithelial mesenchymal transition (EMT). Here, we determined the prognostic significance of L1CAM in cervical cancer and its association with vimentin expression on tumor cells, indicative of EMT. METHODS: Formalin-fixed, paraffin-embedded primary tumor samples from 372 cervical cancer patients were collected for immunohistochemical analysis of L1CAM expression. In 109 FFPE specimens, the percentage of vimentin expressing tumor cells was determined by flow cytometry. RESULTS: Positive L1CAM expression (≥10% of tumor cells) was associated with disease-free survival, validated using RNAseq TCGA data. L1CAM expression was independently associated with locoregional recurrence-free survival (hazard ratio 2.62, 95% CI 1.33 - 5.17, P = 0.006), and strongly associated with percentage of vimentin expressing tumor cells (P = 0.003). Expression of both L1CAM and vimentin indicated a subgroup with the highest risk of recurrence (hazard ratio 3.15, 95% CI 1.25 - 7.92, P = 0.015). CONCLUSION: L1CAM might be a promising new prognostic marker for locoregional recurrences in cervical cancer, and its association with vimentin expression suggests that L1CAM might affect tumor aggressiveness, possibly through EMT.

Genomic Characterization of Vulvar (Pre)cancers Identifies Distinct Molecular Subtypes with Prognostic Significance.

Purpose: Vulvar cancer (VC) can be subclassified by human papillomavirus (HPV) status. HPV-negative VCs frequently harbor TP53 mutations; however, in-depth analysis of other potential molecular genetic alterations is lacking. We comprehensively assessed somatic mutations in a large series of vulvar (pre)cancers.Experimental Design: We performed targeted next-generation sequencing (17 genes), p53 immunohistochemistry and HPV testing on 36 VC and 82 precursors (sequencing cohort). Subsequently, the prognostic significance of the three subtypes identified in the sequencing cohort was assessed in a series of 236 VC patients (follow-up cohort).Results: Frequent recurrent mutations were identified in HPV-negative vulvar (pre)cancers in TP53 (42% and 68%), NOTCH1 (28% and 41%), and HRAS (20% and 31%). Mutation frequency in HPV-positive vulvar (pre)cancers was significantly lower (P = 0.001). Furthermore, a substantial subset of the HPV-negative precursors (35/60, 58.3%) and VC (10/29, 34.5%) were TP53 wild-type (wt), suggesting a third, not-previously described, molecular subtype. Clinical outcomes in the three different subtypes (HPV+, HPV-/p53wt, HPV-/p53abn) were evaluated in a follow-up cohort consisting of 236 VC patients. Local recurrence rate was 5.3% for HPV+, 16.3% for HPV-/p53wt and 22.6% for HPV-/p53abn tumors (P = 0.044). HPV positivity remained an independent prognostic factor for favorable outcome in the multivariable analysis (P = 0.020).Conclusions: HPV- and HPV+ vulvar (pre)cancers display striking differences in somatic mutation patterns. HPV-/p53wt VC appear to be a distinct clinicopathologic subgroup with frequent NOTCH1 mutations. HPV+ VC have a significantly lower local recurrence rate, independent of clinicopathological variables, opening opportunities for reducing overtreatment in VC. Clin Cancer Res; 23(22); 6781-9. ©2017 AACR.

Potential Targets' Analysis Reveals Dual PI3K/mTOR Pathway Inhibition as a Promising Therapeutic Strategy for Uterine Leiomyosarcomas-an ENITEC Group Initiative.

