Heinzen, Ethan P.

e17038 Background: Carcinosarcomas (CS) are aggressive gynecologic malignancies defined by a biphasic tumor histology of carcinomatous and sarcomatous components. High expression of glucocorticoid receptor (GR) is associated with aggressive tumor biology in triple-negative breast, ovarian, and endometrial cancers. GR activation results in transcriptional regulation of epithelial-mesenchymal transition (EMT) genes. Therefore, we hypothesized that the sarcomatous component of Müllerian CS may be associated with relatively higher GR expression compared to the carcinomatous portion. Methods: Müllerian CSs from the University of Chicago tissue bank diagnosed from 5/19/2009 – 8/25/2014 were identified and pathology confirmed (RL). In addition, we evaluated GR expression in a mouse PDX CS model. Immunohistochemical expression of nuclear estrogen (ER), progesterone (PR), androgen (AR), and GR was quantified using the H-score. Scores between the sarcomatous and carcinomatous components were compared using a paired t-test. Results: Mean GR H-score was relatively higher in the sarcomatous vs carcinomatous component, overall (144.44 vs 38.89, P = 0.0014). Mean H-score also differed between sarcomatous and carcinomatous components for ER (3.06 vs 63.06, P = 0.016), PR (1.67 vs 47.22, P = 0.004), and AR (0 vs 2.78, P = 0.056). The significantly higher sarcomatous GR expression was replicated in IHC evaluation of the mouse PDX model. Conclusions: GR expression is significantly higher in the sarcomatous than the carcinomatous component of Müllerian CS, while ER, PR, and AR expression is significantly lower. Recent data suggest that an EMT gene signature is more prominently expressed in the sarcomatous versus carcinomatous component. We hypothesize that GR expression and activity may mediate EMT gene expression and represent a therapeutic target for CS. [Table: see text]

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Abstract Purpose: Here we assess whether molecular subtyping identifies biological features of tumors that correlate with survival and surgical outcomes of high-grade serous ovarian cancer (HGSOC). Experimental Design: Consensus clustering of pooled mRNA expression data from over 2,000 HGSOC cases was used to define molecular subtypes of HGSOCs. This de novo classification scheme was then applied to 381 Mayo Clinic HGSOC patients with detailed survival and surgical outcome information. Results: Five molecular subtypes of HGSOC were identified. In the pooled dataset, three subtypes were largely concordant with prior studies describing proliferative, mesenchymal, and immunoreactive tumors (concordance &gt; 70%), and the group of tumors previously described as differentiated type was segregated into two new types, one of which (anti-mesenchymal) had downregulation of genes that were typically upregulated in the mesenchymal subtype. Molecular subtypes were significantly associated with overall survival (P &lt; 0.001) and with rate of optimal surgical debulking (≤1 cm, P = 1.9E−4) in the pooled dataset. Among stage III-C or IV Mayo Clinic patients, molecular subtypes were also significantly associated with overall survival (P = 0.001), as well as rate of complete surgical debulking (no residual disease; 16% in mesenchymal tumors compared with &gt;28% in other subtypes; P = 0.02). Conclusions: HGSOC tumors may be categorized into five molecular subtypes that associate with overall survival and the extent of residual disease following debulking surgery. Because mesenchymal tumors may have features that were associated with less favorable surgical outcome, molecular subtyping may have future utility in guiding neoadjuvant treatment decisions for women with HGSOC. Clin Cancer Res; 23(15); 4077–85. ©2017 AACR.

Abstract Background: Defects in homologous recombination (HR) DNA repair are common in high grade serous ovarian, peritoneal and fallopian tube carcinoma (HGSOC). Other than mutations in BRCA1 and BRCA2 (BRCA1/2), the molecular causes of HR deficiency have not been well delineated. The frequency of HR deficiency in non-serous ovarian carcinoma is not known. We previously identified that germline and somatic HR mutations in 13 genes in OC was associated with improved survival. We sought to replicate these data in an independent tumor set, while sequencing a larger number of DNA repair genes. Methods: We used BROCA-HR to sequence 65 DNA repair genes in germline (blood) and tumor DNA from 363 women with ovarian (262), peritoneal (91), or fallopian tube (10) carcinoma from an institutional tissue bank. Cox proportional hazards models, adjusted for stage and residual tumor after cytoreduction, were used to assess associations between mutations in DNA repair genes in HR or related pathways and overall survival from diagnosis. Results: In total, 96/363 (25%) patients had 59 germline and 45 somatic deleterious mutations in HR genes, including 34 germline and 22 somatic BRCA1/2 mutations. Damaging mutations found in other HR genes included 25 germline and 23 somatic in the following genes: 5 ATM, 6 ATR, 1 BAP1, 2 BARD1, 6 BLM, 2 BRIP1, 2 BRE, 1 BRCC3, 2 CHEK2, 1 ERCC1, 1 FANCG, 1 FANCI, 2 FANCL, 8 FANCM, 1 NBN, 2 PALB2, 1 RAD51D, 1 RBBP8, 1 SLX4, 1 UIMC1 and 1 XRCC2. Of 322 HGSOC, HR mutations occurred in 86 patients (26.7%) including 54 (16.8%) germline and 32 somatic (9.9%). HR mutation rates were similar across the different non-serous histologies. Of 13 clear cell OC, 2 (15.3%) had germline mutations in non-BRCA1/2 HR genes and 3 (23.1%) had PTEN/PIK3CA mutations. Of 27 endometrioid OC, 3 (11.1%) had HR mutations (1 germline BRCA1, and 2 non-BRCA1/2 somatic HR mutations). PTEN/PIK3CA mutations were identified in 9 (2.8%) HGSOC, 3 (23.1%) clear cell and 2 (7.5%) endometrioid. HR mutations demonstrated a trend towards improved long-term overall survival (hazard ratio = 0.78, 96% CI 0.57-1.05) after accounting for stage and debulking status. Conclusions: These data not only indicate that damaging HR mutations, both germline and somatic, are found in endometrioid and clear cell as well as HGSOC, but also confirm our previous results showing an association between HR mutations and improved overall survival in OC. We are now sequencing 163 additional non-serous OC to augment the current analysis and these data will be available at presentation. Citation Format: Maria I. Harrell, Matthew J. Maurer, Ethan P. Heinzen, Ming K. Lee, Kimberly R. Kalli, Kathy J. Agnew, Ann L. Oberg, Tom Walsh, Barbara M. Norquist, Lynn C. Hartmann, Scott H. Kaufmann, Elizabeth M. Swisher. Homologous recombination mutations and overall survival in high-grade serous, endometrioid, and clear cell ovarian carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A07.