Faculty Bibliography

We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of β-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC.

OBJECTIVES/OBJECTIVE:Emerging data suggests that abnormal (nuclear) β-catenin expression in some settings is associated with poorer outcomes. Our study aimed to verify the significance of abnormal β-catenin expression in early-stage endometrial cancer patients and determine if adjuvant radiation therapy (RT) improves local control. METHODS:We identified 213 patients with FIGO 2018 stage I-II endometrioid endometrial cancer who underwent surgery from 2009 to 2021 with β-catenin expression assessed. Vaginal, regional, and distant recurrences were analyzed using competing risk methods, and overall survival was analyzed using Kaplan-Meier. RESULTS:Median follow up was 53.2 months; 6.9% experienced vaginal, 8.2% regional, and 7.4% distant recurrence. For the entire cohort, abnormal β-catenin expression was significantly associated with vaginal recurrence and remained significant on multivariate analysis (p = 0.03). There were 114 patients in the no specific molecular profile (NSMP) subgroup, and abnormal β-catenin expression was present in 46.5%. In the NSMP subgroup, abnormal β-catenin expression was associated with increased rates of vaginal recurrence (p = 0.06). Abnormal β-catenin expression in the NSMP subgroup was significant on multivariate analysis for vaginal recurrence (p = 0.04). RT significantly decreased vaginal recurrences in the entire cohort in patients with abnormal β-catenin expression (0%) versus wild type expression (17.5%; p = 0.03). In the NSMP subgroup 0% of patients who received RT versus 20.9% of patients who did not receive RT experienced a vaginal recurrence (p = 0.03). CONCLUSION/CONCLUSIONS:Use of adjuvant RT for stage I-II NSMP endometrial cancer with abnormal β-catenin expression improved local control. RT should be considered in these patients to decrease risk of vaginal recurrences.

CONTEXT.—/UNASSIGNED:Clinical testing for tumor cell-free DNA (cfDNA) has evolved rapidly, but no practice guidelines exist. OBJECTIVE.—/UNASSIGNED:To summarize cfDNA laboratory practices based on self-reporting and assess preanalytical, analytical, and postanalytical trends that may influence the quality, accuracy, and consistency of cfDNA testing. DESIGN.—/UNASSIGNED:Data were derived from the College of American Pathologists cfDNA proficiency testing program submitted by 101 participating laboratories from 2018 to 2019. RESULTS.—/UNASSIGNED:Most laboratories performing clinical circulating tumor DNA testing are commercial/nonhospital (71.2%; 72 of 101) and international (77.2%; 78 of 101) laboratories. Commercial laboratories had higher monthly test volumes than hospital-based laboratories (median, 36 versus 7-8) and tended to have larger gene panels (median, 50 versus 11 genes) when panel-based testing was offered. The main clinical indications include therapy selection and treatment/disease monitoring. Plasma is the most commonly accepted specimen, which is predominantly collected in cell-stabilizing tubes. Equal proportions of laboratories use next-generation sequencing (NGS) and non-NGS methods to assess key genes, including EGFR, BRAF, KRAS, NRAS, and IDH1. Most laboratories reported a lower limit of detection (LLOD) of 0.5%, variant allele frequency or less, which did not differ by method, NGS or non-NGS, except for EGFR. Sixty-five percent (17 of 26) of laboratories using the FDA-approved non-NGS EGFR assay report analytical sensitivities higher than 0.5%, as compared to 15% (16 of 104) of laboratories using an alternative NGS or non-NGS method. There is also a wider range in LLODs obtained for the FDA-approved EGFR assay than nonapproved assays. CONCLUSIONS.—/UNASSIGNED:These results highlight emerging practice trends and serve as a foundation to initiate future practice recommendations.

Amplicon-based next-generation sequencing (NGS) assays employ highly sensitive, rapid, and cost-effective methods to detect clinically actionable mutations for the diagnosis, prognosis, and treatment of patients with cancer. However, recognition of certain limitations inherent to amplicon-based NGS assays is crucial for the correct interpretation and reporting of variants in the clinical setting. In this report, we illustrate three different potential pitfalls related to amplicon-based NGS assays based on our institutional experience and highlight how the risk of such events can be minimised.

