Abstract: 10530 Background: Tumor profiling is an emergent technique to determine tissue of origin in CUP patients. However, the value of these predictions in improving treatment efficacy is unknown. In this prospective trial, we used tumor profiling results to direct site-specific therapy for CUP pts. Methods: A 92-gene RT-PCR assay (CancerTYPE ID; bioTheranostics, Inc.) was performed on tumor biopsies from previously untreated CUP pts who consented. When a tissue of origin was predicted, pts who were treatment candidates were assigned standard site-specific first-line therapy. Results: Between 10/08 and 12/11, 289 pts were enrolled, 252 had successful assays performed, and 247 (98%) had a tissue of origin predicted. 224 pts were eligible for treatment; 197 pts received assay-directed treatment. 120 of 224 treated pts (54%) had assay diagnoses of tumor types known to derive substantial benefit from standard site-specific treatment (bladder 27, colorectal 26, NSCLC 24, breast 10, ovary 10, kidney 9, prostate 4, germ cell 4, others 6 (3 sites), while 104 pts (46%) had assay diagnoses of relatively resistant tumors (biliary tract 45, pancreas 12, gastroesophageal 10, liver 7, sarcoma 5, cervix 5, others 20 (8 sites). Median OS for all treated pts was 10.8 months (mos); OS for 197 pts with assay-directed treatment was 12.2 mos (versus 6.0 mos for 27 pts receiving empiric therapy). Median OS was better in the 120 pts with assay diagnoses of more responsive tumor types (12.8 vs 7.4 mos; p = .027). Median OS (mos) in specific subgroups: pancreas 9, kidney 12, colon 12, NSCLC 16, ovary 30. Conclusions: This is the first prospective trial in which molecular profiling has directed site-specific therapy in CUP pts. Assay-directed therapy in 197 pts produced a median OS (12.2 mos) that compares favorably with previous empiric CUP therapy. CUP pts predicted to have more responsive tumor types had longer survival compared to less responsive types, suggesting accurate identification by the assay. These results strengthen the rationale for molecular profiling in CUP management.

Abstract: Liquid biopsies represent an attractive alternative to tissue biopsies, particularly rebiopsies, in determining patient eligibility for targeted therapies. Clinical utility of urine genotyping, however, has not been explored extensively. We evaluated epidermal growth factor receptor ( EGFR) T790M detection in matched urine, plasma, and tissue and the clinical outcomes of patients with advanced non–small-cell lung cancer treated with rociletinib. Methods Tissue (n = 540), plasma (n = 482), and urine (n = 213) were collected from evaluable patients enrolled in TIGER-X, a phase I/II study. Genotyping was performed by therascreen EGFR testing in tissue, BEAMing in plasma, and a quantitative short footprint assay (Trovera) in urine, which was used to further examine discordant samples. Results Positive percent agreement with tissue T790M results was similar for urine (82%; 142 of 173) and plasma (81%; 313 of 387) genotyping. Urine and plasma together identified more patients who were T790M positive (92%) than tissue alone (83%) among matched samples (n = 177). The ability to identify mutations in plasma was strongly associated with M stage ( P 〈 .001); rate of T790M detection for patients with M1a/M0 disease increased from 54% for plasma alone to 85% when urine and plasma were both examined. Objective response rates of patients who were T790M positive were comparable between tumor (34%), plasma (32%), and urine (37%). Conclusion Clinical response to rociletinib was comparable irrespective of whether T790M status was identified by liquid or tissue biopsy. Combined, urine and plasma identified a higher percentage of patients who were T790M positive than tumor genotyping alone and improved detection of T790M, particularly in the absence of distant metastases. These findings support the noninvasive analysis of urine and plasma before tumor rebiopsy when assessing T790M status.

