The term verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN) was coined to describe HPV-independent p53-wildtype lesions with characteristic clinicopathologic characteristics and association with vulvar squamous cell carcinoma (vSCC). We aimed to expand on the molecular landscape of vaVIN using comprehensive sequencing and copy number variation profiling. vaVIN diagnosis in institutional cases was confirmed by a second review, plus negative p16 and wildtype p53 by immunohistochemistry. Multigene next-generation sequencing and shallow-whole genome sequencing were used to survey for single-nucleotide variants (SNV), copy number alterations, and structural variants. Targeted TERT promoter sequencing was also carried out. Nineteen patients with vaVIN were included; 4 had concurrent vSCC. The median patient age was 74 (range 56–90) years. Genomic aberrations were noted in 18 cases (95%) as follows: PIK3CA in 10 (53%), CDKN2A in 7 (37%), HRAS in 6 (32%), FAT1 and NOTCH1-2 in 5 each (26%), TSC2 in 2 (11%), and PTEN, ARID2, and KRAS in 1 (5%) each. TERT promoter variants were detected in 11 of 13 cases successfully tested (85%). Five vaVINs harbored a TP53 variant but showed wild-type p53 immunohistochemical expression. In one of these, the concurrent carcinoma showed abnormal p53 and biallelic TP53 mutations. Out of 15 patients with follow-up (mean: 20, range: 2–50 mo), vaVIN persistence/recurrence was seen in 8 (53%), and subsequent vSCC in 2 (13%). At the last encounter, 3 (20%) patients had persistent disease and 1 (7%) died of vSCC. vaVIN is characterized by a wider molecular spectrum, beyond known alterations in PIK3CA, HRAS, and ARID2, to include TERT promoter, CDKN2A, FAT1, and NOTCH1-2, which are characteristic of HPV-independent vSCC. vaVIN can occur with concurrent or subsequent carcinoma, sometimes with fatal outcomes. These findings support the concept of vaVIN as a neoplastic process within the family of HPV-independent vulvar neoplasia.

Of the 4 molecular subtypes of endometrial cancer (EC), p53-abnormal (p53abn) EC is associated with abundant copy number alterations and the worst clinical outcome. Patients with p53abn EC have the highest risk of disease recurrence and death, independent of tumor grade and histologic subtype. Currently, all invasive p53abn ECs are considered high risk, and no prognostic biomarkers have yet been found that can aid in clinical management. Here, we aimed to test whether loss of retinoblastoma (RB) protein expression using immunohistochemistry has the potential for prognostic refinement of p53abn EC. A large cohort of 227 p53abn ECs collected from the PORTEC-1/2/3 clinical trials and the Medisch Spectrum Twente cohort study was investigated, and RB loss was identified in 7.0% (n = 16/227). RB-lost p53abn ECs were predominantly high-grade endometrioid ECs (n = 6, 37.5%) and carcinosarcomas with endometrioid-type epithelial component (n = 5, 31.3%). Histologically, RB-lost p53abn ECs were typified by high-grade nuclear atypia (n = 16, 100%), predominantly solid growth pattern (n = 15/16, 93.8%), and polypoid growth (n = 9/16, 56.3%). Copy number loss involving the RB1 locus was identified in the majority of RB-lost p53abn EC (n = 13/14, 92.9%), explaining the loss of RB expression. Comparative analysis also showed that RB-lost p53abn ECs were diagnosed at earlier stages than RB-retained p53abn EC (P = .014). Interestingly, RB-lost p53abn EC showed prolonged time to overall recurrence (P = .038), even within stage I alone (P = .040). These findings highlight distinct morphomolecular features in RB-lost p53abn ECs and confirm the utility of RB immunohistochemistry as a surrogate for underlying molecular RB1 alterations. To our knowledge, this is the first study to show the potential use of RB in prognostic refinement of p53abn EC, although validation is warranted. (c) 2024 THE AUTHORS. Published by Elsevier Inc. on behalf of the United States & Canadian Academy of Pathology. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Abstract Copy number changes are defining features in ovarian cancers. Two distinct methods by Steele et al. and Drews et al., published back-to-back in 2022, detail features and categories of copy number signatures. However, these methods have not been benchmarked for ovarian cancer. Mutational signatures are pivotal in understanding cancer biology and serve as predictive and prognostic biomarkers. This study aims to examine the landscape of copy number signatures to characterize the diversity of Homologous Recombination (HR) impairment within ovarian cancer. While assessing Homologous Recombination Deficiency (HRD) is crucial for targeted treatments, current HRD tests lack clarity. Using signature extraction tools SigProfilerExtractor and CINSignature, we analyzed whole-genome sequencing data (mean depth 100x) from 416 ovarian cases from Genomics England (GeL), employing both copy number signature methods. Among the signatures extracted, HRD signature CN17 from Steele et al. methodology was identified in 37% of the samples and primarily found in the high-grade serous ovarian cancer (HGSOC) subtype (77%). Additionally, higher exposure levels (median of 0.4 in n=154 compared to median of 0.29 in n=416) of Drews et al. methodology’s most complex HRD signature, CX3, was detected in samples exhibiting CN17 activity. This ongoing exploration of the copy number signatures landscape works towards identifying the spectrum of HR impairment, aiming to benchmark these two methods for a standardized diagnostic tool. This study contributes to our understanding of the mutational landscape of ovarian cancer and its potential implications for prevention and personalized treatment strategies. Citation Format: Aliah Hawari, Avraam Tapinos, Claire J. Kramer, Andreas J. Gruber, Richard Houlston, James Brenton, David Wedge. Characterizing HRD in ovarian cancer: Insights from copy number signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4355.