Purpose: Uterine sarcomas are rare and heterogeneous tumors characterized by an aggressive clinical behavior. Their high rates of recurrence and mortality point to the urgent need for novel targeted therapies and alternative treatment strategies. However, no molecular prognostic or predictive biomarkers are available so far to guide choice and modality of treatment.Experimental Design: We investigated the expression of several druggable targets (phospho-S6S240 ribosomal protein, PTEN, PDGFR-α, ERBB2, and EGFR) in a large cohort of human uterine sarcoma samples (288), including leiomyosarcomas, low-grade and high-grade endometrial stromal sarcomas, undifferentiated uterine sarcomas, and adenosarcomas, together with 15 smooth muscle tumors of uncertain malignant potential (STUMP), 52 benign uterine stromal tumors, and 41 normal uterine tissues. The potential therapeutic value of the most promising target, p-S6S240, was tested in patient-derived xenograft (PDX) leiomyosarcoma models.Results: In uterine sarcomas and STUMPs, S6S240 phosphorylation (reflecting mTOR pathway activation) was associated with higher grade (P = 0.001) and recurrence (P = 0.019), as shown by logistic regression. In addition, p-S6S240 correlated with shorter progression-free survival (P = 0.034). Treatment with a dual PI3K/mTOR inhibitor significantly reduced tumor growth in 4 of 5 leiomyosarcoma PDX models (with tumor shrinkage in 2 models). Remarkably, the 4 responding models showed basal p-S6S240 expression, whereas the nonresponding model was scored as negative, suggesting a role for p-S6S240 in response prediction to PI3K/mTOR inhibition.Conclusions: Dual PI3K/mTOR inhibition represents an effective therapeutic strategy in uterine leiomyosarcoma, and p-S6S240 expression is a potential predictive biomarker for response to treatment. Clin Cancer Res; 23(5); 1274-85. ©2017 AACR.

Incidence Changes of Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma and Effects on Survival in the Netherlands Cancer Institute, 1980-2009.

AIM: Human papillomavirus (HPV) is a risk factor for oropharyngeal squamous cell carcinoma (OPSCC), with an increasing incidence. The present study aimed to determine the changing incidence of HPV in patients with OPSCC in the period 1980-2009 and its influence on survival. PATIENTS AND METHODS: We randomly sampled 158 patients from a cohort of 828 patients with OPSCC stratified by decade (1980-1989, 1990-1999, 2000-2009). Formalin-fixed paraffin-embedded material was tested for HPV DNA by SPF-10 polymerase chain reaction (PCR) and immunohistochemically stained for p16 and p53. RESULTS: DNA from 146 patients was suitable for HPV detection. HPV DNA was detected in 13/47 (28%), 18/47 (38%), and 20/52 (38%) patients in the cohorts of 1980-1989, 1990-1999, and 2000-2009, respectively (p-value for trend=0.269). Lack of further increase during the most recent decade is inconsistent with the rising incidence and higher prevalence reported in other Western countries. Patients with HPV-positive OPSCC had a better survival in spite of higher tumor stage.

Precise Classification of Cervical Carcinomas Combined with Somatic Mutation Profiling Contributes to Predicting Disease Outcome.

INTRODUCTION: Squamous cell carcinoma (SCC), adenocarcinoma (AC), and adenosquamous carcinoma (ASC) are the most common histological subtypes of cervical cancer. Differences in the somatic mutation profiles of these subtypes have been suggested. We investigated the prevalence of somatic hot-spot mutations in three well-defined cohorts of SCC, AC, and ASC and determined the additional value of mutation profiling in predicting disease outcome relative to well-established prognostic parameters. MATERIALS AND METHODS: Clinicopathological data were collected for 301 cervical tumors classified as SCC (n=166), AC (n=55), or ASC (n=80). Mass spectrometry was used to analyze 171 somatic hot-spot mutations in 13 relevant genes. RESULTS: In 103 (34%) tumors, 123 mutations were detected (36% in SCC, 38% in AC, and 28% in ASC), mostly in PIK3CA (20%) and KRAS (7%). PIK3CA mutations occurred more frequently in SCC than AC (25% vs. 11%, P=0.025), whereas KRAS mutations occurred more frequently in AC than SCC (24% vs. 3%, P<0.001) and ASC (24% vs. 3%, P<0.001). A positive mutation status correlated with worse disease-free survival (HR 1.57, P=0.043). In multivariate analysis, tumor diameter, parametrial infiltration, and lymph node metastasis, but not the presence of a somatic mutation, were independent predictors of survival. CONCLUSION: Potentially targetable somatic mutations occurred in 34% of cervical tumors with different distributions among histological subtypes. Precise classification of cervical carcinomas in combination with mutation profiling is valuable for predicting disease outcome and may guide the development and selection of tumor-specific treatment approaches.