Abstract: In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes. Cancer Res; 70(6); 2158–64

Abstract: Background: Human papillomavirus (HPV) testing from clinician-collected cervical and self-collected cervico-vaginal samples is more sensitive for detecting CIN2+/CIN3+ than cytology-based screening, stimulating interest in HPV testing from urine. The objective was to determine the performance of the Trovagene HPV test for the detection of CIN2+ from urine and PreservCyt cervical samples. Methods: Women referred for colposcopy at St Mary's Hospital (London, United Kingdom), following abnormal cytology, were recruited to this diagnostic accuracy study by convenience sampling (September 2011 to April 2013). A total of 501 paired urine and cervical samples were collected. Primary outcomes were sensitivity for CIN2+/CIN3+ and specificity for & lt;CIN2; secondary outcomes were comparisons with other HPV tests and agreement/kappa values between urine and cervical samples. Results: Trovagene HPV test sensitivity and specificity from PreservCyt were similar to well-established tests [sensitivity for CIN3+ (n = 145) 96.3% (95% confidence interval (CI), 89.6–99.2); CIN2+ (n = 81) 94.5% (95% CI, 89.4–97.6); specificity for & lt;CIN2 25.3% (95% CI, 20.8–30.1)]. Sensitivity from urine was slightly, but not significantly, lower [CIN3+ 91.4% (95% CI, 83.0–96.5), P = 0.3; CIN2+ 88.3% (95% CI, 81.9–93.0), P = 0.06] . Specificity for & lt;CIN2 was similar: 24.7% (95% CI, 20.3–29.5), P = 0.9. A total of 403 Trovagene cervical and 396 urine HPV tests were positive. Overall agreement between paired samples was 82.6% (95% CI, 79.3–86.0). Conclusions: Trovagene HPV test's performance on PreservCyt cervical samples was comparable with established HPV tests. Sensitivity in urine, although slightly lower, may nevertheless be adequate for self-sampling. This referral population's higher HPV positivity rate affects specificity, warranting further studies in a screening population. Impact: This may prove useful for women not attending for cervical screening. Cancer Epidemiol Biomarkers Prev; 26(7); 1053–9. ©2017 AACR.

Abstract: Langerhans Cell Histiocytosis (LCH) and Erdheim-Chester Disease (ECD) are systemic histiocytic disorders characterized by infiltration of histiocytes in multiple tissues of the body leading to organ compromise. Although the underlying etiology of these conditions has long been enigmatic, recent investigations have determined that LCH and ECD are clonal disorders of myeloid-derived precursor cells with a high frequency of BRAFV600E mutations (40-60% of patients). Moreover, treatment of BRAF-mutant LCH and ECD patients with the BRAF inhibitor vemurafenib has demonstrated dramatic efficacy. The above data underline the importance of accurately identifying BRAF mutational status in patients with systemic LCH and ECD. Unfortunately, the scant histiocyte content and marked stromal contamination, which are a hallmark of these disorders, make mutation detection in tissue biopsies challenging. Moreover,the propensity of histiocytic lesions to involve difficult to biopsy locations frequently necessitates the use of bone biopsies further limiting the availability of suitable tumor material for BRAF genotyping. We therefore initiated a prospective, blinded, multicenter study of BRAFV600E mutation detection in the cell-free DNA (cfDNA) from plasma and urine of histiocytosis patients to determine the sensitivity/specificity of cfDNA mutation detection compared with tissue biopsy and to track disease burden serially with therapy. Between January 2013 and June 2014, 30 consecutive patients with ECD (n=25) and LCH (n=5) were enrolled from Memorial Sloan Kettering Cancer Center and MD Anderson Cancer Center. Initial BRAF tissue mutation testing on tissue biopsies was performed by a variety of methods as part of routine care in CLIA-certified molecular diagnostic laboratories. We applied a droplet-digitial PCR assay (RainDrop ddPCR) for quantitative detection of the BRAFV600E mutation in plasma and urine cell-free cfDNA in all patients. Of 30 patients enrolled, initial tissue BRAFV600E genotyping identified 15 patients to be mutant, 6 patients as wildtype, and 9 as indeterminate. There was 100% concordance between tissue and urinary cfDNA genotype in samples from treatment naïve patients (sensitivity 92.9%, specificity 100%, positive predictive value 100%, negative predictive value 85.7% compared to tissue biopsy detection). Urinary cfDNA analysis identified 5 patients as being BRAFV600E mutant that were not known to have the BRAF mutation previously (Figure A). Subsequent tissue biopsy was performed in 2 of these patients and identified the BRAFV600E mutation, allowing both patients to enroll in an ongoing phase II study of vemurafenib. Results from plasma cfDNA for identifying the BRAFV600E mutation were comparable to urinary cfDNA results Next examining serial samples during therapy, a significant decrease in the cfDNA BRAFV600E:BRAF wildtype ratio was seen with therapy compared with pretreatment samples (p 〈 0.0001). In all 10 patients treated with a BRAF inhibitor, serial urinary cfDNA analysis revealed progressive decrements in the BRAFV600E allele burden. Weekly serial urinary cfDNA analysis throughout the course of BRAF inhibitor therapy revealed that the decline in mutant cfDNA burden in response to BRAF inhibitors was consistent with radiographic disease improvement (Figure B). Moreover, in at least one patient where successful RAF inhibitor therapy was discontinued for toxicity, urinary cfDNA BRAFV600E burden increased after vemurafenib discontinuation which mirrored radiographic evidence of disease recurrence. Finally, 2 patients treated with the IL1-receptor antagonist anakinra exhibited substantial decreases in BRAFV600E burden, highlighting an effect of IL-1 receptor antagonism in BRAF-mutant histiocytes. These results indicate that cfDNA BRAFV600E mutational analysis in plasma and urine provides a convenient and reliable method of detecting mutational status and can serve as a non-invasive biomarker to monitor response to therapy in LCH and ECD. Moreover, the dynamic results achieved with cfDNA analysis allowed for monitoring of disease recurrence with treatment cessation. Of note, this study represents the largest prospective study of adult ECD patients to date. Moreover, these data represent the first evidence of the effect of RAF inhibition as well as IL1-receptor antagonism on the BRAF mutant clone in patients with histiocytosis. Figure 1 Figure 1. Disclosures Vibat: Trovagene Inc.: Employment. Hassaine:Trovagene Inc.: Employment. Poole:Trovagene Inc.: Employment. Lu:Trovagene Inc.: Employment. Erlander:Trovagene Inc.: Employment. Janku:Trovagene Inc.: Consultancy, Research Funding.