Determining the replicative DNA polymerase epsilon (POLE) mutation status in endometrial carcinomas (ECs) has important clinical implications given that the majority of "ultramutated" tumors harboring pathogenic exonuclease domain mutations in POLE (POLEmut) have a favorable prognosis, even among high-grade histotypes. Currently, there are no specific morphologic or immunophenotypic features that allow accurate detection of POLEmut tumors without molecular testing. Consequently, identifying POLEmut tumors has been challenging without employing costly and/or time-consuming DNA sequencing approaches. Here we developed a novel SNaPshot assay to facilitate routine and efficient POLE mutation testing in EC. The SNaPshot assay interrogates 15 nucleotide sites within exons 9, 11, 13, and 14 encoding the POLE exonuclease domain. The variant sites were selected based on recurrence, evidence of functional impact, association with high tumor mutation burden and/or detection in EC clinical outcome studies. Based on the pathogenic somatic variants reported in the literature, the assay is predicted to have a clinical sensitivity of 90% to 95% for ECs. Validation studies showed 100% specificity and sensitivity for the variants covered, with expected genotypic results for both the positive (n=11) and negative (n=20) patient controls on multiple repeat tests and dilution series. Analytic sensitivity was conservatively approximated at a 10% variant allele fraction (VAF), with documented detection as low as 5% VAF. As expected, the SNaPshot assay demonstrated greater sensitivity than Sanger sequencing for VAFs below 20%, an important characteristic for somatic mutation detection. Here we have developed and validated the first SNaPshot assay to detect hotspot POLE mutations. While next-generation sequencing and Sanger sequencing-based approaches have also been used to detect POLE mutations, a SNaPshot approach provides useful balance of analytical sensitivity, cost-effectiveness, and efficiency in a high-volume case load setting.

Purpose/Objective(s): There is increasing data about the prognostic significance of molecular profiling for endometrial cancer outcomes and emerging data showing that abnormal (nuclear) beta-catenin expression is associated with an increased risk of recurrence and poorer clinical outcomes. Our study aimed to determine if the addition of adjuvant radiation therapy following surgery improves local control in early-stage endometrial cancer patients with abnormal beta-catenin expression. Materials/Methods: We identified 217 patients with FIGO 2009 stage I and stage II endometrial cancer who underwent surgical resection from 2018 to 2021 at our institution and had molecular data available. We reviewed the beta-catenin expression status and outcomes in these patients. Vaginal cuff recurrences, regional recurrences, and distant metastases were analyzed using competing risk methods, and overall survival was analyzed using Kaplan-Meier.
Result(s): The median follow up time was 22.5 months; 6.9% experienced vaginal, 8.2% regional, and 7.4% distant recurrence. Abnormal beta-catenin expression was present in 36.8% of all patients (47% of grade 1-2 versus 17% of grade 3 patients (p=0.007)) and associated with no specific molecular profile subtype. Abnormal beta-catenin expression was significantly associated with increased rates of vaginal cuff recurrence (p=0.02), was borderline associated with worse overall survival (p=0.07), and not significantly associated with regional recurrence or distant metastases. One third of patients received adjuvant radiation therapy, generally based on other pathologic factors, and radiation was significantly associated with decreased vaginal cuff recurrences in patients with abnormal beta-catenin expression (p=0.01). This decreased incidence of vaginal recurrence was seen with the use of any type of radiation including brachytherapy or external beam radiation therapy.
Conclusion(s): Patients with Stage I-II endometrial cancer with abnormal beta-catenin expression have increased rates of vaginal cuff recurrences, and adjuvant radiation therapy in this group significantly improved local control. Radiation therapy should be considered in patients with endometrial cancers demonstrating abnormal beta-catenin expression to decrease the risk of vaginal cuff recurrences.
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CONTEXT.—/UNASSIGNED:Neoplastic cellularity assessment has become an essential component of molecular oncology testing; however, there are currently no best practice recommendations or guidelines for this potentially variable step in the testing process. OBJECTIVE.—/UNASSIGNED:To describe the domestic and international practices of neoplastic cellularity assessment and to determine how variations in laboratory practices affect neoplastic cellularity assessment accuracy. DESIGN.—/UNASSIGNED:Data were derived from 57 US and international laboratories that participated in the 2019 College of American Pathologists Neoplastic Cellularity Proficiency Testing Survey (NEO-B 2019). NEO-B 2019 included 29 laboratory practice questions and 5 images exhibiting challenging histologic features. Participants assessed the neoplastic cellularity of hematoxylin-eosin-stained digital images, and results were compared to a criterion standard derived from a manual cell count. RESULTS.—/UNASSIGNED:The survey responses showed variations in the laboratory practices for the assessment of neoplastic cellularity, including the definition of neoplastic cellularity, assessment methodology, counting practices, and quality assurance practices. In some instances, variation in laboratory practice affected neoplastic cellularity assessment performance. CONCLUSIONS.—/UNASSIGNED:The results highlight the need for a consensus definition and improved standardization of the assessment of neoplastic cellularity. We put forth an initial set of best practice recommendations to begin the process of standardizing neoplastic cellularity assessment.

