Comprehensive genomic sequencing of paired ovarian cancers reveals discordance in genes that determine clinical trial eligibility.

OBJECTIVE: We analyzed comprehensive genomic sequencing results from paired ovarian cancer samples to identify changes in mutational events over time. METHODS: DNA from paired FFPE tumor samples from 50 ovarian cancer patients in the Clearity Foundation Data Repository was analyzed for genomic mutations (GM), copy number alterations (CNA), microsatellite status (MS), tumor mutation burden (TMB), and loss of heterozygosity (LOH) by hybrid-capture, next-generation sequencing of up to 315 genes. Genomic profiles were compared between samples from the same patient. Poor quality results excluded 6 pairs from all analyses and 9 from CNA or LOH. RESULTS: Forty-four patients with predominantly advanced stage disease (34, 77%) and serous histology (31, 70%) received a median of 3 intervening treatment regimens (range 1-13). Analysis of 22 primary and recurrent sample pairs and 22 recurrent tumor pairs detected a median of 2 GM (range 0-5) and 1 CNA (range 0-6)/sample. TMB, MS, and LOH results were mostly concordant across paired samples. GM were consistent across most pairs [32/44 (73%) concordant], while CNA concordance was less [18/35 (51%)]. No changes were detected in therapeutically relevant GM, but 23% of patients had GM or CNA in the second sample that affect clinical trial eligibility. CONCLUSIONS: Paired ovarian cancer samples demonstrate stable genomic alterations across time. However, discordance was observed for some genes used as eligibility criteria for molecularly targeted clinical trials. Repeat tumor testing may be useful in cases where eligibility for such trials is deemed important after consideration of testing costs and potential clinical benefit.

Constitutively active ESR1 mutations in gynecologic malignancies and clinical response to estrogen-receptor directed therapies.

OBJECTIVE: Endocrine therapy is often considered as a treatment for hormone-responsive gynecologic malignancies. In breast cancer, activating mutations in the estrogen receptor (mutESR1) contribute to therapeutic resistance to endocrine therapy, especially aromatase inhibitors (AIs). The purpose of this study was to evaluate the frequency and clinical relevance of ESR1 genomic alterations in gynecologic malignancies. METHODS: DNA from FFPE tumor tissue obtained during routine clinical care for 9645 gynecologic malignancies (ovary, fallopian tube, uterus, cervix, vagina, vulvar, and placenta) was analyzed for all classes of genomic alterations (base substitutions (muts), insertions, deletions, rearrangements, and amplifications) in ESR1 by hybrid capture next generation sequencing. A subset of alterations was characterized in laboratory-based transcription assays for response to endocrine therapies. RESULTS: A total of 295 ESR1 genomic alterations were identified in 285 (3.0%) cases. mutESR1 were present in 86 (0.9%) cases and were more common in uterine compared to other cancers (2.0% vs <1%, respectively p < 0.001). mutESR1 were enriched in carcinomas with endometrioid versus serous histology (4.4% vs 0.2% respectively, p < 0.0001 in uterine and 3.5% vs 0.3% respectively, p = 0.0004 in ovarian carcinomas). In three of four patients with serial sampling, mutESR1 emerged under the selective pressure of AI therapy. Despite decreased potency of estrogen receptor (ER) antagonists in transcriptional assays, clinical benefit was observed following treatment with selective ER-targeted therapy, in one case lasting >48 months. CONCLUSIONS: While the prevalence of ESR1 mutations in gynecologic malignancies is low, there are significant clinical implications useful in guiding therapeutic approaches for these cancers.

Change in Topoisomerase 1-Positive Circulating Tumor Cells Affects Overall Survival in Patients with Advanced Breast Cancer after Treatment with Etirinotecan Pegol.

Purpose: Preplanned exploratory analyses were performed to identify biomarkers in circulating tumor cells (CTC) predictive of response to the topoisomerase 1 inhibitor etirinotecan pegol (EP).Experimental Design: The BEACON trial treated patients with metastatic breast cancer (MBC) with EP or treatment of physician's choice (TPC). Blood from 656 of 852 patients (77%) was processed with ApoStream to enrich for CTCs. A multiplex immunofluorescence assay measured expression of candidate response biomarkers [topoisomerase 1 (Top1), topoisomerase 2 (Top2), Ki67, RAD51, ABCG2, γH2AX, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)] in CTCs. Patients were classified as Top1 low (Top1Lo) or Top1 high (Top1Hi) based on median CTC Top1 expression. Correlation of CTC biomarker expression at baseline, cycle 2 day 1 (C2D1), and cycle 4 day 1 with overall survival (OS) was investigated using Cox regression and Kaplan-Meier analyses.Results: Overall, 98% of samples were successfully processed, of which 97% had detectable CTCs (median, 47-63 CTCs/mL; range, 0-2,020 CTCs/mL). Top1, Top2, and TUNEL expression was detected in 52% to 90% of samples; no significant associations with OS were observed in pretreatment samples for either group. EP-treated patients with low C2D1Top1+ CTCs had improved OS compared with those with higher positivity (14.1 months vs. 11.0 months, respectively; HR, 0.7; P = 0.02); this difference was not seen in TPC-treated patients (HR, 1.12; P = 0.48). Patients whose CTCs decreased from Top1Hi to Top1Lo at C2D1 had the greatest OS benefit from EP (HR, 0.57; P = 0.01).Conclusions: CTC Top1 expression following EP treatment may identify patients with MBC most likely to have an OS benefit. Clin Cancer Res; 24(14); 3348-57. ©2018 AACR.

