Kalari, Krishna R.

Abstract Background: Multiple studies have confirmed the central role of preexisting immune response measured by stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). Emerging studies showed that not only the number of TILs but also the location of TILs is important. There are 3 distinct immune architectures described based on the amount and locations of TILs, namely immune enriched (IN), immune excluded (IE), and immune desert (ID). Here we evaluated outcomes and characteristics associated with each immune landscape. Methods: NanoString Digital Spatial Profiling (DSP) and CosMx, a spatial multi-omics single-cell imaging platform, were performed in 75 samples from the Mayo Clinic (MC) TNBC cohort (Leon-Ferre BCRT 2018). NanoString IO360 was performed in 114 samples from the FinXX trial ( NCT00114816) . Firstly, tumors with sTIL quantified by H&E ≤ 30% were classified as ID. The rest of the tumors with high sTIL > 30% were categorized according to the intratumoral CD8 protein expression by DSP, with IE having intratumoral CD8 in the lower median and IN having intratumoral CD8 in the upper median. Chi-square test, gene set enrichment, Cox regression, and Kaplan-Meier analysis were used. Differential expression listed as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: ID is associated with low Ki67 < 5% (23.3% vs. 9.6% ID, p 0.02) as well as apocrine (11/13, 84.6%) and metaplastic histology (10/12, 83%). In both univariate and multivariate analysis, patients with IN had significantly improved recurrence-free survival (RFS) compared to those with ID (HR 0.37, 95%CI 0.18-0.74, p 0.005). Despite having high sTILs, IE had poor outcomes similar to ID (HR 0.84, 95%CI 0.36-1.98). Strikingly, we identified that IE patients had significantly lower plasmacytoid dendritic cells (pDCs) compared to IN (mean 0 vs. 0.26/100 tumor cells, 95%CI 0.08-0.43 , p 0.01). Using Gene Set Enrichment Analysis to evaluate differential hallmarks between IN and IE, we identified IF Nɑ and IFNγ (FDR < 0.001) responses as significantly enriched in IN group, consistent wi th the function of pDCs, which are a subset of dendritic cells specialized in secreting high levels of type I interferon. To validate this finding, we further evaluated the 11 leading edge gene IFNɑ signature in the FinXX trial. A high IFNɑ signature score was associated with significantly improved outcomes in the FinXX trial (HR 0.21, 95%CI 0.09-0.51 , p < 0.001). Similar findings were observed using Kaplan-Meier analysis in the FinXX trial with significantly improved RFS (p 0.0006) and overall survival (p 0.0001) in patients with high IFNɑ signature scores. Furthermore, we evaluated the differential gene expression unique to IN tumors in the Mayo cohort. Expressions of MHC class I and class II in tumor cells, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DPA1, and HLA-E, were associated with IN and significantly improved outcomes (p < 0.05) compared to ID and IE. Conclusions: Highlighting the importance of spatial context, we identified that patients with IE tumors had poor outcomes despite having high TILs. Moreover, u sing an in-depth analysis with spatially defined context, we identified the central role of pDC and the significance of IF Nɑ in TNBC. Support: Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17) W81XWH-15-1-0292, P50CA015083, R35CA253187 Citation Format: Saranya Chumsri, Yi Liu, Yaohua Ma, Jodi Carter, Mark Gregory, Sarah Church, Jason Reeves, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith Perez, Roberto Leon-Ferre, David Hillman, Judy Boughey, James Ingle, Krishna Kalari, Fergus Couch, Matthew Goetz, Keith Knutson, E. Thompson. The spatially resolved single-cell atlas of the tumor immune architecture revealed the central role of IFN-alpha and plasmacytoid dendritic cells in triple-negative breast cancer in the Mayo Clinic cohort and FinXX trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-03.