Abstract: Background: PLK1 is a serine/threonine kinase and master regulator of G2/M cell-cycle progression. PLK1 is overexpressed in numerous cancer types including AML and inhibition induces mitotic arrest and cell death. ONV (PCM-075) is an orally active, highly selective PLK1 inhibitor with significant activity in preclinical AML models as a single agent and in combination with cytarabine. Here we provide an updated analysis of the ongoing Phase 1b dose-escalation trial (NCT03303339), evaluating the safety, efficacy and correlative analyses of ONV in combination with either LDAC or DEC in R/R AML. Methods: R/R AML pts are treated with ONV for 5 days in combination with LDAC (20 mg/m2 SC qd x 10d) or DEC (20 mg/m2 IV qd x 5d) within a 21 to 28-day cycle. Dose escalation is in 50% increments, and dose limiting toxicities (DLTs) are evaluated at the end of cycle 1. Efficacy is evaluated using modified International Working Group 2003 criteria. We evaluated PLK1 inhibition and gene expression changes with treatment. Based on preclinical studies, PLK1 activity can be monitored in vivo through changes in phosphorylation of its direct substrate: the translationally controlled tumor protein (TCTP). Blood samples are collected on day 1 at pre-dose and 3h post-dose (corresponding to ONV ≈ Cmax), pTCTP and TCTP are assessed by capillary Western-Blot in isolated peripheral mononuclear cells and biomarker positivity is defined as ≥ 35% decrease in pTCTP/TCTP at 3h versus pre-dose. In addition, blood samples are collected on day 1 at pre-dose, 3h and 24h post-dose for gene expression analysis (RNA-seq). Results: As of 22Jul2019, 37 pts were treated (20 DEC, 17 LDAC) with the ONV dose escalated in 3-subject cohorts from 12, 18, 27, 40, 60 to an ongoing 90mg/m2 cohort. No DLTs or drug-related deaths were reported. Adverse events (AEs, all grades) reported in ≥10% pts (n=37) included anemia (11 pts), fatigue (11), thrombocytopenia (10), febrile neutropenia (9), neutropenia (9), dyspnea (8) and nausea (8). Grade 3 and 4 AEs possibly related to ONV were hematological AEs (12), elevated bilirubin (1) and stomatitis (1). Preliminary efficacy for the 30 evaluable patients in the Phase 1b showed 4 pts achieving objective responses: Complete response (CR,n=2) or CR with incomplete count recovery (CRi,n=1) in the DEC arm (27 and 40mg/m2) and CR with incomplete platelet count recovery (CRp,n=1) in the LDAC arm (40mg/m2). Among responders, 3 pts remain on treatment with ongoing responses for ≥100 days. Fourteen (45%) of the 31 evaluable pts in the Phase 1b were biomarker positive (achieved a ≥ 35% reduction in pTCTP upon treatment). pTCTP inhibition was not correlated with ONV dose level, individual pts pharmacokinetics (AUC0-24), or % circulating blasts. However, response to treatment was significantly associated with biomarker positivity with all 4 responding pts being in the biomarker positive group (n=11). In contrast, no responses were observed in the biomarker negative group (n=15) (p=0.02). Whole transcriptomic comparison between biomarker negative (n=11) and positive pts (n=11) indicated a significant enrichment for increased expression of oxidative phosphorylation (OXPHOS) hallmark genes within the biomarker positive pts (p = 0.009). Interestingly, OXPHOS gene expression was downregulated at 3h and 24h after treatment in the biomarker positive but not the biomarker negative group. Finally, to determine whether the underlying tumor biology that is classified as biomarker positive is associated with pt outcome, a predictive gene expression signature for biomarker positive pts was developed and subsequently tested as a prognostic for R/R pts from the BeatAML dataset; this predictive signature identified patients with decreased overall survival (p=0.0005). Conclusion: In the Phase 1b, the combination of ONV with either LDAC or DEC demonstrated safety, tolerability and preliminary efficacy. Biomarker positivity was observed in a subset of pts, possibly related to dependency of the tumor on PLK1 activity, and was associated with objective response. In addition, gene expression data suggest that biomarker positive pts have a worse prognosis and an underlying OXPHOS-dependency of the tumor. The OXPHOS-dependent AML population has been shown to be resistant to chemotherapy (Farge et al., Cancer Discov. 