Abstract Copy-number (CN)-high endometrial carcinoma (EC) is characterized by abundant CN-alterations, thought to be driven by TP53 aberrations, and is the subgroup with the poorest prognosis. CN-high EC exhibit substantial heterogeneity in histotypes, molecular markers, and mutational profiles. Here, we aimed to refine this subgroup by assessing the evolution and identifying CN-signatures (Drews et al. Nature) to establish CN-clusters. We used whole-genome sequencing (WGS;100X) data from 146 CN-high cases from Genomics England (GeL) as discovery cohort. We then performed shallow WGS (sWGS;0.1X) of FFPE tumor DNA of n=307 CN-high EC from (non-)trial cohorts. Timing of canonical driver mutations by MutationTimer in CN-high GeL revealed that TP53 mutations were: (i) not ubiquitous and (ii) not consistently identified as early clonal events (Table), challenging conventional belief that TP53 inactivation is necessary for initiation of CN-high EC. Evolutionary analyses (Plackett-Luce) are ongoing to identify carcinogenesis trajectories and assess how driver mutations other than TP53 influence CN-phenotypes. Unsupervised clustering of 307 EC by CN-signatures identified five clusters (E1: 19%; E2: 2%; E3; 20%; E4: 19%; E5: 40%), characterized by CN-signatures linked to biological phenomena. Clusters E1/E4 reflect homologous recombination deficiency (CX3), while cluster E5 seems driven by chromosomal missegregation (CX1). Notably, there was no one-to-one relationship between CN-clusters and histotypes, suggesting CN-evolution is independent of morphology. In conclusion, we have identified TP53-dependent and -independent pathways in CN-high EC evolution. Potentially clinically relevant CN-clusters were identified using cost-effective sWGS. sWGS of additional n=475 CN-high EC are ongoing. Furthermore, we will analyze in-depth clinicopathological relationships of clusters, including prognosis/prediction, using PORTEC trials prior to the meeting. Table. Timing of canonical driver mutations in CN-high EC Clonal - early Clonal - late Subclonal Clonal - NA Total 1. TP53 87 (67%) 4 (3%) 3 (2%) 36 (28%) 130 (100%) 2. PIK3CA 42 (76%) 1 (2%) 1 (2%) 11 (20%) 55 (100%) 3. PPP2R1A 18 (47%) 1 (3%) 2 (5%) 17 (45%) 38 (100%) 4. PTEN 17 (59%) 1 (3%) 2 (7%) 9 (31%) 29 (100%) 5. PIK3R1 11 (46%) 0 (0%) 3 (13%) 10 (42%) 24 (100%) 6. FBXW7 14 (61%) 0 (0%) 0 (0%) 9 (39%) 23 (100%) 7. KRAS 9 (64%) 0 (0%) 2 (14%) 3 (21%) 14 (100%) Abbreviation: NA, timing not applicable. Citation Format: Claire J. Kramer, Dina Ruano, Aliah Hawari, Felix Blanc-Durand, Cor D. de Kroon, Judith R. Kroep, Gitte Ortoft, Estrid Hogdall, Alexandra Leary, Carien L. Creutzberg, Maaike P. Vreeswijk, Nanda Horeweg, David N. Church, David C. Wedge, Tjalling Bosse. Evolutionary characterization and refinement of copy-number high endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5051.

