B. Schlappe

Loss of SMARCA4 Expression Is Both Sensitive and Specific for the Diagnosis of Small Cell Carcinoma of Ovary, Hypercalcemic Type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare ovarian neoplasm that occurs in young women and has a poor prognosis. The histologic diagnosis of SCCOHT can be challenging due to its rarity and relatively nonspecific histologic features, which range from the classic, first-described small cell morphology to a pattern in which there are large cells with abundant eosinophilic cytoplasm. Many entities can be in the differential diagnosis and to date, immunohistochemical stains have shown no distinctive profile and have been of limited aid. SMARCA4 (also known as BRG1) mutations have recently been reported at high frequency in these tumors. SMARCA4 is an important component of the SWI/SNF complex that regulates gene expression through alteration of nucleosome conformation. Studies to date have suggested that immunohistochemical loss of expression of SMARCA4 is associated with the presence of a SMARCA4 mutation in most cases. In this study, the sensitivity and specificity of the immunohistochemical loss of SMARCA4 expression for the diagnosis of SCCOHT is examined in the context of the differential diagnosis with other primary or metastatic ovarian tumors. All but one of the SCCOHT showed loss of SMARCA4 expression (16/17; 94%), while of 279 other tumors tested, only two tumors (one clear cell carcinoma and one ovarian melanoma) showed loss of SMARCA4 expression. We conclude that SMARCA4 immunohistochemistry is highly sensitive and specific for a diagnosis of SCCOHT and is of clinical utility in the differential diagnosis of poorly differentiated ovarian tumors.

Characteristics of 10-year survivors of high-grade serous ovarian carcinoma

OBJECTIVE High-grade serous carcinoma (HGSC) generally presents at an advanced stage with poor long-term (LT) survival. Here we describe clinical features found in women surviving HGSC for ten or more years. METHODS A multi-center research consortium was established between five participating academic centers. Patient selection criteria included high-grade serous ovarian, fallopian tube, or peritoneal carcinoma with at least ten years of follow up. Non-serous, borderline tumors and low-grade serous subtypes were excluded. RESULTS The 203 identified LT ten-year survivors with HGSC were diagnosed at a median age of 57years (range 37-84years). The majority of patients had stage IIIC (72.4%) disease at presentation. Of those who underwent primary cytoreductive surgery, optimal cytoreduction was achieved in 143 (85.6%) patients. After a median follow up of 144months, 88 (46.8%) patients did not develop recurrent disease after initial treatment. Unexpected findings from this survey of LT survivors includes 14% of patients having had suboptimal cytoreduction, 11% of patients having an initial platinum free interval of <12months, and nearly 53% of patients having recurrent disease, yet still surviving more than ten years after diagnosis. CONCLUSIONS LT survivors of HGSC of the ovary generally have favorable clinical features including optimal surgical cytoreduction and primary platinum sensitive disease. The majority of patients will develop recurrent disease, however many remained disease free for more than 10years. Future work will compare the clinical features of this unusual cohort of LT survivors with the characteristics of HGSC patients having less favorable outcomes.

Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type.

Small cell carcinoma of the ovary, hypercalcemic type is an aggressive tumor generally affecting young women with limited treatment options. Mutations in SMARCA4, a catalytic subunit of the SWI/SNF chromatin remodeling complex, have recently been identified in nearly all small cell carcinoma of the ovary, hypercalcemic type cases and represent a signature molecular feature for this disease. Additional biological dependencies associated with small cell carcinoma of the ovary, hypercalcemic type have not been identified. SMARCA2, another catalytic subunit of the SWI/SNF complex mutually exclusive with SMARCA4, is thought to be post-translationally silenced in various cancer types. We analyzed 10 archival small cell carcinoma of the ovary, hypercalcemic type cases for SMARCA2 protein expression by immunohistochemistry and found that SMARCA2 expression was lost in all but one case. None of the 50 other tumors that primarily or secondarily involved the ovary demonstrated concomitant loss of SMARCA2 and SMARCA4. Deep sequencing revealed that this loss of SMARCA2 expression is not the result of mutational inactivation. In addition, we established a small cell carcinoma of the ovary, hypercalcemic type patient-derived xenograft and confirmed the loss of SMARCA2 in this in vitro model. This patient-derived xenograft model, established from a recurrent tumor, also had unexpected mutational features for this disease, including functional mutations in TP53 and POLE. Taken together, our data suggest that concomitant loss of SMARCA2 and SMARCA4 is another hallmark of small cell carcinoma of the ovary, hypercalcemic type—a finding that offers new opportunities for therapeutic interventions.

