p53-Abnormal "Fields of Dysplasia" in Human Papillomavirus-Independent Vulvar Squamous Cell Carcinoma Impacts Margins and Recurrence Risk.

Abnormal p53 (p53abn) immunohistochemical (IHC) staining patterns can be found in vulvar squamous cell carcinoma (VSCC) and differentiated vulvar intraepithelial neoplasia (dVIN). They can also be found in the adjacent skin that shows morphology that falls short of the traditional diagnostic threshold for dVIN. Vulvectomy specimens containing human papillomavirus-independent p53abn VSCC with margins originally reported as negative for invasive and in situ disease were identified. Sections showing the closest approach by invasive or in situ neoplasia to margins were stained with p53 IHC stains. We evaluated the following: (1) detection of morphologically occult p53abn in situ neoplasia, (2) rates of margin status change after p53 IHC staining, and (3) effect of p53abn IHC staining at margins on the 2-year local recurrence rates. Seventy-three human papillomavirus-independent p53abn VSCCs were included. Half (35/73, 48%) had documented an in situ lesion in the original report. The use of p53 IHC staining identified 21 additional cases (29%) with the p53abn in situ lesions that were originally unrecognized. The histology of in situ lesions in the p53abn "field" varied and became more subtle (morphologically occult) farther away from the VSCC. Fifteen (21%) cases had a morphologically occult and previously unrecognized p53abn in situ lesion present at a resection margin, which conferred an increased risk of local recurrence (5/7 [71.4%] vs 6/22 [27.3%], P = .036). The p53abn in situ lesions at a margin were confirmed to have TP53 mutations by sequencing. p53 IHC staining identified morphologically occult p53abn in situ lesions surrounding human papillomavirus-independent VSCC. p53abn IHC staining at a margin was associated with a 3-fold increased risk of local recurrence.

Classification of Vulvar Squamous Cell Carcinoma and Precursor Lesions by p16 and p53 Immunohistochemistry: Considerations, Caveats, and an Algorithmic Approach.

There is emerging evidence that vulvar squamous cell carcinoma (VSCC) can be prognostically subclassified into 3 groups based on human papillomavirus (HPV) and p53 status: HPV-associated (HPV+), HPV-independent/p53 wild-type (HPV-/p53wt), or HPV-independent/p53 abnormal (HPV-/p53abn). Our goal was to assess the feasibility of separating VSCC and its precursors into these 3 groups using p16 and p53 immunohistochemistry (IHC). A tissue microarray containing 225 VSCC, 43 usual vulvar intraepithelial neoplasia (uVIN/HSIL), 10 verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), and 34 differentiated VIN (dVIN), was stained for p16 and p53. Noncomplementary p16 and p53 patterns were resolved by repeating p53 IHC and HPV RNA in situ hybridization (ISH) on whole sections, and sequencing for TP53. Of 82 p16-positive VSCC, 73 (89%) had complementary p16 and p53 patterns and were classified into the HPV+ group, 4 (4.9%) had wild-type p53 staining, positive HPV ISH and were classified into the HPV+ group, whereas 5 (6.1%) had p53 abnormal IHC patterns (1 null, 4 overexpression), negativity for HPV ISH, and harbored TP53 mutations (1 splice site, 4 missense); they were classified as HPV-/p53abn. Of 143 p16-negative VSCC, 142 (99.3%) had complementary p53 and p16 patterns: 115 (80.4%) HPV-/p53abn and 27 (18.9%) HPV-/p53wt. One had a basal-sparing p53 pattern, positivity for HPV ISH and was negative for TP53 mutations-HPV+ category. The use of IHC also led to revised diagnoses-HSIL to dVIN (3/43), dVIN to vaVIN (8/34), and dVIN to HSIL (3/34). Overall, 215/225 VSCC (95.6%) could be easily classifiable into 3 groups with p16 and p53 IHC. We identified several caveats, with the major caveat being that "double-positive" p16/p53 should be classified as HPV-/p53abn. We propose an algorithm that will facilitate the application of p16 and p53 IHC to classify VSCC in pathology practice.

Grade and Estrogen Receptor Expression Identify a Subset of No Specific Molecular Profile Endometrial Carcinomas at a Very Low Risk of Disease-Specific Death.