HER2 is an established therapeutic biomarker in advanced or recurrent endometrial serous carcinoma. Current clinical guidelines recommend HER2 testing exclusively in this endometrial carcinoma (EC) subtype; however, the full spectrum of ECs harboring HER2 amplification remains ill-defined. The present study characterizes the clinicopathologic and molecular features of HER2-amplified ECs across all histologic subtypes. Retrospective analysis of our institutional cohort of 2,042 ECs subjected to targeted clinical massively parallel sequencing identified 77 (3.8%) cases with HER2 amplification, a group comprised of serous (n = 29), endometrioid (low-grade, n = 2, high-grade, n = 1) and clear cell (n = 4) carcinomas, carcinosarcomas (n = 18) and high-grade ECs with ambiguous features (HGEC, n = 23). A co-existing TP53 mutation was identified in 94% (72/77) of HER2-amplified ECs. Other recurrent genetic alterations included amplification of CCNE1 (22%) and ERBB3 (10%), FBXW7 mutations or deletions (13%), and mutations in PIK3CA (40%) and PPP2R1A (13%). The HER2 immunohistochemistry score was 2+ or 3+ for all evaluable cases (n = 61). Apart from carcinosarcomas, which often showed lower HER2 expression, particularly in the sarcomatous component, HER2 immunohistochemical staining pattern and intensity were similar across EC subtypes. Intratumor heterogeneity in HER2 expression was common and correlated with genetic heterogeneity as detected by fluorescence in-situ hybridization. These results demonstrate the frequent co-occurrence of HER2 amplification with TP53 mutation and high-grade histology, rather than being specific to serous carcinoma, per se. Overall, these findings suggest that HER2 targeted therapy may be more broadly applicable to all high-grade EC histotypes and consideration should be given to expanding therapeutic eligibility.

Fumarate hydratase-deficient leiomyomas (dFH leiomyomas) often display atypical pathologic features yet exhibit a benign clinical course. Recent data suggest that dFH leiomyomas may be misclassified as smooth muscle tumors of uncertain malignant potential, a category that encompasses a heterogenous subgroup of uterine neoplasms with smooth muscle differentiation and atypical features that impart ambiguity regarding their expected clinical behavior. dFH leiomyomas can be seen in the context of hereditary leiomyomatosis and renal cell carcinoma syndrome or in the sporadic setting. In this retrospective study, we sought to examine the prevalence and clinicopathologic characteristics of dFH leiomyomas in 48 tumors previously diagnosed as smooth muscle tumors of uncertain malignant potential from 38 patients. Of these 48 tumors, 3 (6.3%) occurring in 2 patients were found to be deficient for FH by immunohistochemistry, including 1 uterine and 2 extrauterine (abdominopelvic) tumors. The 3 tumors showed histologic features typical of dFH leiomyomas, including hemangiopericytoma-like vessels, edema, macronucleoli, and atypia. Neither patient developed recurrent leiomyomas or renal cell carcinoma, and both were alive without disease at last follow-up. Our data suggest that dFH leiomyomas should be considered in the differential diagnosis of smooth muscle tumors of uncertain malignant potential, even in the context of extrauterine disease. Identification of FH deficiency in these tumors supports their classification as dFH leiomyomas despite their atypical morphologic features and/or clinical presentation. Importantly, detection of dFH in these cases may identify women at increased risk for hereditary leiomyomatosis and renal cell carcinoma who would benefit from genetic counseling and consideration for FH germline testing.

The comprehensive genomic analysis of endometrial carcinoma (EC) by The Cancer Genome Atlas (TCGA) led to the discovery of four distinct and prognostically significant molecular subgroups. Molecular classification has the potential to improve risk-stratification when integrated with clinicopathologic features and has recently been included in national and international patient management EC guidelines. Thus, the adoption of molecular classification into routine pathologic and clinical practice is likely to grow significantly in the upcoming years. Establishing an efficient and standardized workflow for performing molecular classification on ECs, and reporting both the molecular and histologic findings in an integrative manner, is imperative. Here we describe our effort to implement rapid and routine molecular classification on all ECs diagnosed at our institution. To this effect, we performed immunohistochemistry as a surrogate marker for identifying genetic and/or epigenetic alterations in DNA mismatch repair (e.g., MLH1, PMS2, MSH6, MSH2), and TP53 genes. In addition, we have developed and employed a single-gene POLE SNaPshot assay, which is a rapid and analytically sensitive method for detecting select POLE exonuclease domain mutations (EDMs). We report our molecular testing workflow and integrative reporting system as well as the clinicopathologic and molecular features of 310 ECs that underwent routine molecular classification at our institution. The 310 ECs were molecularly classified as follows: 15 (5%) POLE mutant (POLEmut), 79 (25%) mismatch repair-deficient (MMRd), 135 (44%) no specific molecular profile (NSMP), and 81 (26%) p53 abnormal (p53abnl). This work provides an initial framework for implementing routine molecular classification of ECs.