Rationale. Homologous recombination deficiency (HRD), defined as BRCA1/2 mutation (BRCAmut) or high genomic instability, is used to identify ovarian cancer (OC) patients most likely to benefit from PARP inhibitors. While these tests are useful, they are imperfect. Another approach is to measure the capacity of tumor cells to form RAD51 foci in the presence of DNA damage using an immunofluorescence assay (IF). We aimed to describe for the first time this assay in OC and correlate it to platinum response and BRCAmut.Methods. Tumor samples were prospectively collected from the randomized CHIVA trial of neoadjuvant plat-inum +/- nintedanib. IF for RAD51, GMN and gH2AX was performed on FFPE blocks. Tumors were considered RAD51-low if <= 10% of GMN-positive tumor cells had >= 5 RAD51 foci. BRCAmut were identified by NGS.

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.

Supplementary material and methods

Endometrial cancer (EC) can be classified into four molecular subgroups: POLE mutant, MSI/dMMR, non-specific profiles and P53 mutant (P53mut). P53mut EC comprise ∼20% of cases and have the worst prognosis. There is an urgent medical need to better understand P53mut EC in order to propose effective new therapeutic strategies. We conducted a retrospective analysis of P53abn EC patients from PORTEC3 (NCT00411138) with available DNA for a large-scale panel sequencing (Discovery Cohort). Results were confirmed on an independent cohort of EC patients (Gustave Roussy, France and National University Cancer Institute, Singapore) identified by their molecular profile using FoundationOneCDX or FoundationOne Liquid CDX panel (Validation Cohort). Molecular findings were correlated with clinicopathologic features from medical record review. 39 P53abn cases were included in the discovery cohort. Molecular profiling was able to distinguish 4 mutually exclusive subgroups: CCNE1 amplified (15%), ERBB2 amplified (21%), PTEN alteration (21%) and a non-specific group. In the Validation Cohort, 71 P53mut EC patients were included. 40 patients were molecularly characterized on tumor tissue and 31 on circulating tumor DNA. Median age was 66 years, 38% presented with primary metastatic diseases, 40% were serous, 30% endometrioid and 20% carcinosarcoma. We detected the same four molecular subgroups defined by CCNE1amp (13%), ERBB2amp (16%), and PTEN mutation or loss (34%). These subgroups were also almost exclusive to one another. Only two patients (3%) harbored co-alterations. In terms of outcomes, we did not observe any overall survival difference between these subgroups. Among P53mut EC, we detected 3 nearly mutually-exclusive molecular subgroups: CCNE1 amplified, ERBB2 amplified and PTEN loss, accounting together for 60% of cases. Whether these subgroups might benefit from personalized therapeutic strategies is currently being explored.