Abstract Background: Previous studies demonstrated that obesity is associated with poor outcomes and significantly worse pathological complete response (pCR) in triple-negative breast cancer (TNBC) treated with conventional chemotherapy. Emerging studies described a phenomenon termed the obesity paradox, which referred to improved outcomes observed in obese patients treated with immune checkpoint inhibitors (ICIs). Our study aimed to determine distinct immune features in TNBC across body mass index (BMI). Methods: Characteristics and outcomes of TNBC patients treated with neoadjuvant pembrolizumab-based chemotherapy at Mayo Clinic were collected. Normal BMI was defined as 18-25 kg/m2, overweight 25-30, and obese BMI ≥ 30. NanoString CosMx, a spatial multi-omics single-cell imaging (SMI), was performed in 75 samples from the Mayo Clinic TNBC cohort (Leon-Ferre BCRT 2018). Differential expression as log 2-fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p< 0.05. Results: Between 2021-2023, there were 108 TNBC patients treated with neoadjuvant pembrolizumab. 25.9% were obese, 32.4% were overweight, and 41.7% had normal BMI. In contrast to previous studies with conventional chemotherapy, obese patients numerically had a higher pCR rate of 71.4% with pembrolizumab-based chemotherapy compared to 55.6% in normal and 62.9% in overweight patients. However, this difference did not reach statistical significance (obese vs. normal weight, OR 2.26, 95%CI 0.89-6.06, p 0.094). We further evaluated spatially-resolved immune features associated with obesity using CosMx SMI in 75 samples from the MC TNBC cohort. Obese patients had significantly higher intratumoral B cells (log2FC 1.88, p 0.017), stromal B cells (log2FC 2.79, p 0.001), stromal plasmablasts (log2FC 1.94, p 0.047), and stromal macrophages (log2FC 1.80, p 0.017) compared to patients with normal BMI. Obese patients also had significantly lower intratumoral myeloid dendritic cells (mDC) (log2FC -1.27, p 0.037). Similar trends were observed after standardizing cell counts per 100 tumor or stromal cells, including higher intratumoral B cells (log2FC 3.00, p 0.001), intratumoral plasmablasts (log2FC 2.00, p 0.006), intratumoral natural killer (NK) cells (log2FC 1.66, p 0.044), and stromal B cells (log2FC 1.50, p 0.008). Differential gene analyses for intratumoral B cells revealed that genes (CD74, HLA-C, HLA-DRB1, HLA-DRA, HLA-DRB) associated with the positive regulation of type 2 immune or chemokine response were upregulated. Conclusions: Using in-depth analysis with spatially defined context, our study showed that obese TNBC patients had significantly higher B cell infiltration, which is associated with improved outcomes with ICIs. Future research on how obesity alters the immune microenvironment is needed to identify potential pathways to improve outcomes with ICIs in TNBC. Citation Format: Songhan Pang, Yi Liu, Yaohua Ma, Zhuo Li, Roberto A. Leon-Ferre, David Zahrieh, David W. Hillman, Judy C. Boughey, James N. Ingle, Krishna R. Kalari, Fergus J. Couch, Matthew P. Goetz, Keith L. Knutson, Saranya Chumsri, E. Aubrey Thompson. Tumor-immune microenvironment associated with obesity in triple-negative breast cancer in the Mayo Clinic cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1162.

