Treatment recommendations to cancer patients in the context of FDA guidance for next generation sequencing.

BACKGROUND: Regulatory approval of next generation sequencing (NGS) by the FDA is advancing the use of genomic-based precision medicine for the therapeutic management of cancer as standard care. Recent FDA guidance for the classification of genomic variants based on clinical evidence to aid clinicians in understanding the actionability of identified variants provided by comprehensive NGS panels has also been set forth. In this retrospective analysis, we interpreted and applied the FDA variant classification guidance to comprehensive NGS testing performed for advanced cancer patients and assessed oncologist agreement with NGS test treatment recommendations. METHODS: NGS comprehensive genomic profiling was performed in a CLIA certified lab (657 completed tests for 646 patients treated at Roswell Park Comprehensive Cancer Center) between June 2016 and June 2017. Physician treatment recommendations made within 120 days post-test were gathered from tested patients' medical records and classified as targeted therapy, precision medicine clinical trial, immunotherapy, hormonal therapy, chemotherapy/radiation, surgery, transplant, or non-therapeutic (hospice, surveillance, or palliative care). Agreement between NGS test report targeted therapy recommendations based on the FDA variant classification and physician targeted therapy treatment recommendations were evaluated. RESULTS: Excluding variants contraindicating targeted therapy (i.e., KRAS or NRAS mutations), at least one variant with FDA level 1 companion diagnostic supporting evidence as the most actionable was identified in 14% of tests, with physicians most frequently recommending targeted therapy (48%) for patients with these results. This stands in contrast to physicians recommending targeted therapy based on test results with FDA level 2 (practice guideline) or FDA level 3 (clinical trial or off label) evidence as the most actionable result (11 and 4%, respectively). CONCLUSIONS: We found an appropriate "dose-response" relationship between the strength of clinical evidence supporting biomarker-directed targeted therapy based on application of FDA guidance for NGS test variant classification, and subsequent treatment recommendations made by treating physicians. In view of recent changes at FDA, it is paramount to define regulatory grounds and medical policy coverage for NGS testing based on this guidance.

Evaluation of the National Surgical Quality Improvement Program Universal Surgical Risk Calculator for a gynecologic oncology service.

OBJECTIVES: The National Surgical Quality Improvement Program is aimed at preventing perioperative complications. An online calculator was recently published, but the primary studies used limited gynecologic surgery data. The purpose of this study was to evaluate the performance of the National Surgical Quality Improvement Program Universal Surgical Risk Calculator (URC) on the patients of a gynecologic oncology service. STUDY DESIGN: We reviewed 628 consecutive surgeries performed by our gynecologic oncology service between July 2012 and June 2013. Demographic data including diagnosis and cancer stage, if applicable, were collected. Charts were reviewed to determine complication rates. Specific complications were as follows: death, pneumonia, cardiac complications, surgical site infection (SSI) or urinary tract infection, renal failure, or venous thromboembolic event. Data were compared with modeled outcomes using Brier scores and receiver operating characteristic curves. Significance was declared based on P < 0.05. RESULTS: The model accurately predicated death and venous thromboembolic event, with Brier scores of 0.004 and 0.003, respectively. Predicted risk was 50% greater than experienced for urinary tract infection; the experienced SSI and pneumonia rates were 43% and 36% greater than predicted. For any complication, the Brier score 0.023 indicates poor performance of the model. CONCLUSIONS: In this study of gynecologic surgeries, we could not verify the predictive value of the URC for cardiac complications, SSI, and pneumonia. One disadvantage of applying a URC to multiple subspecialties is that with some categories, complications are not accurately estimated. Our data demonstrate that some predicted risks reported by the calculator need to be interpreted with reservation.

LINE1 and Alu repetitive element DNA methylation in tumors and white blood cells from epithelial ovarian cancer patients.