Successful use of next generation genomic sequencing (NGS)-directed therapy of clear cell carcinoma of the ovary (CCCO) with trametinib and metformin in a patient with chemotherapy-refractory disease.

PURPOSE: Compared to other subtypes of epithelial ovarian cancer, clear cell carcinoma of the ovary bears an ominous reputation for chemotherapy resistance, increased relapse rate, and diminished survival. Among patients with distinct histopathologic subtypes, molecular analyses have identified a variety of known drivers of the malignant behavior, and depict a striking heterogeneity. METHODS: A patient with rapidly metastatic CCCO that was refractory to taxane, platinum, pemetrexed, and bevacizumab-based strategies underwent molecular profiling which disclosed dual MAPK and PI3K/AKT/mTOR pathway mutations. RESULTS: Combined targeted therapy with trametinib and metformin resulted in a dramatic disease regression without toxicity. CONCLUSION: The case highlights the utility of precision medicine combining individual molecular diagnosis with rational therapeutic intervention with targeted agents.

Treatment-related protein biomarker expression differs between primary and recurrent ovarian carcinomas.

The molecular characteristics of recurrent ovarian cancers following chemotherapy treatment have been poorly characterized. Such knowledge could impact salvage therapy selection. Since 2008, we have profiled 168 patients' ovarian cancers to determine the expression of proteins that may predict chemotherapy response or are targets for drugs that are in clinical trials for ovarian cancer treatment. Expression of epidermal growth factor receptor (EGFR), HER2, VEGF, ER, c-Met, IGF1R, Ki67, COX2, PGP/MDR1, BCRP, MRP1, excision repair complementation group 1 (ERCC1), MGMT, TS, RRM1, TOPO1, TOP2A, and SPARC was measured by immunohistochemical analyses at Clinical Laboratory Improvement Amendments-certified laboratories. Our univariate analysis of 56 primary and 50 recurrent tumors from patients with advanced stage ovarian serous carcinoma revealed that PGP and ERCC1 were significantly upregulated in recurrent lesions (P < 0.05). To determine whether these or any of the other markers were differentially expressed in specimens obtained from the same individual at diagnosis and at recurrence, we analyzed 43 matched tumor specimens from 19 advanced stage ovarian carcinoma patients. We confirmed the expression differences in PGP and ERCC1 that were observed in the cohort analysis but discovered that the expression levels of BCRP, RRM1, and COX2 were also discordant in more than 40% of the matched tumor specimens. These results may have implications both for the use of biomarkers in therapy selection as well as for their discovery and validation. Expression of these and other candidate response biomarkers must be evaluated in much larger studies and, if confirmed, support the need for profiling of recurrent tumor specimens in future clinical trials.

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.

Anti-EphA2 antibodies decrease EphA2 protein levels in murine CT26 colorectal and human MDA-231 breast tumors but do not inhibit tumor growth.

The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb) that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of EphA2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose cross reactivity with murine EphA2 is unknown, we generated a mAb (Ab20) that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to approximately 12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7). No inhibition of humanMDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies.

Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) in human pancreatic and ovarian tumor xenograft models.

1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) is a deoxycytidine analog that has been shown previously to have impressive anti-proliferative and cytotoxic effects in vitro and in vivo toward colorectal and gastric tumors. In our present studies, the pharmacokinetic behavior in nude mice and the effectiveness of 4'-thio-FAC against human pancreatic and ovarian tumor growth were assessed in comparison with standard chemotherapeutic agents. Potent in vitro anti-proliferative effects were observed against pancreatic (Capan-1, MIA-PaCa-2, BxPC-3) and ovarian (SK-OV-3, OVCAR-3, ES-2) cancer cell lines with IC(50) of 0.01-0.2 microM. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously (s.c.) implanted human pancreatic tumor xenografts or intraperitoneally (i.p.) disseminated human ovarian xenografted tumors. Oral daily administration of 4'-thio-FAC for 8-10 days significantly inhibited the growth of gemcitabine-resistant BxPC-3 pancreatic tumors and induced regression of gemcitabine-refractory Capan-1 tumors. 4'-Thio-FAC was also a highly effective inhibitor of ovarian peritoneal carcinomatosis. In the SK-OV-3 and ES-2 ovarian cancer models, 4'-thio-FAC prolonged survival to a greater extent than that observed with gemcitabine. Furthermore, the superiority of 4'-thio-FAC to carboplatin and paclitaxel was demonstrated in the ES-2 clear cell ovarian carcinoma model. Studies provide evidence that 4'-thio-FAC is a promising new alternative to gemcitabine and other chemotherapeutic drugs in the treatment of a variety of tumor indications, including pancreatic and ovarian carcinoma.

Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.

Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.