Abstract Ovarian cancer is the most fatal of all female reproductive cancers due to its late detection, aggressive nature, and propensity to develop resistance to current standards of care. Despite initial responses to first line platinum- and paclitaxel-based chemotherapies, >70% of ovarian cancers recur with increasingly resistant disease. Targeted therapies, including PARP-inhibitors (PARPi) and VEGF-inhibitors, are being included in the front-line setting; and while they have improved progression-free survival in a subset of patients, they have thus far failed to improve overall survival in patients without specific genetic signatures. With the broad uptake of PARPi, PARPi-resistance is becoming common. Once resistance develops, nearly all patients die of their disease and there is an obvious need to elucidate the basis for chemotherapy- and PARPi-resistance and devise novel approaches to overcome resistance. Using a small molecule drug screen, we identified lestaurtinib, a known tyrosine kinase inhibitor as a potent inhibitor of treatment naive and highly advanced models of ovarian cancer. Lestaurtinib was originally developed to target FLT3, a protein not expressed in any ovarian cancer models we’ve tested. To identify lestaurtinib targets, we preformed phospho-proteomics in parallel with RNAseq and discovered the JAK/STAT signally pathway as a top hit. We confirmed that essential components of JAK/STAT signaling are constitutively activated in chemotherapy- and PARPi-resistant cells, suggesting that induction of this pathway may drive resistance. Importantly, we found that lestaurtinib-sensitive patient-derived-xenografts (PDXs) exhibited higher expression of multiple JAK/STAT signaling pathway members compared to PDXs that did not respond to lestaurtinib. Genetic knockdown of STAT1 and STAT3 via siRNA, or knockout via CRISPR/Cas9, resulted in significant growth inhibition of sensitive and resistant models, confirming their importance in maintaining cell viability and progression. Combining lestuartinib with standard-of-care cisplatin or olaparib, a PARPi, was shown to be synergistic in multiple models, indicating that pharmacological inhibition of JAK/STAT signaling has the potential to alleviate drug resistance. Finally, we assessed a panel of JAK/STAT inhibitors that interfere at various levels of the pathway and found profound differences in their ability to block STAT-phosphorylation and inhibit cell proliferation. Thus, it is critical to further define the mechanisms by which these drugs function to selectively suppress JAK/STAT signaling in order to elucidate specific nodes of the pathway that drive ovarian cancer progression. Ongoing studies are aimed at identifying the unique roles of STAT1 and STAT3, the specific contributions of their tyrosine and serine phosphorylation, and the relevance of up-/down-stream mediators of the JAK/STAT pathway on ovarian cancer cell growth with the goal of informing future clinical trials. Citation Format: Esther Rodman, Michael Emch, Archit Bajaj, Xiaojia Tang, Xiaonan Hou, Scott Kaufmann, Krishna Kalari, John Weroha, John Hawse. Exploiting JAK/STAT signaling to inhibit highly advanced and resistant forms of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B002.

Background: A strong body of evidence suggests that cerebrovascular pathologies augment the onset and progression of Alzheimer’s disease (AD). One distinctive aspect of this cerebrovascular dysfunction is the degeneration of brain pericytes—often overlooked supporting cells of blood-brain barrier endothelium. Objective: The current study investigates the influence of pericytes on gene and protein expressions in the blood-brain barrier endothelium, which is expected to facilitate the identification of pathophysiological pathways that are triggered by pericyte loss and lead to blood-brain barrier dysfunction in AD. Methods: Bioinformatics analysis was conducted on the RNA-Seq expression counts matrix (GSE144474), which compared solo-cultured human blood-brain barrier endothelial cells against endothelial cells co-cultured with human brain pericytes in a non-contact model. We constructed a similar cell culture model to verify protein expression using western blots. Results: The insulin resistance and ferroptosis pathways were found to be enriched. Western blots of the insulin receptor and heme oxygenase expressions were consistent with those observed in RNA-Seq data. Additionally, we observed more than 5-fold upregulation of several genes associated with neuroprotection, including insulin-like growth factor 2 and brain-derived neurotrophic factor. Conclusions: Results suggest that pericyte influence on blood-brain barrier endothelial gene expression confers protection from insulin resistance, iron accumulation, oxidative stress, and amyloid deposition. Since these are conditions associated with AD pathophysiology, they imply mechanisms by which pericyte degeneration could contribute to disease progression.