Massively parallel sequencing analysis of mucinous ovarian carcinomas: genomic profiling and differential diagnoses

OBJECTIVE Mucinous ovarian cancer (MOC) is a rare type of epithelial ovarian cancer resistant to standard chemotherapy regimens. We sought to characterize the repertoire of somatic mutations in MOCs and to define the contribution of massively parallel sequencing to the classification of tumors diagnosed as primary MOCs. METHODS Following gynecologic pathology and chart review, DNA samples obtained from primary MOCs and matched normal tissues/blood were subjected to whole-exome (n = 9) or massively parallel sequencing targeting 341 cancer genes (n = 15). Immunohistochemical analysis of estrogen receptor, progesterone receptor, PTEN, ARID1A/BAF250a, and the DNA mismatch (MMR) proteins MSH6 and PMS2 was performed for all cases. Mutational frequencies of MOCs were compared to those of high-grade serous ovarian cancers (HGSOCs) and mucinous tumors from other sites. RESULTS MOCs were heterogeneous at the genetic level, frequently harboring TP53 (75%) mutations, KRAS (71%) mutations and/or CDKN2A/B homozygous deletions/mutations (33%). Although established criteria for diagnosis were employed, four cases harbored mutational and immunohistochemical profiles similar to those of endometrioid carcinomas, and one case for colorectal or endometrioid carcinoma. Significant differences in the frequencies of KRAS, TP53, CDKN2A, FBXW7, PIK3CA and/or APC mutations between the confirmed primary MOCs (n = 19) and HGSOCs, mucinous gastric and/or mucinous colorectal carcinomas were found, whereas no differences in the 341 genes studied between MOCs and mucinous pancreatic carcinomas were identified. CONCLUSIONS Our findings suggest that the assessment of mutations affecting TP53, KRAS, PIK3CA, ARID1A and POLE, and DNA MMR protein expression may be used to further aid the diagnosis and treatment decision-making of primary MOC.

Robotic Para-aortic Lymph Node Dissection

Use of the robotic surgical platform in gynecologic oncology, including para-aortic lymph node dissection, has steadily increased due to improved dexterity, better visualization, increased primary surgeon independence, and increased comfort over conventional laparoscopic equipment. With patient positioning and robotic docking techniques to improve access to the infrarenal para-aortic lymph nodes, robotic para-aortic lymph node dissection may be safely performed without diminishing the number of para-aortic lymph nodes removed. In this chapter, we review the published data on the safety and feasibility of robotic-assisted para-aortic lymphadenectomy in gynecologic oncology as well as describe the procedure itself, including trocar placement, patient positioning, and robot docking.

Cited rationale for variance in the use of primary intraperitoneal chemotherapy following optimal cytoreduction for stage III ovarian carcinoma at a high intraperitoneal chemotherapy utilization center