OBJECTIVE: We determined whether DNA methylation of repetitive elements (RE) is altered in epithelial ovarian cancer (EOC) patient tumors and white blood cells (WBC), compared to normal tissue controls. METHODS: Two different quantitative measures of RE methylation (LINE1 and Alu bisulfite pyrosequencing) were used in normal and tumor tissues from EOC cases and controls. Tissues analyzed included: i) EOC, ii) normal ovarian surface epithelia (OSE), iii) normal fallopian tube surface epithelia (FTE), iv) WBC from EOC patients, obtained before and after treatment, and v) WBC from demographically-matched controls. RESULTS: REs were significantly hypomethylated in EOC compared to OSE and FTE, and LINE1 and Alu methylation showed a significant direct association in these tissues. In contrast, WBC RE methylation was significantly higher in EOC cases compared to controls. RE methylation in patient-matched EOC tumors and pre-treatment WBC did not correlate. CONCLUSIONS: EOC shows robust RE hypomethylation compared to normal tissues from which the disease arises. In contrast, RE are generally hypermethylated in EOC patient WBC compared to controls. EOC tumor and WBC methylation did not correlate in matched patients, suggesting that RE methylation is independently controlled in tumor and normal tissues. Despite the significant differences observed over the population, the range of RE methylation in patient and control WBC overlapped, limiting their specific utility as an EOC biomarker. However, our data demonstrate that DNA methylation is deranged in normal tissues from EOC patients, supporting further investigation of WBC DNA methylation biomarkers suitable for EOC risk assessment.

Expression and clinical significance of the transforming growth factor-ß signalling pathway in endometrial cancer.

AIMS: To evaluate the components of the transforming growth factor (TGF)-ß-Smad signalling pathway in human endometrial cancer (EC). METHODS AND RESULTS: TGF-ß1, TGF-ß receptor type I, TGF-ß receptor type II, Smad2, Smad3, Smad4, Skil and Disabled-2 (DAB2) mRNA levels were determined by reverse transcriptase polymerase chain reaction on EC cell lines and in 70 EC tissues. Immunohistochemistry for Skil and DAB2 antibodies was performed on 362 EC cases. Decreased mRNA levels of all eight components of the TGF-ß pathway tested were found in the majority of 70 cases. For DAB2, the mRNA level was correlated with protein expression level (P = 0.04). The Skil mRNA level was associated with tumour stage (P = 0.03), and the Smad2/3/4 mRNA level with tumour grade (P = 0.03, P = 0.02, and P = 0.00, respectively). The Smad4 mRNA level was also associated with tumour size (P = 0.05), subtype (P = 0.04), and disease-free survival (DFS) (P = 0.05). The TGF-ß1 mRNA level was associated with DFS (P = 0.04). Finally, tumours with positive Skil protein expression had a shorter recurrence time, whereas, those with positive DAB2 protein expression had a longer recurrence time. CONCLUSIONS: Down-regulation of the TGF-ß-Smad signalling pathway might be responsible for the pathogenesis of human EC, and some of its components appeared to be prognostic factors. Exploration of future therapy targeting the TGF-ß-Smad pathway is warranted in EC.

Efficacy and Safety of Pembrolizumab in Combination With Bevacizumab and Oral Metronomic Cyclophosphamide in the Treatment of Recurrent Ovarian Cancer: A Phase 2 Nonrandomized Clinical Trial.

IMPORTANCE: Treatment options for recurrent ovarian cancer are of limited clinical benefit and adversely affect patient quality of life, representing an unmet need for tolerable effective therapies. OBJECTIVE: To assess the efficacy and safety of a combination of pembrolizumab with bevacizumab and oral metronomic cyclophosphamide in patients with recurrent platinum-sensitive, platinum-resistant, or refractory epithelial ovarian, fallopian tube, or primary peritoneal cancer. DESIGN, SETTING, AND PARTICIPANTS: This open-label, single-arm phase 2 cohort study enrolled patients from September 6, 2016, to June 27, 2018, at a single institution in the United States. Eligible patients had recurrent ovarian cancer, measurable disease per immune-related Response Evaluation Criteria In Solid Tumors (irRECIST), and Eastern Cooperative Oncology Group performance status of 0 to 1. Data were analyzed from September 6, 2016, to February 20, 2020. INTERVENTIONS: Patients received intravenous pembrolizumab, 200 mg, and bevacizumab, 15 mg/kg, every 3 weeks and oral cyclophosphamide, 50 mg, once daily during the treatment cycle until disease progression, unacceptable toxic effects, or withdrawal of consent. MAIN OUTCOMES AND MEASURES: Primary outcomes were objective response rate (ORR) and progression-free survival (PFS). RESULTS: Of the 40 women enrolled, 30 (75.0%) had platinum-resistant and 10 (25.0%) had platinum-sensitive ovarian cancer with a mean (SD) age of 62.2 (9.4) years. Three women (7.5%) had complete responses, 16 (40.0%) had partial responses, and 19 (47.5%) had stable disease in response to treatment based on irRECIST criteria, with an ORR of 47.5%, clinical benefit in 38 (95.0%), and durable response in 10 (25.0%). Median PFS was 10.0 (90% CI, 6.5-17.4) months. The most common grade 3 to 4 treatment-related adverse events were hypertension (6 [15.0%]) and lymphopenia (3 [7.5%]). The most frequently reported adverse events included fatigue (18 [45.0%]), diarrhea (13 [32.5%]), and hypertension (11 [27.5%]). CONCLUSIONS AND RELEVANCE: In this phase 2 nonrandomized clinical trial, the combination of pembrolizumab with bevacizumab and oral cyclophosphamide was well tolerated and demonstrated clinical benefit in 95.0% and durable treatment responses (>12 months) in 25.0% of patients with recurrent ovarian cancer. This combination may represent a future treatment strategy for recurrent ovarian cancer. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02853318.