AbstractPurpose: The clinical significance of the p53-abnormal (p53abn) molecular subtype in stage I low-grade endometrioid endometrial carcinoma (EEC) is debated. We aimed to review pathologic and molecular characteristics, and outcomes of stage I low-grade p53abn EEC in a large international cohort. Experimental Design: Previously diagnosed stage I p53abn EC (POLE–wild-type, mismatch repair–proficient) low-grade EEC from Canadian retrospective cohorts and PORTEC-1&2 trials were included. Pathology review was performed by six expert gynecologic pathologists blinded to p53 status. IHC profiling, next-generation sequencing, and shallow whole-genome sequencing was performed. Kaplan–Meier method was used for survival analysis. Results: We identified 55 stage I p53abn low-grade EEC among 3,387 cases (2.5%). On pathology review, 17 cases (31%) were not diagnosed as low-grade EEC by any pathologists, whereas 26 cases (47%) were diagnosed as low-grade EEC by at least three pathologists. The IHC and molecular profile of the latter cases were consistent with low-grade EEC morphology (ER/PR positivity, patchy p16 expression, PIK3CA and PTEN mutations) but they also showed features of p53abn EC (TP53 mutations, many copy-number alterations). These cases had a clinically relevant risk of disease recurrence (5-year recurrence-free survival 77%), with pelvic and/or distant recurrences observed in 12% of the patients. Conclusions: A subset of p53abn EC is morphologically low-grade EEC and exhibit genomic instability. Even for stage I disease, p53abn low-grade EEC are at substantial risk of disease recurrence. These findings highlight the clinical relevance of universal p53-testing, even in low-grade EEC, to identify women at increased risk of recurrence.

Representative examples of cases diagnosed as low-grade EEC by ≤1 pathologists.

H&E, immunohistochemistry stains and relative copy number plot of a case diagnosed as low-grade EEC by ≥3 pathologists.

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.

Introduction Endometrial cancer (EC) can be classified into four molecular subgroups: POLE mutant, MSI/dMMR, non-specific profiles and P53 mutant (P53mut). P53mut EC comprise ~20% of cases and have the worst prognosis. There is an urgent medical need to better understand P53mut EC in order to propose effective new therapeutic strategies. Methods We conducted a retrospective analysis of P53abn EC patients from PORTEC3 (NCT00411138) with available DNA for a large-scale panel sequencing (Discovery Cohort). Results were confirmed on an independent cohort of EC patients (Gustave Roussy, France and National University Cancer Institute, Singapore) identified by their molecular profile using FoundationOneCDX or FoundationOne Liquid CDX panel (Validation Cohort). Molecular findings were correlated with clinicopathologic features from medical record review. Results 39 P53abn cases were included in the discovery cohort. Molecular profiling was able to distinguish 4 mutually exclusive subgroups: CCNE1 amplified (15%), ERBB2 amplified (21%), PTEN alteration (21%) and a non-specific group. In the Validation Cohort, 71 P53mut EC patients were included. Median age was 66 years, 40% were serous, 30% endometrioid and 20% carcinosarcoma. 38% presented with primary metastatic diseases. We detected the same four molecular subgroups defined by CCNE1amp (13%), ERBB2amp (16%), and PTEN mutation or loss (34%). Only two patients (3%) harbored co-alterations. We did not observe any overall survival difference between these subgroups. Conclusion/Implications Among P53mut EC, we detected 3 nearly mutually-exclusive molecular subgroups: CCNE1 amplified, ERBB2 amplified and PTEN loss, accounting together for 60% of cases. Whether these subgroups might benefit from personalized therapeutic strategies is currently being explored.