Endometrial carcinoma (EC) can be divided into 4 prognostic molecular subtypes, and no specific molecular profile (NSMP) type is the most commonly occurring type (∼50%). Although described as having an intermediate to favorable prognosis, this subtype encompasses pathologically and molecularly diverse tumors. We aimed to identify factors associated with outcomes within the NSMP ECs that might be used to stratify prognosis and direct treatment. Clinicopathologic, immunohistochemical, and genetic features of a large series of NSMP EC were used to identify parameters that could identify the subset associated with a very favorable outcome (disease-specific death rate <5% at 5 years, termed low-risk NSMP). A total of 1110 NSMP ECs were profiled. In a univariate analysis, stage, grade, lymphovascular invasion, estrogen receptor (ER) and progesterone receptor (PR) expression, L1CAM overexpression, and mutations in PIK3CA were associated with disease-specific survival. Two critical features, grade and ER expression, identified a low-risk NSMP subset (grade 1-2, ER-positive [>1%], 84% of cases), which showed a 5-year disease-specific death rate of 1.6% across all stages and 1.4% within stage I. The remaining cases (high-risk NSMPs, grade 3, and/or ER-negative status) were responsible for most of the disease-specific deaths (disease-specific death rate at 5 years, 22.9%; hazard ratio compared with that of low-risk NSMPs: 16.3; 95% CI, 8.4-31.7). Within NSMP EC, the low-risk and high-risk categories were of prognostic significance independent of the stage on a multivariate analysis. Low-grade and ER-positive NSMP ECs are a homogeneous low-risk group associated with an exceptionally favorable prognosis in which de-escalation and/or endocrine therapy strategies can be applied. Grade 3 and/or ER-negative status identifies a high-risk NSMP subset, including rare high-grade histotypes (eg, clear cell, dedifferentiated, and mesonephric-like), responsible for most NSMP-related deaths. Subclassification of NSMPs allows for the category of low-risk EC molecular subtypes to be dramatically expanded because it now includes both POLEmut and the much more common low-risk NSMP EC.

Subclonal p53 immunostaining in the diagnosis of endometrial carcinoma molecular subtype.

AIMS: The significance of subclonal expression of p53 (abrupt transition from wild-type to mutant-pattern staining) is not well understood, and the arbitrary diagnostic cut-off of 10% between NSMP and p53abn molecular subtypes of endometrial carcinoma (EC) has not been critically assessed. Our aim was to characterise subclonal p53 and discrepant p53 expression/TP53 sequencing results in EC and assess their clinical significance. METHODS AND RESULTS: Subclonal p53 immuostaining on whole sections from 957 ECs was recorded. Agreement between TP53 mutational assessment and p53 immunostaining was evaluated. Subclonal p53 IHC staining was seen in 4.0% (38 of 957) of cases, with 23 of 957 (2.4%) showing mutant-pattern p53 staining in ≥10% of tumour cells. It was most commonly seen in POLEmut (nine of 65, 14%) and MMRd (13 of 274, 4.7%) EC ('multiple classifier' ECs), where subclonal p53 staining does not impact the molecular subtype diagnosis. Excluding POLEmut and MMRd EC, 11 of 957 (1.1%) showed ≥10% subclonal p53 from which four patients died of disease, while there were no deaths due to disease in the five patients with <10% mutant-pattern p53 staining. Agreement between p53 immunostaining and TP53 sequencing was 92.6%; most of the discrepant results were in the ultramutated POLEmut or hypermutated MMRd ECs. In NSMP and p53abn EC the agreement between IHC and sequencing was 95.8%. CONCLUSIONS: Subclonal p53 staining ≥10% is present in only 1.1% of EC after excluding 'multiple classifier' ECs. The cut-off of ≥10% subclonal p53 staining identified patients at increased risk of dying from EC, supporting its use to diagnose p53abn molecular subtype.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool.