Regulation of cancer germline antigen gene expression: implications for cancer immunotherapy.

Cancer germline (CG; also known as cancer-testis) antigen genes are normally expressed in germ cells and trophoblast tissues and are aberrantly expressed in a variety of human malignancies. CG antigen genes have high clinical relevance as they encode a class of immunogenic and highly selective tumor antigens. CG antigen-directed immunotherapy is undergoing clinical evaluation for the treatment of a number of solid tumor malignancies and has been demonstrated to be safe, provoke immune responses and be of therapeutic benefit. Achieving an improved understanding of the mechanisms of CG antigen gene regulation will facilitate the continued development of targeted therapeutic approaches against tumors expressing these antigens. Substantial evidence suggests epigenetic mechanisms, particularly DNA methylation, as a primary regulator of CG antigen gene expression in normal and cancer cells as well as in stem cells. The roles of sequence-specific transcription factors and signal transduction pathways in controlling CG antigen gene expression are less clear but are emerging. A combinatorial therapeutic approach involving epigenetic modulatory drugs and CG antigen immunotherapy is suggested based on these data and is being actively pursued. In this article, we review the mechanisms of CG antigen gene regulation and discuss the implications of these mechanisms for the development of cancer immunotherapy approaches targeting CG antigens.

Global DNA Hypomethylation in Epithelial Ovarian Cancer: Passive Demethylation and Association with Genomic Instability.

A hallmark of human cancer is global DNA hypomethylation (GDHO), but the mechanisms accounting for this defect and its pathological consequences have not been investigated in human epithelial ovarian cancer (EOC). In EOC, GDHO was associated with advanced disease and reduced overall and disease-free survival. GDHO (+) EOC tumors displayed a proliferative gene expression signature, including FOXM1 and CCNE1 overexpression. Furthermore, DNA hypomethylation in these tumors was enriched within genomic blocks (hypomethylated blocks) that overlapped late-replicating regions, lamina-associated domains, PRC2 binding sites, and the H3K27me3 histone mark. Increased proliferation coupled with hypomethylated blocks at late-replicating regions suggests a passive hypomethylation mechanism. This hypothesis was further supported by our observation that cytosine DNA methyltransferases (DNMTs) and UHRF1 showed significantly reduced expression in GDHO (+) EOC after normalization to canonical proliferation markers, including MKI67. Finally, GDHO (+) EOC tumors had elevated chromosomal instability (CIN), and copy number alterations (CNA) were enriched at the DNA hypomethylated blocks. Together, these findings implicate a passive DNA demethylation mechanism in ovarian cancer that is associated with genomic instability and poor prognosis.

Oncologist uptake of comprehensive genomic profile guided targeted therapy.

We describe the extent to which comprehensive genomic profiling (CGP) results were used by oncologists to guide targeted therapy selection in a cohort of solid tumor patients tested as part of standard care at Roswell Park Comprehensive Cancer Center June 2016-June 2017, with adequate follow up through September 2018 (n = 620). Overall, 28.4% of CGP tests advised physicians about targeted therapy use supported by companion diagnostic or practice guideline evidence. Post-test targeted therapy uptake was highest for patients in active treatment at the time of order (86% versus 76% of treatment naïve patients), but also took longer to initiate (median 50 days versus 7 days for treatment naïve patients), with few patients (2.6%) receiving targeted agents prior to testing. 100% of patients with resistance variants did not receive targeted agents. Treatment naïve patients received immunotherapy as the most common alternative. When targeted therapy given off-label or in a trial was the best CGP option, (7%) of patients received it. Our data illustrate the appropriate and heterogeneous use of CGP by oncologists as a longitudinal treatment decision tool based on patient history and treatment needs, and that some patients may benefit from testing prior to initiation of other standard treatments.

