Sosa, Carlos

201 Background: Previous studies have evaluated peripheral whole blood mRNA to identify prognostic biomarkers in metastatic prostate cancer, with no consistent genes of prognostic significance between studies and sample collection done at various points of therapy. We aim to identify the prognostic value of 20 previously identified genes in patients mCRPC from a prospective clinical trial. Methods: Between June 2013 and August 2015, whole blood was prospectively collected in PAXgene RNA tubes from 92 men with mCRPC (NCT01953640). Gene expression from samples with RIN > 4.0 were quantified using a customized NanoString CodeSet of 20 candidate genes (C1QA, MCM2, STOM, GABARAPL2, R10K3, HMBS, TFDP1, CDKN1A, ITGAL, TMCC2, TERF2IP, MMP9, SNCA, TIMP1, SLC4A1, SELENBP1, TMEM66, SEMA4D, PROS1, CD22). The raw data from the nCounter Analysis system were normalized to the positive controls and log2 transformed. Univariable Cox proportional hazards models were performed on each target gene sufficiently above negative control (5 times the maximum value of the negative control) to evaluate associations with overall survival (OS). For each gene we considered a linear, quadratic, or categorical model with quantile cutpoints. Categorical models with cutpoints at the 25th percentile for the genes CD22, GABARAPL2, R10K3, and STOM were used as there was a nonlinear relationship between gene expression and death hazard. The remaining genes were modeled with a linear model. Findings were considered statistically significant based on p-values adjusted using the Holm-Bonferroni correction. Results: 18/20 genes were identified and passed quality control (QC) measures, which excluded C1QA and MCM2. 72/92 men had an evaluable pretreatment specimen that passed QC. The median age was 72 years. The study cohort was evenly balanced in terms of Gleason score ( < = 7 v. > 7) and 60% had high volume disease. The median follow-up time was 5.73 years (IQR: 5.39, 6.49) and 58 patients had died. Of the 18 genes evaluated, STOM (Stomatin) [hazard ratio (HR) 3.00, adjusted p = 0.007] and GABARAPL2 (GABA Type A Receptor Associated Protein Like 2) [HR 2.62, adjusted p = 0.033] were significantly associated with overall survival in univariate analysis. In multivariate analysis, after adjusting for volume of metastatic disease and Gleason score, STOM [HR 2.89, adjusted p = 0.014] was significantly associated with overall survival. Conclusions: Elevated whole blood mRNA expression of STOM is adversely associated with OS in mCRPC. Further studies investigating elucidating the molecular significance of STOM in mCRPC are planned. Clinical trial information: NCT01953640 .

e16559 Background: ARVs that develop early during treatment with abiraterone acetate/ prednisone (AA/P) may play a role in treatment resistance. We evaluated metastatic site and whole blood mRNA expression of full-length AR (AR-FL) and ARVs to characterize treatment-emergent ARVs (TE-ARVs) from men with mCRPC pre- and post- 12 weeks of AA/P collected in a prospective clinical trial (NCT#01953640). Methods: RNAseq performed on paired metastatic site biopsy (n = 40) and whole blood (n = 25) passed quality control. Reads were aligned to the GRCh38 reference genome with the spliced-alignment TopHat2 package. AR-FL or ARVs were detected if ≥ 2 splice reads aligned to unique splice junctions for AR-FL, AR-8, AR-45, AR-23, AR-V3, AR-V5V6, AR-V7, AR-V8, AR-V9, AR-V10, AR-V12, AR-V13, and AR-V14, and normalized to splice reads per million (SRPM). Cox proportional hazard regression analysis was performed on AR-FL and AR-Vs with ≥1 SRPM for association with time to treatment change (TTTC). Results: In metastatic site biopsies post-AA/P, the average number of splice reads was 27,376,541 (range 7,753,998 to 62,456,773). The median number of ARVs detected was 2 (range 0-8), with a total of total of 110 ARVs identified (Table). Dynamic shifts in ARV profiles were observed post-AAP, with 41 TE-ARVs identified in 17 unique patients. The most common TE-ARV was AR-8 (n = 8), followed by AR-45 (n = 5), AR-23 (n = 5), and AR-V7 (n = 4). The presence of AR-V7 post-AAP was adversely associated with TTTC (hazard ratio 2.46, p = 0.013). The identification of early TE- ARV was not associated with TTTC. In whole blood samples, post-AA/P detection of AR-FL was low (n = 3) and no ARVs were detected. Conclusions: No specific patterns were observed in ARV profiles obtained in the metastatic biopsies after 12 weeks of treatment with AA/P. Clinical trial information: 01953640. [Table: see text]