Abstract Background: Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would thus be warranted, and in this report, we have established such a classifier. Methods: To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated and reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by g-secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent data sets. Results: An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2) and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. Conclusions: The 20-gene transcriptional signature has the potential to better stratify patients and to evaluate the response of future Notch-based therapies for breast cancer. Citation Format: Eike-Benjamin Braune, Felix Geist, Xiaojia Tang, Krishna Kalari, Judy Boughey, Liewei Wang, Roberto A. Leon-Ferre, Antonino B. D’Assoro, James N. Ingle, Matthew P. Goetz, Kang Wang, Theodoros Foukakis, Anita Seshire, Dirk Wienke, Urban Lendahl. Identification of a Notch transcriptomic signature for breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B013.

581 Background: Several studies have established the crucial role of preexisting immune response measured by tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC). Emerging studies showed that not only the number of TILs but also the location of TILs is as critical. There are 3 distinct immune landscapes described based on the locations of TILs, namely immune enriched (IN), immune excluded (IE), and immune desert (ID), which are associated with outcomes in TNBC treated with immune-checkpoint inhibitors. Here we evaluated characteristics associated with each immune landscape. Methods: NanoString IO360, Digital Spatial Profiling (DSP), and CosMx, a spatial multi-omics single-cell imaging platform, were used. DSP was used to quantify 39 immune-related proteins in stromal and tumor-enriched segments from 44 TNBC samples from the FinXX trial (NCT00114816) and 276 samples from the Mayo Clinic (MC) TNBC cohort (Leon-Ferre BCRT 2018). CosMx was performed in 75 samples from the MC TNBC cohort. First, tumors with TIL quantified by H&E ≤ 30% were classified as ID. The rest of the tumors were categorized according to the intratumoral CD8 protein expression by DSP, with IE having intratumoral CD8 in the lower median and IN having intratumoral CD8 in the upper median. Differential expression listed as log fold change (FC) was estimated from the linear mixed model with significance defined as two-sided p < 0.05. Results: Using DSP in the FinXX trial, intratumoral and stromal higher HLA-DR (FC 1.68, p = 0.001), B2M (FC 0.8, p = 0.005), CD4 (FC 0.74, p = 0.01), and CD40 (FC 1.56, p = 0.001) were associated with IN compared to ID. When comparing IE and IN, higher intratumoral CD11c (FC 0.97, p = 0.01) and stromal CD4 (FC 0.89, p = 0.047), CD20 (FC 0.85, p = 0.016), CD40 (FC 0.95, p = 0.045), and CD27 (FC 0.84, p = 0.024) were associated with IN. Similar findings were observed in the MC cohort. Moreover, intratumoral NY-ESO-1 expression (FC 0.55, p = 0.03) was associated with IN. Using GSEA with IO360 in the FinXX trial, PI3K-Akt signaling was associated with ID compared to IN (p = 0.01). We further evaluated the differential gene expression in a spatially resolved manner using CosMx with single-cell sequencing in the MC cohort. Expressions of MHC class I and class II in tumor cells, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DPA1, and HLA-E, were associated with IN compared to ID and IE. Conclusions: Using an in-depth analysis with spatially defined context, we identified characteristics associated with distinct immune landscapes in TNBC. Our study highlights the potential future implications of intratumoral MHC expression, CD40, and PI3K-AKT as biomarkers and therapeutic targets. Clinical trial information: NCT00114816 .