OBJECTIVES: Despite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. METHODS: DNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. RESULTS: The new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. CONCLUSION: ProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Significance of p53 immunostaining in mesothelial proliferations and correlation with TP53 mutation status.

p53 immunohistochemistry has long been proposed for the separation of benign from malignant mesothelial proliferations, with the older literature suggesting that any degree of positivity supported a diagnosis of mesothelioma. However, using modern immunohistochemistry platforms in other organ systems, notably gynecologic tumors, it has become clear that p53 staining can represent wild-type protein, and only specific staining patterns (absent, overexpression, or cytoplasmic expression) are indicative of a TP53 mutation. We applied these principles to two tissue microarrays containing 94 mesotheliomas and 66 reactive mesothelial proliferations. Seven/65 (11%) epithelioid mesotheliomas showed aberrant staining (four absent and three overexpression patterns) as did 5/29 (17%) of sarcomatoid mesotheliomas (all overexpression patterns). We sequenced the TP53 gene (exons 2-11) in five of the epithelioid and three of the sarcomatoid cases with aberrant staining as well as 12 epithelioid and eight sarcomatoid mesotheliomas with wild-type staining. All three sarcomatoid cases with aberrant staining showed mutated TP53, as did three of the epithelioid cases; in two of the epithelioid cases no mutation was detected, most likely because of large deletions not detected by this assay. In contrast, none of the 20 mesotheliomas with wild-type staining contained mutated TP53. We conclude that absent or overexpression p53 staining patterns can be used as a marker of a malignant vs. a benign mesothelial proliferation. The sensitivity of p53 staining by itself is low, but here addition of p53 to BAP1/MTAP staining increased sensitivity from 72 to 81% for epithelioid and 38 to 50% for sarcomatoid mesotheliomas.

Variability in endometrial carcinoma pathology practice: opportunities for improvement with molecular classification.

We assessed the landscape of diagnostic pathology practice and how molecular classification could potentially impact management of patients with endometrial cancer by collecting patient samples, clinicopathologic data, and patient outcomes from EC patients diagnosed in 2016 at 10 Canadian tertiary cancer centers and 19 community hospitals. ProMisE molecular subtype (POLEmut, MMRd, p53abn, No Specific Molecular Profile (NSMP)) was assigned retrospectively. 1357 patients were fully evaluable including 85 POLEmut (6.3%), 380 MMRd (28.0%), 643 NSMP (47.4%), and 249 p53abn ECs (18.3%). Immunohistochemistry (IHC) for MMR proteins was undertaken at the time of primary diagnosis in 2016 in only 42% of the cohort (570/1357; range 3.5-95.4%/center). p53 IHC had only been performed in 21.1% of the cohort (286/1357; range 10.1-41.9%/center). Thus, based on the retrospective molecular subtype assignment, 54.7% (208/380) of MMRd EC had not been tested with MMR IHC (or MSI) and 48.2% (120/249) of p53abn ECs were not tested with p53 IHC in 2016. Molecular subtype diversity within histotypes was profound; most serous carcinomas were p53abn (91.4%), but only 129/249 (51.8%) p53abn EC were serous. Low-grade (Gr1-2) endometrioid carcinomas were mostly NSMP (589/954, 61.7%) but included all molecular subtypes, including p53abn (19/954, 2.0%). Molecular subtype was significantly associated with clinical outcomes (p < 0.001) even in patients with stage I disease (OS p = 0.006, DSS p < 0.001, PFS p < 0.001). Assessment of national pathologic practice in 2016 shows highly variable use of MMR and p53 IHC and demonstrates significant opportunities to improve and standardize biomarker reporting. Inconsistent, non-reflexive IHC resulted in missed opportunities for Hereditary Cancer Program referral and Lynch Syndrome diagnosis, and missed potential therapeutic implications (e.g., chemotherapy in p53abn EC, immune blockade for MMRd EC). Routine integration of molecular subtyping into practice can improve the consistency of EC pathology assessment and classification.

Significance of p53 and presence of differentiated vulvar intra-epithelial neoplasia (dVIN) at resection margin in early stage human papillomavirus-independent vulvar squamous cell carcinoma.