The role of pathology in patient management has evolved over time from the retrospective review of cells, tissue, and disease (‘what happened’) to a prospective outlook (‘what will happen’). Examination of a static, two‐dimensional hematoxylin and eosin (H&E)‐stained tissue slide has traditionally been the pathologist's primary task, but novel ancillary techniques enabled by technological breakthroughs have supported pathologists in their increasing ability to predict disease status and behaviour. Nevertheless, the informational limits of 2D, fixed tissue are now being reached and technological innovation is urgently needed to ensure that our understanding of disease entities continues to support improved individualized treatment options. Here we review pioneering work currently underway in the field of cancer pathology that has the potential to capture information beyond the current basic snapshot. A selection of exciting new technologies is discussed that promise to facilitate integration of the functional and multidimensional (space and time) information needed to optimize the prognostic and predictive value of cancer pathology. Learning how to analyse, interpret, and apply the wealth of data acquired by these new approaches will challenge the knowledge and skills of the pathology community. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Introduction/Background A popular dogma is that in epithelial ovarian carcinomas (EOCs) mutations in BRCA1/2 and other homologous recombination (HR)-genes are exclusive to high-grade serous ovarian carcinomas (HGSOC). Nevertheless, the European guidelines recommend germline or tumor screening, regardless of histotype. Here, we report on the results of five-year prospective, universal ‘tumor-first’ screening of BRCA1/2 and HR-genes in EOC and assess the relationship between identified mutations and histotypes. Methodology EOCs were prospectively sequenced between September 2017 and December 2021 in two university medical centers in The Netherlands. The gene-panel included BRCA1/2 and for a large subset of cases the panel was expanded with EOC-susceptibility HR-genes BRIP1, PALB2, RAD51C and RAD51D. All mutations (class 4 and 5 variants) were reported. Prior to sequencing, all EOC underwent a central pathology review by expert gynecopathologists. Results The universal ‘tumor-first’ screening strategy was executed in the two centers on a total of 831 EOCs (table 1). In total, 73% were HGSOCs and 27% were EOC-cases of other histologies. The overall yield for BRCA1/2 mutations in EOC was 13%, and the vast majority of mutations (94.5%) were identified in HGSOCs (yield of 17%; table 1). Intriguingly, 6/221 (2.7%) non-HGSOCs, i.e., n=3 high-grade endometroid, n=1 low-grade endometrioid and n=2 low-grade serous OC, harboured a BRCA2 mutation. No BRCA1/2 or HR-gene mutations were identified in clear cell and mucinous carcinomas. The results of extensive HRD testing of these six outliers will be presented at the meeting, including loss of heterozygosity, functional RAD51 assay and copy-number signatures. Conclusion This large ‘real-world’ cohort of centrally revised and prospectively sequenced EOCs confirmed that BRCA1/2 mutations are almost exclusively identified in HGSOC. Extensive HRD testing will inform us about the clinical relevance of the identified BRCA1/2 and HR-gene mutations beyond HGSOCs and whether histotype-directed HRD-screening, after central pathology revision, may be justified.

Introduction/Background* RAD51 protein has been proposed as a functional readout of homologous recombination (HR) status using formalin-fixed paraffin embedded (FFPE) tumour tissue blocks. Recently, few laboratories have assessed the performance of RAD51 as a predictive biomarker. However, the robustness of the test when applied in different laboratories has not been systematically investigated so far. In this study, we performed an interlaboratory (n = 4) analytical validation to determine the interobserver variability and the effect of (subtle) differences in the co-immunofluorescence (co-IF) protocol and microscope technicalities on RAD51 scores. Methodology The RING trial cohort comprised of 12 high-grade serous ovarian cancer cases. On unstained serial sections of FFPE tumour tissue blocks, a co-IF staining with RAD51 and geminin was performed: (1) centrally in Vall d'Hebron Institute of Oncology and (2) locally in participating centers. The centrally stained slides were distributed among participating centers for local RAD51 scoring. For the scope of the RING trial, a predefined and uniform scoring methodology was applied. Scoring was performed blinded for genetic and clinical data. Specific features in the analysis of the co-IF, including the number of RAD51 foci per nucleus and the presence of RAD51 subclonality, i.e., distinct RAD51 positive and negative areas, were incorporated in the RAD51 scoring form. For non-normally distributed data, variability was analyzed using the median, 25th percentile (Q1) and 75th percentile (Q3). Result(s)* Median variability in RAD51 scores between observers in centrally stained slides was 21% (Q1: 15%; Q3: 24%) (figure 1). For the majority of cases (n = 10/12), a limited interobserver variability, defined as ≥ 3 observers with scores in a narrow range, was detected. In contrast, in cases where observers noted granular pannuclear RAD51 staining or RAD51 background, there was a substantial variability in scores (figure 1; case 6 and 8). Median variability in RAD51 scores between centrally and locally stained IF slides was 7.7% (Q1: 4.1%; Q3: 11.7%). Conclusion* This is the first cross-European interlaboratory assessment of the performance of RAD51/Geminin co-IF. We show that subtle local protocol differences do not impact final RAD51 scores. Furthermore, we elucidated features that may negatively impact RAD51 score accuracy.