Ultrarestrictive Opioid Prescription Protocol for Pain Management After Gynecologic and Abdominal Surgery.

Importance: Opioids are routinely prescribed for postoperative home pain management for most patients in the United States, with limited evidence of the amount needed to be dispensed. Opioid-based treatment often adversely affects recovery. Prescribed opioids increase the risk of chronic opioid use, abuse, and diversion and contribute to the current opioid epidemic. Objective: To evaluate whether after hospital discharge, postsurgical acute pain can be effectively managed with a markedly reduced number of opioid doses. Design, Setting, and Participants: In this case-control cohort study, an ultrarestrictive opioid prescription protocol (UROPP) was designed and implemented from June 26, 2017, through June 30, 2018, at a single tertiary-care comprehensive cancer center. All patients undergoing gynecologic oncology surgery were included. Patients undergoing ambulatory or minimally invasive surgery (laparoscopic or robotic approach) were not prescribed opioids at discharge unless they required more than 5 doses of oral or intravenous opioids while in the hospital. Patients who underwent a laparotomy were provided a 3-day opioid pain medication supply at discharge. Main Outcomes and Measures: Total number of opioid pain medications prescribed in the 60-day perioperative period, requests for opioid prescription refills, and postoperative pain scores and complications were evaluated. Factors associated with increased postoperative pain, preoperative and postoperative pain scores, inpatient status, prior opioid use, and all opioid prescriptions within the 60-day perioperative window were monitored among the case patients and compared with those from consecutive control patients treated at the center in the 12 months before the UROPP was implemented. Results: Patient demographics and procedure characteristics were not statistically different between the 2 cohorts of women (605 cases: mean [SD] age, 56.3 [14.5] years; 626 controls: mean [SD] age, 55.5 [13.9] years). The mean (SD) number of opioid tablets given at discharge after a laparotomy was 43.6 (17.0) before implementation of the UROPP and 12.1 (8.9) after implementation (P < .001). For patients who underwent laparoscopic or robotic surgery, the mean (SD) number of opioid tablets given at discharge was 38.4 (17.4) before implementation of the UROPP and 1.3 (3.7) after implementation (P < .001). After ambulatory surgery, the mean (SD) number of opioid tablets given at discharge was 13.9 (16.6) before implementation of the UROPP and 0.2 (2.1) after implementation (P < .001). The mean (SD) perioperative oral morphine equivalent dose was reduced to 64.3 (207.2) mg from 339.4 (674.4) mg the year prior for all opioid-naive patients (P < .001). The significant reduction in the number of dispensed opioids was not associated with an increase the number of refill requests (104 patients [16.6%] in the pre-UROPP group vs 100 patients [16.5%] in the post-UROPP group; P = .99), the mean (SD) postoperative visit pain scores (1.1 [2.2] for the post-UROPP group vs 1.4 [2.3] for pre-UROPP group; P = .06), or the number of complications (29 cases [4.8%] in the post-UROPP group vs 42 cases [6.7%] in the pre-UROPP group; P = .15). Conclusions and Relevance: Implementation of a UROPP was associated with a significant decrease in the overall amount of opioids prescribed to patients after gynecologic and abdominal surgery at the time of discharge for all patients, and for the entire perioperative time for opioid-naive patients without changes in pain scores, complications, or medication refill requests.

DNA Methylome Analyses Implicate Fallopian Tube Epithelia as the Origin for High-Grade Serous Ovarian Cancer.

UNLABELLED: High-grade serous ovarian cancer (HGSC) is the most common and lethal form of epithelial ovarian cancer (EOC). Two distinct tissues have been suggested as the tissue of origin: ovarian surface epithelia (OSE) and fallopian tube epithelia (FTE). We hypothesized that the DNA methylome of HGSC should more closely resemble the methylome of its tissue of origin. To this end, we profiled HGSC (n = 10), and patient-matched OSE and FTE (n = 5) primary fresh-frozen tissues, and analyzed the DNA methylome using Illumina 450K arrays (n = 20) and Agilent Sure Select methyl-seq (n = 7). Methylomes were compared using statistical analyses of differentially methylated CpG sites (DMC) and differentially methylated regions (DMR). In addition, methylation was evaluated within a variety of different genomic contexts, including CpG island shores and Homeobox (HOX) genes, due to their roles in tissue specification. Publicly available HGSC methylome data (n = 628) were interrogated to provide additional comparisons with FTE and OSE for validation. These analyses revealed that HGSC and FTE methylomes are significantly and consistently more highly conserved than are HGSC and OSE. Pearson correlations and hierarchal clustering of genes, promoters, CpG islands, CpG island shores, and HOX genes all revealed increased relatedness of HGSC and FTE methylomes. Thus, these findings reveal that the landscape of FTE more closely resembles HGSC, the most common and deadly EOC subtype. IMPLICATIONS: DNA methylome analyses support the hypothesis that HGSC arise from the fallopian tube and that due to its tissue-specificity and biochemical stability, interrogation of the methylome may be a valuable approach to examine cell/tissue lineage in cancer. Mol Cancer Res; 14(9); 787-94. ©2016 AACR.