OBJECTIVE: Vulvar squamous cell carcinoma and in situ lesions can be stratified by human papillomavirus (HPV) and TP53 status into prognostic risk groups using p16 and p53 immunohistochemistry. We assessed the significance of vulvar squamous cell carcinoma resection margin positivity for either differentiated vulvar intra-epithelial neoplasia (dVIN) or abnormal p53 immunohistochemistry, and other pathologic variables, in a cohort of patients with HPV-independent (HPV-I) p53 abnormal (p53abn) vulvar squamous cell carcinomas. METHODS: Patients with stage I-II HPV-I p53abn vulvar squamous cell carcinoma with negative invasive margins who did not receive adjuvant radiation from a single institution were included. Tumors underwent margin reassessment using p53 immunohistochemistry. Cases were segregated into (1) morphologic dVIN at margin; or (2) abnormal p53 immunohistochemistry staining at margin without morphologic dVIN (p53abn immunohistochemistry); or (3) margins negative by morphology and p53 immunohistochemistry. Clinicopathologic/outcome data were collected. RESULTS: A total of 51 patients were evaluated: (1) 12 with dVIN on margin; (2) 12 with p53abn immunohistochemistry on margin without morphologic dVIN; and (3) 27 with margins negative for morphologic dVIN and p53abn immunohistochemistry. The recurrence rate for patients with dVIN or p53abn immunohistochemistry on the margin was equally high at 75% each, compared with 33% with margins negative for morphologic dVIN and p53abn immunohistochemistry (p=0.009). On multivariate analysis, positive in situ margins maintained an association with disease recurrence (p=0.03) whereas invasive margin distance (radial and deep), lymphovascular invasion, and tumor size did not. CONCLUSIONS: Patients with stage I-II HPV-I vulvar squamous cell carcinoma with margins positive for either dVIN or p53abn immunohistochemistry without morphologic dVIN showed increased disease recurrence, regardless of invasive margin distance. These findings show that p53 immunohistochemistry is a useful adjunct for evaluating margin status in HPV-I vulvar squamous cell carcinoma and may support repeat excision for positive in situ margins (dVIN or p53abn immunohistochemistry).

Molecular subclassification of vulvar squamous cell carcinoma: reproducibility and prognostic significance of a novel surgical technique.

OBJECTIVES: Vulvar squamous cell carcinoma is subclassified into three prognostically relevant groups: (i) human papillomavirus (HPV) associated, (ii) HPV independent p53 abnormal (mutant pattern), and (iii) HPV independent p53 wild type. Immunohistochemistry for p16 and p53 serve as surrogates for HPV viral integration and TP53 mutational status. We assessed the reproducibility of the subclassification based on p16 and p53 immunohistochemistry and evaluated the prognostic significance of vulvar squamous cell carcinoma molecular subgroups in a patient cohort treated by vulvar field resection surgery. METHODS: In this retrospective cohort study, 68 cases treated by vulvar field resection were identified from the Leipzig School of Radical Pelvic Surgery. Immunohistochemistry for p16 and p53 was performed at three different institutions and evaluated independently by seven pathologists and two trainees. Tumors were classified into one of four groups: HPV associated, HPV independent p53 wild type, HPV independent p53 abnormal, and indeterminate. Selected cases were further interrogated by (HPV RNA in situ hybridization, TP53 sequencing). RESULTS: Final subclassification yielded 22 (32.4%) HPV associated, 41 (60.3%) HPV independent p53 abnormal, and 5 (7.3%) HPV independent p53 wild type tumors. Interobserver agreement (overall Fleiss' kappa statistic) for the four category classification was 0.74. No statistically significant differences in clinical outcomes between HPV associated and HPV independent vulvar squamous cell carcinoma were observed. CONCLUSION: Interobserver reproducibility of vulvar squamous cell carcinoma subclassification based on p16 and p53 immunohistochemistry may support routine use in clinical practice. Vulvar field resection surgery showed no significant difference in clinical outcomes when stratified based on HPV status.

Redefinition of familial intestinal gastric cancer: clinical and genetic perspectives.