Prognostic significance of POLE proofreading mutations in endometrial cancer.

BACKGROUND: Current risk stratification in endometrial cancer (EC) results in frequent over- and underuse of adjuvant therapy, and may be improved by novel biomarkers. We examined whether POLE proofreading mutations, recently reported in about 7% of ECs, predict prognosis. METHODS: We performed targeted POLE sequencing in ECs from the PORTEC-1 and -2 trials (n = 788), and analyzed clinical outcome according to POLE status. We combined these results with those from three additional series (n = 628) by meta-analysis to generate multivariable-adjusted, pooled hazard ratios (HRs) for recurrence-free survival (RFS) and cancer-specific survival (CSS) of POLE-mutant ECs. All statistical tests were two-sided. RESULTS: POLE mutations were detected in 48 of 788 (6.1%) ECs from PORTEC-1 and-2 and were associated with high tumor grade (P < .001). Women with POLE-mutant ECs had fewer recurrences (6.2% vs 14.1%) and EC deaths (2.3% vs 9.7%), though, in the total PORTEC cohort, differences in RFS and CSS were not statistically significant (multivariable-adjusted HR = 0.43, 95% CI = 0.13 to 1.37, P = .15; HR = 0.19, 95% CI = 0.03 to 1.44, P = .11 respectively). However, of 109 grade 3 tumors, 0 of 15 POLE-mutant ECs recurred, compared with 29 of 94 (30.9%) POLE wild-type cancers; reflected in statistically significantly greater RFS (multivariable-adjusted HR = 0.11, 95% CI = 0.001 to 0.84, P = .03). In the additional series, there were no EC-related events in any of 33 POLE-mutant ECs, resulting in a multivariable-adjusted, pooled HR of 0.33 for RFS (95% CI = 0.12 to 0.91, P = .03) and 0.26 for CSS (95% CI = 0.06 to 1.08, P = .06). CONCLUSION: POLE proofreading mutations predict favorable EC prognosis, independently of other clinicopathological variables, with the greatest effect seen in high-grade tumors. This novel biomarker may help to reduce overtreatment in EC.

CDKN2A(p16) and HRAS are frequently mutated in vulvar squamous cell carcinoma.

BACKGROUND: Two etiologic pathways of vulvar cancer are known, a human papillomavirus (HPV)- and a TP53-associated route, respectively, but other genetic changes may also play a role. Studies on somatic mutations in vulvar cancer other than TP53 are limited in number and size. In this study, we investigated the prevalence of genetic mutations in 107 vulvar squamous cell carcinomas (VSCCs). METHODS: A total of 107 paraffin-embedded tissue samples of primarily surgically treated VSCCs were tested for HPV infection and screened for mutations in 14 genes (BRAF, CDKN2A(p16), CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A, PTEN, and TP53) using Sanger sequencing and mass spectrometry. RESULTS: Mutations were detected in 7 genes. Of 107 VSCCs, 66 tumors (62%) contained at least one mutation (TP53=58, CDKN2A(p16)=14, HRAS=10, PIK3CA=7, PPP2R1A=3, KRAS=1, PTEN=1). Mutations occurred most frequently in HPV-negative samples. Five-year survival was significantly worse for patients with a mutation (47% vs 59%, P=.035), with a large effect from patients carrying HRAS-mutations. CONCLUSION: Somatic mutations were detected in 62% of VSCCs. As expected, HPV infection and TP53-mutations play a key role in the development of VSCC, but CDKN2A(p16), HRAS, and PIK3CA-mutations were also frequently seen in HPV-negative patients. Patients with somatic mutations, especially HRAS-mutations, have a significantly worse prognosis than patients lacking these changes, which could be of importance for the development of targeted therapy.