5058 Background: The detection of full length AR (AR-FL) or AR variants (AR-Vs) in blood and association with outcomes in mCRPC is unknown. We compared whole blood mRNA expression of AR-FL and AR-Vs to circulating tumor cells (CTCs) for predicting OS and time to treatment failure (TTF). Methods: We isolated RNA from whole blood collected in PAXgene RNA tubes and concurrent metastatic tissue biopsy from 51 men with mCRPC prior to initiation of abiraterone acetate in a prospective clinical trial (NCT#01953640). Whole transcriptome sequencing (RNAseq) was performed on blood samples and paired biopsies to detect AR-FL, ARV1, ARV3, ARV7, ARV8, AR12, ARV14, and ARV45. Reads were aligned to the GRCh38 reference genome with the spliced-alignment TopHat2 package. The Pearson correlation coefficient was calculated between AR-FL in blood and matched bone biopsy. CTCs were determined using the CELLSEARCH assay. Cox proportional hazard regression analysis was performed on AR-FL, each AR-V, and CTCs for association with OS and TTF. We compared the area under the curve (AUC) using CTCs alone to a multivariable model that included AR-Vs for predicting OS. Results: The median follow up was 3.0 years, (range 0.3-3.5); the median CTC count was 3 (range 0-372); 34/53 men were deceased. Blood based AR-FL or AR-Vs were detected in 50/53 patients with following distribution: AR-FL (41/53), ARV3 (9/53), ARV45 (8/53), ARV12 (4/53), ARV14 (4/53), ARV7 (2/53), and ARV8 (2/53). Whole blood AR-FL transcripts were highly correlated to paired bone biopsy (r2= 0.76). Elevated transcripts of either ARV12 or ARV14 were associated with decreased OS [hazard ratio (HR) 3.46, p = 0.006]. CTC count ≥5 was associated with poorer OS [HR 3.42, p = 0.02] and shorter TTF [HR 3.52, p = < 0.001]. Adjusting for CTC counts, in a multivariate model, blood AR12 expression was associated with poor OS [HR = 6.33, p = 0.009]. AR12 and CTCs trended toward improved AUC compared to CTC alone (0.78 vs 0.71, p = 0.07). Conclusions: AR-FL and AR-Vs are detectable in whole blood and are highly correlated with metastatic bone AR-FL expression. AR-Vs may add to prognostication in mCRPC and further validation is needed. Clinical trial information: NCT#01953640.

Abstract Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5726.

Abstract The Hippo pathway effector, Yes-associated protein (YAP), is a transcriptional coactivator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2–LATS1/2) culminating in phosphorylation of YAP at Serine 127 and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression, including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Mol Cancer Res; 16(10); 1556–67. ©2018 AACR.

350 Background: In prostate cancer, overexpression of EZH2 is associated with disease recurrence, androgen independent growth, cell invasion, and metastases. We evaluated the prognostic significance of EZH2 gene expression in metastatic biopsies from men with CRPC. Methods: EZH2 (RNA) expression was evaluated in two serial metastatic site biopsies in patients (pts) prior to treatment with abiraterone acetate and prednisone (pre-AA/P) and after 12 weeks of treatment (post-AA/P). All pts were enrolled and prospectively followed for clinical outcomes. The primary endpoint was overall survival (OS), defined as the time from CRPC to death or last follow-up. Cox proportional hazard regression analysis was performed on EZH2 expression using normalized fragments per kilobase million (FPKM) > 1 for association with OS and time to treatment failure (TTF). Results: Of 92 enrolled pts, 59 pts had (pre-AA/P) gene expression using RNA-seq, 45 had paired pre and post-AA/P RNA-seq available for analysis. In pre-AA/P samples, the median EZH2 expression was 4.82 FPKMs (range 1.05-38.8). Elevated expression of EZH2 was associated with shorter OS [hazard ratio (HR) 2.58, p = 0.016]. Radiographic progression at 12 weeks was more frequent in high EZH2 tumors compared to low EZH2 tumors (48% vs 20%, p = 0.03, Fisher’s exact test) No statistically significant associations were identified between EZH2 expression and TTF. In paired post-AA/P biopsies, EZH2 expression increased in 13/45 tumors and decreased in 32/45 tumors. The median log2 fold change in EZH2 expression was -1.38 (range -6.7 to 3.1). In post-AA/P biopsies, no associations were identified between an increase in EZH2 expression and OS or TTF. Conclusions: EZH2 expression represents a potential novel prognostic biomarker in mCRPC and warrants further exploration. Clinical trial information: 01953640.