OBJECTIVE Studies have demonstrated improved ovarian cancer survival with the administration of a combination of intravenous (IV) and intraperitoneal (IP) chemotherapy following optimal cytoreduction. Despite this, IV/IP chemotherapy is not uniformly used. In this retrospective cohort study, we assessed the documented reasons for giving IV-only chemotherapy. METHODS All patients who had optimal primary cytoreductive surgery for stage III ovarian, fallopian tube, or primary peritoneal carcinoma, met eligibility criteria for GOG-172, and received primary chemotherapy at our institution between 2006 and 2013 were identified. Patients who received at least one cycle of adjuvant IV/IP therapy were included in the IP group. Patient characteristics, treatment information, and reason cited for not administering IP therapy were collected. RESULTS Of the patients evaluated, 330 met inclusion criteria. The majority (n=261, 79%) received at least one IV/IP cycle (median, 6; range, 1-6), and 62% completed 6cycles. The most common reason for giving IV-only therapy was postoperative status (i.e., delayed wound healing, performance status), accounting for 18 (26%) of the 69 IV-only patients (5% of the entire cohort). Other cited reasons were baseline comorbidities (15%) and IP port complications (12%). Receipt of ≥1cycle of IP chemotherapy (HR 0.51; 95% CI, 0.32-0.80) and no gross residual disease (HR 0.47; 95% CI, 0.31-0.71) were associated with improved overall survival. CONCLUSION Potentially modifiable factors identified as leading to the use of IV-only chemotherapy were postoperative status and IP port complications, which if altered, could potentially lead to increased IP chemotherapy use.

Abstract B15: Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type.

Objective: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive subtype of ovarian cancer with limited treatment options. Inactivating SMARCA4 germline and somatic mutations are present in nearly all SCCOHT cases, representing a signature molecular feature of this disease. Concomitant loss of SMARCA2, a mutually exclusive catalytic subunit of the SWI/SNF chromatic remodeling complex, has been attributed to worse overall survival in non-small cell lung carcinoma (NSCLC) when compared to cases with loss of SMARCA4 only. The objective of this study was to evaluate the mutational status and expression of SMARCA2 in SCCOHT. Methods: We evaluated 10 archival cases of SCCOHT and 1 newly established case from a PDX model of recurrent disease. Specialty gynecologic pathologists reviewed all cases to confirm the diagnosis. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumors with at least 50% tumor cell nuclei. Using the MSK-IMPACT assay, samples were deep-sequenced for a panel of 341 known oncogenes and tumor suppressor genes frequently altered in cancer. Identified SMARCA4 mutations were validated using Sanger sequencing. Sequencing of the SMARCA2 coding regions and splice sites was done using AmpliSeq. We performed immunoblotting for SMARCA4 and SMARCA2 on available frozen tumor samples using standard protocol. To confirm loss of protein expression, immunohistochemistry (IHC) for the SMARCA4 and SMARCA2 was performed on FFPE slides of all cases; the absence of tumor cell nuclear staining in the presence of internal positive controls scored as loss-of-expression. For cell-line over-expression studies, SCCOHT BIN67 and NSCLC H1299 cells were transiently transfected with an expression plasmid containing cDNA for SMARCA4 using FuGene reagent (Invitrogen) according to manufacturer9s instructions. Immunoblotting for SMARCA2 and SMARCA4 was performed on protein lysates extracted from cell pellets. Results: IHC demonstrated loss of SMARCA2 expression in 9 (90%) of 10 archival SCCOHT cases and the PDX model. Sequencing of the SMARCA2 coding region and splice sites revealed no mutations, suggesting a non-mutational etiology of SMARCA2 protein loss. Previous work revealed biallelic SMARCA4 mutations in the 9 archival cases with SMARCA2 loss. SMARCA4 mutations were confirmed in both the PDX model and the one additional archival case, resulting in universal SMARCA4 mutations. The only case with normal SMARCA2 expression was also the only case with retained SMARCA4 expression. To explore the relationship between SMARCA2 and SMARCA4, we re-introduced SMARCA4 into BIN67 SCCOHT and H1299 NSCLC cell lines that lack expression. Wild-type BIN67 cells have very low levels of SMARCA2 expression, while wild-type H1299 cells express SMARCA2 normally. After transfection with plasmid cDNA and confirmation of SMARCA4 expression by immunoblotting, there was no increase in SMARCA2 expression in either BIN67 or H1299 cells. Conclusions: Concomitant loss of SMARCA2 and SMARCA4 expression was seen in nearly all SCCOHT cases and our PDX model. While loss of SMARCA4 expression is due to biallelic mutations, the absence of SMARCA2 expression is non-mutational and potentially the result of epigenetic or post-translational silencing. Our data suggest that concomitant loss of SMARCA2 and SMARCA4 is another hallmark of SCCOHT. The reversal of SMARCA2 loss may serve as a potential novel therapeutic approach for SCCOHT. Citation Format: Jill H. Tseng, Petar Jelinic, Brooke A. Schlappe, Niamh Conlon, Narciso Olvera, Fanny Dao, Jennifer J. Mueller, Yaser Hussein, Robert A. Soslow, Douglas A. Levine. Concomitant loss of SMARCA2 and SMARCA4 expression in small cell carcinoma of the ovary, hypercalcemic type. [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 B15.