2017 Jul 1;7(7):716-35 ), therefore this hard-to treat population could potentially benefit from the addition of ONV. Disclosures Zeidan: BeyondSpring: Honoraria; Seattle Genetics: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Agios: Honoraria; Ariad: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Astellas: Honoraria; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding. Schiller:Amgen: Other, Research Funding; Agios: Research Funding, Speakers Bureau; Astellas: Research Funding; Biomed Valley Discoveries: Research Funding; Bristol Myer Squibb: Research Funding; Celgene: Research Funding, Speakers Bureau; Constellation Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding; Eli Lilly and Company: Research Funding; FujiFilm: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; Incyte: Research Funding; J & J: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Onconova: Research Funding; Pfizer Pharmaceuticals: Equity Ownership, Research Funding; Sangamo Therapeutics: Research Funding. Lin:Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Becker:AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; The France Foundation: Honoraria; Accordant Health Services/Caremark: Consultancy. Patel:France Foundation: Honoraria; Dava Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Wang:Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role. Spira:MedImmune: Research Funding; Abbvie: Research Funding; Newlink Genetics: Research Funding; Astellas Pharma: Research Funding; Virginia Cancer Specialists: Employment; Novartis: Research Funding; BMS: Consultancy; Incyte: Research Funding; Roche: Research Funding; ADC Therapeutics: Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Ridinger:Trovagene: Employment. Croucher:Trovagene: Employment. Erlander:Trovagene: Employment. Silberman:Trovagene: Consultancy; Moleculin Biotech: Employment.

Abstract: 369 Background: The majority of metastatic castration-resistant prostate cancer (mCRPC) patients will become resistant to abiraterone. Drug combinations with abiraterone offer the possibility to extend patient benefit to anti-androgen therapy. PLK1, an essential mitotic kinase for onset of G2/M transition and cytokinesis, is over-expressed in prostate cancer, correlated positively with Gleason grade and PLK1 RNAi induces G2/M arrest and apoptosis in prostate cancer cells. PCM-075 (formerly NMS-1286937) is a potent, highly selective PLK1 inhibitor currently in clinical trials for the treatment of acute myeloid leukemia. Combination of PCM-075 with abiraterone was examined in mCRPC models and other cancer types for synergistic interaction. Methods: We used a variety of tumor cell lines (C4-2, LNCaP, BT-20, AU565, SK-OV-3), animal and computational modeling techniques to elucidate the mechanism and assess the potential clinical utility of synergistic tumor cell killing by PLK1 inhibitors (PLK1i), including PCM-075, with abiraterone. Results: Synergy was observed between abiraterone and PLK1i, including PCM-075, in models of CRPC (C4-2). In addition, PLK1i and abiraterone were synergistic in murine C4-2 xenografts. Unexpectedly, PLK1i sensitized a subset of non-prostate cancer cells to abiraterone, including AR-negative breast and ovarian cancer cells. In contrast to PLK1i, other mitotic arrest agents do not synergize with abiraterone. To elucidate the molecular basis this synergy, we screened a panel of cell lines and implemented a new computational method that links RNA expression patterns to drug combination synergy. This analysis implicated signaling through the retinoic acid (RA) nuclear receptors as a component of this synergy, which was validated experimentally. Increased basal expression of genes involved in RA metabolism in cancer cells is associated with increased synergy. Conclusions: Abiraterone has AR-independent effects that provide a mechanism and biomarkers predictive for synergistic killing when combined with the PLK1i PCM-075. This combination has potential to extend mCRPC patient benefit to abiraterone regardless of AR status.