BACKGROUND: Familial intestinal gastric cancer (FIGC) remains genetically unexplained and without testing/clinical criteria. Herein, we characterised the age of onset and disease spectrum of 50 FIGC families and searched for genetic causes potentially underlying a monogenic or an oligogenic/polygenic inheritance pattern. METHODS: Normal and tumour DNA from 50 FIGC probands were sequenced using Illumina custom panels on MiSeq, and their respective germline and somatic landscapes were compared with corresponding landscapes from sporadic intestinal gastric cancer (SIGC) and hereditary diffuse gastric cancer cohorts. RESULTS: The most prevalent phenotype in FIGC families was gastric cancer, detected in 138 of 208 patients (50 intestinal gastric cancer probands and 88 unknown gastric cancer histology relatives), followed by colorectal and breast cancers. After excluding benign and intronic variants lacking impact in splicing, 12 rare high-quality variants were found exclusively in 11 FIGC probands. Only two probands carried potentially deleterious variants, but lacked somatic second-hits, weakly supporting the monogenic hypothesis for FIGC. However, FIGC probands developed gastric cancer at least 10 years earlier and carried more TP53 germline common variants than SIGC (p=4.5E-03); FIGC and SIGC could be distinguished by specific germline and somatic variant profiles; there was an excess of FIGC tumours presenting microsatellite instability (38%); and FIGC tumours displayed significantly more somatic common variants than SIGC tumours (p=4.2E-06). CONCLUSION: This study proposed the first data-driven testing criteria for FIGC families, and supported FIGC as a genetically determined, likely polygenic, gastric cancer-predisposing disease, with earlier onset and distinct from patients with SIGC at the germline and somatic levels.

Cancer-Associated Mutations in Endometriosis without Cancer.

BACKGROUND: Endometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive-age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis. METHODS: We analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer-driver targeted sequencing (3 patients). Mutations were validated with the use of digital genomic methods in microdissected epithelium and stroma. Epithelial and stromal components of lesions from an additional 12 patients were analyzed by means of a droplet digital polymerase-chain-reaction (PCR) assay for recurrent activating KRAS mutations. RESULTS: Exome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients harbored known cancer driver mutations in ARID1A, PIK3CA, KRAS, or PPP2R1A, which were validated by Safe-Sequencing System or immunohistochemical analysis. The likelihood of driver genes being affected at this rate in the absence of selection was estimated at P=0.001 (binomial test). Targeted sequencing and a droplet digital PCR assay identified KRAS mutations in 2 of 3 patients and 3 of 12 patients, respectively, with mutations in the epithelium but not the stroma. One patient harbored two different KRAS mutations, c.35G→T and c.35G→C, and another carried identical KRAS c.35G→A mutations in three distinct lesions. CONCLUSIONS: We found that lesions in deep infiltrating endometriosis, which are associated with virtually no risk of malignant transformation, harbor somatic cancer driver mutations. Ten of 39 deep infiltrating lesions (26%) carried driver mutations; all the tested somatic mutations appeared to be confined to the epithelial compartment of endometriotic lesions.

Molecular subtypes of clear cell carcinoma of the endometrium: Opportunities for prognostic and predictive stratification.

OBJECTIVE: Our aim was to characterize the pathological, molecular and clinical outcomes of clear cell carcinoma of the endometrium (CCC). METHODS: CCC underwent ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) classification identifying four molecular subtypes: i) 'POLEmut' for ECs with pathogenic POLE mutations, ii) 'MMRd', if there is loss of mismatch repair proteins by immunohistochemistry (IHC), iii) 'p53wt' or iv) 'p53abn' based on p53 IHC staining. Clinicopathologic parameters, immune markers (CD3, CD8, CD79a, CD138, PD-1), ER, L1CAM, and outcomes were assessed. RESULTS: 52 CCCs were classified, including 1 (2%) POLEmut, 5 (10%) MMRd, 28 (54%) p53wt and 18 (35%) p53abn. Women with p53abn and p53wt CCCs were older than women with MMRd and POLEmut subtypes. p53wt CCC were distinct from typical p53wt endometrioid carcinomas; more likely to arise in older, thinner women, with advanced stage disease, LVSI and lymph node involvement, and a higher proportion ER negative, L1CAM overexpressing tumors with markedly worse outcomes. High levels of immune infiltrates (TILhigh) were observed in 75% of the CCC cohort. L1CAM overexpression was highest within p53abn and p53wt subtypes of CCC. CONCLUSION: CCC is a heterogeneous disease encompassing all four molecular subtypes and a wide range of clinical outcomes. Outcomes of patients with POLEmut, MMRd and p53abn CCC are not distinguishable from those of other patients with these respective subtypes of EC; p53wt CCC, however, differ from endometrioid p53wt EC in clinical, pathological, molecular features and outcomes. Thus, p53wt CCC of endometrium appear to be a distinct clinicopathological entity within the larger group of p53wt ECs.

Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Molecular classification defines outcomes and opportunities in young women with endometrial carcinoma.

OBJECTIVE: Approximately 15% of endometrial carcinomas (ECs) arise in young women who may wish to avoid surgical menopause and/or preserve fertility. Our aim was to evaluate the prognostic significance of Proactive Molecular risk classifier for Endometrial Carcinoma (ProMisE) in young (<50 yo) women with EC. METHODS: ProMisE was applied to a retrospective cohort of women with ECs <50 yo at diagnosis, and associations between the four ProMisE molecular subtypes (MMR deficient (MMRd), POLE mutated (POLE), p53 wild type (p53wt), and p53 abnormal (p53abn)) and clinicopathological parameters, including outcomes, were assessed. RESULTS: Of 257 ECs, there were 48 (19%) MMRd, 34 (13%) POLE, 164 (64%) p53wt and 11 (4%) p53abn. ProMisE subtypes were associated with differences in all measured clinicopathological parameters except for presence of synchronous ovarian tumours and fertility. Age at diagnosis was youngest and BMI highest in women with p53wt ECs. MMRd and p53abn tumours were more likely to be advanced stage (III/IV), high-risk (ESMO), and receive chemotherapy. ProMisE subtypes were strongly associated with outcomes (overall, disease-specific, and progression-free survival (p < 0.0001 for all)). Advanced stage, grade, LVSI, myometrial invasion and ESMO risk groups showed associations with some but not all survival parameters. ProMisE maintained a strong association with OS and DSS on multivariable analysis. CONCLUSIONS: ProMisE molecular classification is prognostic in young women with EC, enabling early stratification and risk assignment to direct care. Further studies can assess response to therapy, fertility, and cancer-related outcomes within the framework of molecular subtype.

Extrauterine high-grade serous carcinomas with bilateral adnexal involvement as the only two disease sites are clonal based on tp53 sequencing results: implications for biology, classification, and staging.

A previous multicenter study of 67 cases of Stage I/II tubo-ovarian high-grade serous carcinoma with complete tubal sampling identified 7 cases in which there were only two disease sites, comprising tumor involving opposite adnexa with no extra-adnexal involvement. This study aimed to determine whether such low-stage extrauterine high-grade serous carcinomas with only two sites of involvement, located on opposite adnexa, have identical or different TP53 mutations in order to investigate their clonal relationship. DNA extracted from both sites of involvement was subjected to TP53 sequencing (n=6) or sequencing of one site and mutation confirmation by droplet digital PCR for the other site (n=1). Of the 7 cases analyzed, 1 case had unilateral serous tubal intraepithelial carcinoma with contralateral ovarian high-grade serous carcinoma, 3 had tubal high-grade serous carcinomas (±serous tubal intraepithelial carcinoma) with contralateral ovarian high-grade serous carcinoma, 2 had bilateral ovarian high-grade serous carcinomas with normal tubes, and 1 had bilateral fallopian tube high-grade serous carcinoma with normal ovaries. All 7 cases showed identical TP53 mutations in tumor from both disease sites. Therefore, these rare cases of high-grade serous carcinoma confined to opposite adnexa all show clonal identity between the two sites of involvement, suggesting unifocal origin and metastasis rather than multifocal origin. Our results suggest that serous tubal intraepithelial carcinoma or adnexal high-grade serous carcinoma can metastasize to the contralateral adnexa without peritoneal involvement. Given the clonal relationship between the two sites, such cases should be considered stage II, with stage I reserved for cases with unilateral and unifocal adnexal involvement. Furthermore, serous tubal intraepithelial carcinoma without invasion should be taken to constitute a disease site for staging purposes.

DICER1 hot-spot mutations in ovarian gynandroblastoma.