Abstract B14: Molecular characterization of mucinous ovarian carcinoma.

Mucinous ovarian carcinoma (MOC) is a rare, chemoresistant tumor known to share pathologic features with tumors of the gastrointestinal and pancreaticobiliary tracts. To better understand the genomic and proteomic landscapes of invasive MOCs, we identified somatic mutations and proteins expression in a single institution cohort and compared these results with data from TCGA tumor projects. Twenty-six tumors consistent with primary invasive MOC after expert pathology review and with available paired tumor and normal tissue were identified from institutional databases between July 2001 and July 2012. DNA extracted from FFPE or fresh frozen samples underwent next generation sequencing with a combination of a candidate gene assay (37 genes), the MSK-IMPACT assay (341 genes), transcriptome sequencing, and whole exome sequencing. Copy number alterations were identified using data from the MSK-IMPACT assay, whole exome sequencing or Affymetrix SNP 6.0 arrays. Immunohistochemistry (IHC) was performed using optimized antibodies for six proteins to confirm the diagnosis of MOC (ER, PR, CK7, CK20, CDX-2, PAX8) and seven proteins to correlate with mutation status and copy number alterations (p53, ARID1A[Baf250a], PTEN, PMS2, MSH6, HER2, p16). Mutation data for other TCGA tumor types was obtained from the cBio Cancer Genomics Portal (cbioportal.org). The median age of the cohort was 58 years (range 20-86 years). Most (19/26, 73%) of the tumors were stage I. Somatic TP53 and KRAS mutations were the most common seen and were identified in 18 (69%) cases each, with a co-mutation rate of 50% (13/26). Other commonly mutated genes include ARID1A , PTEN , and PIK3CA . Homozygous deletions of CDKN2A were found in 27% (7/26). ERBB2 alterations were identified in 19% (5/26) and consisted of three amplifications and two mutations. Mutations in at least one potentially targetable gene were identified in 42% (11/26) of tumors. IHC was concordant with sequencing results in 154/182 (85%) of stained cases. Pancreatic, colorectal, lung adenocarcinoma, endometrial, and stomach cancers have the highest frequency of KRAS mutations. Co-mutations of KRAS and TP53 occur most commonly in pancreatic (59%) and colorectal (21%) carcinomas. CDKN2A homozygous deletions are also found at a similar frequency in pancreatic adenocarcinomas (28%). When evaluating the mutation rates of the five most commonly mutated genes in our MOC cohort with colorectal, pancreatic, gastric and high-grade serous ovarian carcinomas (HGSOC) in the TCGA datasets, pancreatic adenocarcinoma showed the most similarity. HGSOC showed little similarity to the MOCs. KRAS and TP53 co-mutation are common in invasive MOCs. Other commonly mutated genes include ARID1A , PTEN , and PIK3CA . Potentially targetable ERBB2 alterations were identified in several cases. Despite anatomic distinctions, the mutational landscape of MOC shares similarities with that of pancreatic adenocarcinoma including frequent CDKN2A deletions and KRAS / TP53 co-mutation. The suggested shared molecular pattern with pancreatic adenocarcinoma offers potential to guide future developmental therapeutics. Citation Format: Brooke Schlappe, Jennifer J. Mueller, Narciso Olvera, Fanny Dao, Cyriac Kandoth, Faina Bogomolniy, Agnes Viale, Kety Huberman, Gouri Nanjangud, Yaser Hussein, Barry Taylor, Robert Soslow, Douglas A. Levine. Molecular characterization of mucinous ovarian carcinoma. [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 B14.