The Memorial Sloan Kettering Cancer Center (MSKCC) prognostic score is based on clinical parameters. We analyzed whole blood mRNA expression in metastatic clear cell renal cell carcinoma (mCCRCC) patients and compared it to the MSKCC score for predicting overall survival. In a discovery set of 19 patients with mRCC, we performed whole transcriptome RNA sequencing and selected eighteen candidate genes for further evaluation based on associations with overall survival and statistical significance. In an independent validation of set of 47 patients with mCCRCC, transcript expression of the 18 candidate genes were quantified using a customized NanoString probeset. Cox regression multivariate analysis confirmed that two of the candidate genes were significantly associated with overall survival. Higher expression of BAG1 [hazard ratio (HR) of 0.14, p < 0.0001, 95% confidence interval (CI) 0.04–0.36] and NOP56 (HR 0.13, p < 0.0001, 95% CI 0.05–0.34) were associated with better prognosis. A prognostic model incorporating expression of BAG1 and NOP56 into the MSKCC score improved prognostication significantly over a model using the MSKCC prognostic score only (p < 0.0001). Prognostic value of using whole blood mRNA gene profiling in mCCRCC is feasible and should be prospectively confirmed in larger studies.

500 Background: The Memorial Sloan Kettering Cancer Center (MSKCC) prognostic score is based on clinical parameters and has several limitations. We analyzed whole blood mRNA expression in mCCRCC patients (pts) and compared it to MSKCC for predicting overall survival (OS). Methods: A prospective collection of whole blood in PAXgene RNA tubes from mCCRCC pts was performed between 2011 and 2015 with uniform processing. A discovery set (DS) of 19 randomly selected pt samples was used to generate whole transcriptome RNA sequencing and signatures describing differential gene expression patterns associated with OS. We calculated the fragment per kilobase of transcript per million (FPKM) of each transcript and gene to identify correlations between the differentially expressed genes and MSKCC score. Candidate genes were selected based on statistical significance (p<0.05) and associations with OS. In an independent validation set (VS) of 47 pts with mCCRCC, we used the nCounter Analysis System (NanoString Technologies, Seattle, WA, USA) to quantify gene transcript expression selected from the DS. Cox regression and Kaplan-Meier analysis were used for association of candidate mRNAs in the VS with OS (time of metastatic recurrence to death or last follow-up). We compared our gene signature to the MSKCC prognostic score for poor versus good survival. Results: In the DS, at a median follow-up of 4.13 years (yrs) (range: 1.3-4.5), 11 patients had died. We identified eighteen genes in the DS with statistically significant differential gene expression patterns and ability to discriminate patient OS. In the VS at a median follow-up of 4.9 yrs (range: 0.1-11.3), twenty-five patients had died. We confirmed two of the eighteen selected genes that had strong prognostic significance: BAG1 [hazard ratio (HR) 0.31, p=0.007] and NOP56 [HR 0.28, p=0.004]. Comparing OS between good and poor outcome groups for these 2 scores, our two-gene panel had higher prognostic ability (HR 20.07, p <0.0001) than the MSKCC prognostic score (HR 3.19, p 0.04). Conclusions: Prognostic value of using whole blood mRNA gene profiling in mCCRCC is feasible and should be prospectively confirmed in larger studies.