Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis Consortium Study.

BACKGROUND: Recently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC. METHODS: Two prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis. RESULTS: Consistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67-0.91, p < 0.0001). GMNN protein expression was not significantly associated with overall HSGC patient survival. However, HGSCs with >35% GMNN expression showed a trend for better outcomes, though this was not significant. CONCLUSION: We provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

International Endocervical Adenocarcinoma Criteria and Classification (IECC): An Independent Cohort With Clinical and Molecular Findings.

Recently, the International Endocervical Adenocarcinoma Criteria and Classification (IECC) has reorganized the classification of endocervical adenocarcinomas (ECAs), separating them into human papilloma virus (HPV)-associated (HPVA) and HPVA independent (HPVI) categories. In this study, we sought to revalidate the IECC clinical findings in an independent cohort and assess the mutational differences between HPVA and HPVI ECAs using next generation sequencing. Consecutive cases of ECAs were reclassified under the IECC. Clinicopathologic information was collected and tissue was sent for targeted next-generation sequencing in 33 genes. Associations between HPV status, clinicopathologic parameters and mutation status, with survival were evaluated. The series comprised of 85/100 HPVA (63 HPVA-usual type, 4 villoglandular, 3 mucinous intestinal, 15 mucinous not otherwise specified) and 15/100 HPVI (9 gastric, 4 mesonephric, 1 clear cell, 1 not otherwise specified). HPVA ECAs presented at a lower age (P=0.001), smaller tumor sizes (P=0.011), less margin positivity (P=0.027), less Silva pattern C (P=0.002), and lower FIGO stages (P=0.020). HPVA had superior survival compared with HPVI ECA [overall survival (P=0.0026), disease-specific survival (P=0.0092), and progression-free survival (P=0.0041)]. Factors that correlated with worse prognosis irrespective of HPV status were FIGO stage, positive margins and lymphovascular invasion (P<0.05). TP53 mutations were detected in a significantly higher proportion of HPVIs than HPVAs (P<<0.001). The study revalidates the IECC system by reaffirming the clinical and prognostic differences between HPVA and HPVI ECAs in an independent dataset.

Oncogenic mutations in histologically normal endometrium: the new normal?

The advent of next generation sequencing has vastly improved the resolution of mutation detection, thereby both increasing the resolution of the analysis of cancer tissues and shining light on the existence of somatic driver mutations in normal tissues, even in the absence of cancer. Studies have described somatic driver mutations in normal skin, blood, peritoneal washings, and esophageal epithelium. Such findings prompt speculation on whether such mutations exist in other tissues, such as the eutopic endometrium in particular, due to the highly regenerative nature of the endometrium and the recent observation of recurrent somatic driver mutations in deep infiltrating and iatrogenic endometriosis (tissues believed to be derived from the eutopic endometrium) by our group and others. In the current study we investigated the presence of somatic driver mutations in histologically normal endometrium from women lacking evidence of gynecologic malignancy or endometrial hyperplasia. Twenty-five women who underwent hysterectomies and 85 women who underwent endometrial biopsies were included in this study. Formalin-fixed, paraffin-embedded tissue specimens were analyzed by means of targeted sequencing followed by orthogonal validation with droplet digital PCR. PTEN and ARID1A immunohistochemistry (IHC) was also performed as surrogates for inactivating mutations in the respective genes. Overall, we observed somatic driver-like events in over 50% of normal endometrial samples analyzed, including hotspot mutations in KRAS, PIK3CA, and FGFR2 as well as PTEN-loss by IHC. Analysis of anterior and posterior samplings collected from women who underwent hysterectomies was consistent with the presence of somatic driver mutations within clonal pockets spread throughout the uterus. The prevalence of such oncogenic mutations also increased with age (OR: 1.05 [95% CI: 1.00-1.10], p = 0.035). These findings have implications on our understanding of aging and so-called 'normal tissues', thereby necessitating caution in the utilization of mutation-based early detection tools for endometrial or other cancers. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hereditary Breast and Ovarian Cancer Syndrome: Moving Beyond BRCA1 and BRCA2.

The recent implementation of next generation sequencing and multigene platforms has expanded the spectrum of hereditary breast and ovarian cancer syndrome, beyond the traditional genes BRCA1 and BRCA2. A large number of other moderate penetrance genes have now been uncovered, which also play critical roles in repairing double stranded DNA breaks through the homologous recombination pathway. This review discusses the landmark discoveries of BRCA1 and BRCA2, the homologous repair pathway and new genes discovered in hereditary breast and ovarian cancer syndrome, as well as their clinicopathologic significance and implications for genetic testing. It also highlights the new role of PARP inhibitors in the context of synthetic lethality and prophylactic surgical options.

Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms.

BACKGROUND: Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. METHODS: Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. RESULTS: We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell populations, while consistency of KRAS allelic frequency in both ERBB2 amplified and non-amplified regions suggests this mutation occurred in advance of the amplification event. CONCLUSIONS: Overall, the prevalence of RAS-alteration and striking co-occurrence of pathway "double-hits" supports a critical role for tumor progression in this ovarian malignancy. Given the spectrum of RAS-activating mutations, it is clear that targeting this pathway may be a viable therapeutic option for patients with recurrent or advanced stage mucinous ovarian carcinoma, however caution should be exercised in selecting one or more personalized therapeutics given the frequency of non-redundant RAS-activating alterations.

Developing ALK Immunohistochemistry and In Situ Hybridization Proficiency Testing for Non-Small Cell Lung Cancer in Canada: Canadian Immunohistochemistry Quality Control Challenges and Successes.

Intrachromosomal rearrangements involving the ALK gene are found in 3% to 5% of non-small cell lung cancers. Crizotinib is a tyrosine kinase inhibitor that has been shown to prolong progression-free survival in patients with advanced non-small cell lung cancer harboring ALK gene rearrangements. In Canada, ALK immunohistochemistry (IHC) is used as a screening test before confirmation by fluorescence in situ hybridization (FISH). Canadian Immunohistochemistry Quality Control (CIQC) provides ALK (Lung Cancer) proficiency testing (PT) for Canadian IHC laboratories. Samples included 32 previously characterized cases (IHC and FISH) either from the Canadian ALK (CALK) project or from CIQC reference laboratories. The same design was used for both runs. A total of 20 laboratories participated in Run 1 and 22 in Run 2. Some laboratories participated in the anticipation of future need and used the PT exercise as a part of test development and validation. Results of the IHC testing were first self-reported using the CIQC TMA Scorer and then evaluated by expert assessment. FISH results were self-reported only. Participants also reported details about IHC and FISH protocols. The κ-values were calculated, for which values >0.80 were used as acceptable results, respectively. The pass rate between the 2 runs and between different primary antibodies were compared. Six of the 22 protocols (27%) in Run 1 and 15 of the 22 (68%) protocols in Run 2 passed the CIQC PT criteria for IHC testing. The increase in the pass rate for Run 2 was significant (P=0.03, Wilcoxon signed-rank test). All reported FISH results were correct. CALK laboratories had significantly higher κ-values than non-CALK laboratories (P=0.002, t test). PT for IHC for rare diseases such as ALK-positive lung cancer is feasible, but challenging. The academic nature of the CIQC program and collaboration on a national level facilitated the development of appropriate PT samples. Participating laboratories made use of the PT exercise either to confirm that their testing was properly calibrated or to improve their protocols, which was confirmed by the achievement of significantly better results in Run 2. They also used CIQC's PT program for new test development and optimization.

A standardized protocol for identifying and counting lymph nodes harvested by pelvic lymph node dissection at the time of radical cystectomy.

INTRODUCTION: Lymph node counts have become a surrogate measure for the extent and quality of pelvic lymph node dissection (PLND) at radical cystectomy, but little consideration has been given to the methodology of lymph node processing. We report results from a prospective series comparing a conventional protocol for processing PLND specimens to a fat-emulsifying protocol. We hypothesized that the rate of node positivity would increase with the fat-emulsifying protocol. METHODS: Patients undergoing radical cystectomy for cTis-T4aN0-1M0 urothelial carcinoma of the bladder were eligible for this trial. Palpable lymph nodes were isolated from the PLND specimens in the conventional protocol. The remaining tissue was then processed with fat-emulsifying solution to identify further nodes visually. Nodal counts were compared between techniques. RESULTS: The median number of nodes counted in the PLND specimens of 26 patients was 24.5 (range: 20-40) with conventional processing and 37 (range: 24-52) with the fat-emulsifying solution (p < 0.001). Three patients had lymph node positive disease detected by conventional means, and a single patient was found to have a single positive node by the fat-emulsifying solution alone. The study was closed early after conducting a futility analysis. CONCLUSIONS: A fat-emulsifying protocol identified more lymph nodes than a conventional protocol and may be an appropriate method to standardize lymph node processing following PLND. However, we were unable to show that such a standardized approach significantly increased the rate of node positivity in patients undergoing radical cystectomy.