High concordance of molecular tumor alterations between pre-operative curettage and hysterectomy specimens in patients with endometrial carcinoma.

OBJECTIVE: Molecular alterations in endometrial cancer have been shown to be prognostically significant but have not yet been implemented in the current clinical risk assessment. Few studies have investigated the reliability of molecular alterations in pre-operative specimens. Therefore, the objective was to determine whether molecular analysis of pre-operative endometrial cancer samples accurately reflects those alterations in the subsequent hysterectomy specimens. METHODS: Paired pre-operative and hysterectomy specimens of 48 patients diagnosed with endometrial carcinoma, 42 endometrioid (EEC) and 6 non-endometrioid (NEEC) carcinomas, were analyzed for immunohistochemical expression of p53, PTEN and ß-catenin. Tumor DNA was isolated and analyzed for microsatellite instability (MSI), TP53 mutations and somatic hot spot mutations in 13 genes. RESULTS: In EEC patients, loss of PTEN, nuclear ß-catenin and p53-mutant expression was found in 43%, 7% and 12%, respectively. No nuclear ß-catenin was found in 5 of 6 NEEC patients, all serous cancers, whereas a p53-mutant expression was present in all serous cases. MSI was found in 19.5%, all EEC. Concordance for PTEN, ß-catenin, p53 expression and MSI status was found in 79%, 92%, 79% and 93.5%, respectively. We detected 65 hot spot mutations in 39/48 (81%) tumors. Overall concordance of the GynCarta multigene analysis was 99.8%. CONCLUSIONS: The results confirm the reliability of immunohistochemical and DNA-based techniques in the evaluation of molecular alterations in pre-operative endometrial specimens and high concordance rates with the definitive hysterectomy specimens. The resulting molecular signature provides initial pre-operative diagnostic information on the status of oncogenic pathways, which may contribute to individualized treatment strategies.

Loss of heterozygosity and copy number alterations in flow-sorted bulky cervical cancer.

Treatment choices for cervical cancer are primarily based on clinical FIGO stage and the post-operative evaluation of prognostic parameters including tumor diameter, parametrial and lymph node involvement, vaso-invasion, infiltration depth, and histological type. The aim of this study was to evaluate genomic changes in bulky cervical tumors and their relation to clinical parameters, using single nucleotide polymorphism (SNP)-analysis. Flow-sorted tumor cells and patient-matched normal cells were extracted from 81 bulky cervical tumors. DNA-index (DI) measurement and whole genome SNP-analysis were performed. Data were analyzed to detect copy number alterations (CNA) and allelic balance state: balanced, imbalanced or pure LOH, and their relation to clinical parameters. The DI varied from 0.92-2.56. Pure LOH was found in ≥40% of samples on chromosome-arms 3p, 4p, 6p, 6q, and 11q, CN gains in >20% on 1q, 3q, 5p, 8q, and 20q, and losses on 2q, 3p, 4p, 11q, and 13q. Over 40% showed gain on 3q. The only significant differences were found between histological types (squamous, adeno and adenosquamous) in the lesser allele intensity ratio (LAIR) (p = 0.035) and in the CNA analysis (p = 0.011). More losses were found on chromosome-arm 2q (FDR = 0.004) in squamous tumors and more gains on 7p, 7q, and 9p in adenosquamous tumors (FDR = 0.006, FDR = 0.004, and FDR = 0.029). Whole genome analysis of bulky cervical cancer shows widespread changes in allelic balance and CN. The overall genetic changes and CNA on specific chromosome-arms differed between histological types. No relation was found with the clinical parameters that currently dictate treatment choice.