AIMS: Gynandroblastoma is a rare ovarian sex cord-stromal tumour characterised by the presence of both male (Sertoli and/or Leydig cells) and female (granulosa cells) components. We investigated the mutational status of DICER1, FOXL2 and AKT1 genes at hot-spot regions that are known to be the key driving events in the development of Sertoli-Leydig cell tumour (SLCT), adult granulosa cell tumour (aGCT) and juvenile granulosa cell tumour (jGCT), respectively, to gain insights into the molecular pathogenesis of gynandroblastoma. METHODS AND RESULTS: Sixteen cases of gynandroblastoma were studied. All contained SLCT or Sertoli cell tumour components. aGCT and jGCT components were identified in seven and 10 cases, respectively, with one presenting both components. Heterozygous hot-spot mutations in the RNase IIIb domain of DICER1 were discovered in three cases, including one case with heterologous mucinous elements, all of which were composed of moderately or poorly differentiated SLCT and jGCT components, and harboured the mutations in both histological components. None of the 16 cases displayed mutations at the p.C134W (c.402C→G) of FOXL2 or within the pleckstrin-homology domain of AKT1. All cases showed FOXL2 immunostaining in both male and female components. CONCLUSION: DICER1 hot-spot mutation is the key-driving event in a subset of gynandroblastomas containing components of SLCT and jGCT. Gynandroblastomas composed of SLCT and jGCT may represent morphological variants of SLCT. The molecular basis of gynandroblastoma containing a component of aGCT is different from pure aGCT.

Confirmation of ProMisE: A simple, genomics-based clinical classifier for endometrial cancer.

BACKGROUND: Classification of endometrial carcinomas (ECs) by morphologic features is irreproducible and imperfectly reflects tumor biology. The authors developed the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), a molecular classification system based on The Cancer Genome Atlas genomic subgroups, and sought to confirm both feasibility and prognostic ability in a new, large cohort of ECs. METHODS: Immunohistochemistry (IHC) for the presence or absence of mismatch repair (MMR) proteins (to identify MMR deficiency [MMR-D]), sequencing for polymerase-ɛ (POLE) exonuclease domain mutations (POLE EDMs), and IHC for tumor protein 53 (p53) (wild type vs null/missense mutations; p53 wt and p53 abn, respectively) were performed on 319 new EC samples. Subgroups were characterized and assessed relative to outcomes. The prognostic ability of ProMisE was compared with that of current risk-stratification systems (European Society of Medical Oncology [ESMO]). RESULTS: ProMisE decision-tree classification achieved categorization of all cases and identified 4 prognostic subgroups with distinct overall, disease-specific, and progression-free survival (P < .001). Tumors with POLE EDMs had the most favorable prognosis, and those with p53 abn the worst prognosis, and separation of the 2 middle survival curves (p53 wt and MMR-D) was observed. There were no significant differences in survival between the ESMO low-risk and intermediate-risk groups. ProMisE improved the ability to discriminate outcomes compared with ESMO risk stratification. There was substantial overlap (89%) between the p53 abn and high-risk ESMO subgroups; but, otherwise, there were no predictable associations between molecular and ESMO risk groups. CONCLUSIONS: Molecular classification of ECs can be achieved using clinically applicable methods and provides independent prognostic information beyond established clinicopathologic risk factors available at diagnosis. Consistent, biologically relevant categorization enables stratification for clinical trials and/or targeted therapy, identification of women who are at increased risk of having Lynch syndrome, and may guide clinical management. Cancer 2017;123:802-13. © 2016 American Cancer Society.

LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated ovarian cancers.

OBJECTIVE: Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic elements activated in many cancers capable of moving neighboring DNA through 3' transductions. Here we investigated the involvement of specific L1-mediated transductions in EAOCs. METHODS: Through whole genome sequencing, we identified active L1-mediated transductions originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations detected using multiplex PCR and next generation sequencing. RESULTS: TTC28-L1 mediated transductions were identified in at least three tumor samplings in all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites while other selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. CONCLUSION: The TTC28-L1 transductions along with classical driver mutations were near ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal relationships and to track ovarian cancer progression.