Synuclein-γ (SNCG) expression in ovarian cancer is associated with high-risk clinicopathologic disease.

BACKGROUND: Synuclein gamma (SNCG) expression is associated with advanced disease and chemoresistance in multiple solid tumors. Our goal was to determine if SNCG protein expression in ovarian cancer was correlated with clinicopathologic variables and patient outcomes. METHODS: Tissue microarrays from primary tumors of 357 ovarian, fallopian tube, and primary peritoneal cancer patients, who underwent primary surgery at Roswell Park Cancer Institute between 1995 and 2007, were immunohistochemically stained for SNCG. A pathologist blinded to patient data scored tumors as positive if ≥10 % of the sample stained for SNCG. Medical records were reviewed for clinicopathologic and demographic variables. Between the positive and negative groups, Wilcoxon rank-sum test was used to compare the median ages and Fisher's exact test was used to compare groups in categorical variables. Cox proportional hazard models examined associations between SNCG and overall and progression-free survival. RESULTS: The median follow-up was 36 months, median overall survival was 39 months, and median progression-free survival was 18 months. SNCG presence was associated with clinical variables of serous histology, grade 3 disease, suboptimal debulking, ascites at surgery, FIGO stage III-IV cancer, or initial CA-125 level >485. There was no significant difference in overall survival (HR 1.06 95 % CI 0.81-1.39 P 0.69) or progression-free survival (HR 1.16 95 % CI 0.89-1.50 P 0.28) for patients with or without SNCG expression. CONCLUSIONS: SNCG expression in ovarian cancer is frequent in patients with high-risk features, but it does not correlate with chemotherapy response, overall survival, or progression-free survival.

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.

Epigenetic potentiation of NY-ESO-1 vaccine therapy in human ovarian cancer.

The cancer-testis/cancer-germline antigen NY-ESO-1 is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase (DNMT) inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Pre-clinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1-specific CTL-mediated responses. Based on these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome (doxorubicin) chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in 6/10 evaluable patients. Based on these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted.

Coordinated cancer germline antigen promoter and global DNA hypomethylation in ovarian cancer: association with the BORIS/CTCF expression ratio and advanced stage.

PURPOSE: Cancer germline (CG) antigens are frequently expressed and hypomethylated in epithelial ovarian cancer (EOC), but the relationship of this phenomenon to global DNA hypomethylation is unknown. In addition, the potential mechanisms leading to DNA hypomethylation, and its clinicopathologic significance in EOC, have not been determined. EXPERIMENTAL DESIGN: We used quantitative mRNA expression and DNA methylation analyses to determine the relationship between expression and methylation of X-linked (MAGE-A1, NY-ESO-1, XAGE-1) and autosomal (BORIS, SOHLH2) CG genes, global DNA methylation (5mdC levels, LINE-1, Alu, and Sat-α methylation), and clinicopathology, using 75 EOC samples. In addition, we examined the association between these parameters and a number of mechanisms proposed to contribute to DNA hypomethylation in cancer. RESULTS: CG genes were coordinately expressed in EOC and this was associated with promoter DNA hypomethylation. Hypomethylation of CG promoters was highly correlated and strongly associated with LINE-1 and Alu methylation, moderately with 5mdC levels, and rarely with Sat-α methylation. BORIS and LINE-1 hypomethylation, and BORIS expression, were associated with advanced stage. GADD45A expression, MTHFR genotype, DNMT3B isoform expression, and BORIS mRNA expression did not associate with methylation parameters. In contrast, the BORIS/CTCF expression ratio was associated with DNA hypomethylation, and furthermore correlated with advanced stage and decreased survival. CONCLUSIONS: DNA hypomethylation coordinately affects CG antigen gene promoters and specific repetitive DNA elements in EOC, and correlates with advanced stage disease. The BORIS/CTCF mRNA expression ratio is closely associated with DNA hypomethylation and confers poor prognosis in EOC.