Abstract Background: Cancer is usually treated with a combination of drugs rather than a single agent. Treating cancer with a drug combination decreases the likelihood of the cancer acquiring resistance to therapy. It also allows for lower dosages of individual drugs, which reduces the occurrence and severity of side effects. Using public datasets, we investigated potential anti-cancer drug combinations using a novel framework, ReCorDE (Recurrent Correlation of Drugs with Enrichment), which identifies correlated drug response patterns for drugs with different primary mechanisms of action. Methods: 250 drugs from CTRPv2, GDSCv2, and PRISM datasets were examined using normalized logIC50 or AUC measurements. ReCorDE consists of a correlation and an enrichment step. For each dataset, we constructed pairwise drug-drug relationships using Spearman’s correlation. Combinations significant (p &lt; 0.05) in less than two datasets after Benjamini-Hochberg (BH) correction were pruned. To turn each drug combination into a drug class combination, all drugs were mapped to their corresponding fourth-level Anatomical Therapeutic Chemical (ATC) codes. Then, using a hypergeometric test, for each class combination in the subset of significant drug combinations (the “inside” set), we tested for enrichment of that class combination in the inside set compared to the class combination universe, the distribution of class combinations for all unique pairwise drug-drug associations (250 unique drugs; 31,125 distinct drug combinations mapped to 1596 distinct class combinations). Multiple testing during enrichment was corrected using the BH method with a significance cutoff of p&lt; 0.05. Results: We found 2764 pairs to be significant at a = 0.05 using the AUC measure. With a correlation coefficient of 0.77, vincristine and YK-4-279, an EWS-FLI1/RNA Helicase A inhibitor, had the lowest computed p-value of of 1.39 × 10−120 Adjusted p-values for &gt;15% of these combinations had p-values &lt; 5 × 10−6. Class combination enrichment on the same set showed 132 class pairs were enriched in the inside set compared to the class combination universe after BH correction. Taxanes/Plk1 inhibitors (OR = 69.5, adjusted p = 5.6 × 10−11); Aurora Kinase inhibitors/histone modifying agents (OR=9.2, adjusted p = 6.13 × 10−11); and pyrimidine analogues/CDK inhibitors (OR=3.43, adjusted p = 0.02) are a few notable enriched class combinations. The enrichment of these class combinations in the inside set suggests that combinations of drugs from these classes may be particularly effective in treating cancer compared to other drug combinations. Conclusions: Our framework, ReCorDE, demonstrates that finding potential drug combinations and characterizing novel, frequently perturbed pathways outside of a drug's primary mechanisms of action can be accomplished by identifying correlated drug-drug pairs from large, publicly accessible databases. Citation Format: Emily T. Ghose, August John, Huanyao Gao, Krishna R. Kalari, Liewei Wang. ReCorDE: A novel computational framework to discover potential combinations of anti-cancer drugs. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4290.

Abstract Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide. Triple negative breast cancer (TNBC) represents 15-20% of all breast cancers and is an aggressive subtype. Chemotherapy-based treatments remain the standard of care for TNBC. Unfortunately, chemotherapy resistance is common, and for these patients, outcomes are poor and alternative treatment strategies remain an unmet need. circRNAs are a newly identified class of noncoding RNA molecules with covalently closed circular structures. An increasing number of recent studies including ours have indicated that circRNAs play crucial roles in regulating tumor development and chemoresistance. However, the role of circRNAs in the process of chemotherapy resistance and TNBC progression is not clear. Materials and Methods: As a first step towards identifying circRNAs that participate in the development of chemoresistance in TNBC cells, and to determine if targeting such circRNAs is a novel and efficacious therapeutic strategy, doxorubicin-resistant (Doxo-R), paclitaxel-resistant (PTX-R), and double-resistant (DP-R) cell lines were generated from MDA-MB-231. Human circRNA microarrays were utilized to profile the expression of approximately 14,000 known circRNAs in normal breast tissue, matched patient-derived xenografts (PDX) generated prior to and following neoadjuvant chemotherapy (NAC), and TNBC chemosensitive and chemoresistant cell lines. Top hits were validated using RT-PCR. Results: circRNA microarray profiling identified 429 and 310 transcripts differentially expressed in doxorubicin and paclitaxel resistant cells, respectively, compared to parental chemosensitive cell lines (|FC| ≥ 1.5; p value &lt; 0.05). In comparison to pre-NAC derived xenografts, 1,396 circRNAs were dysregulated among post-NAC PDX models. Further, three circRNAs (hsa_circ_001388, hsa_circ_104652, and hsa_circ_061260) were upregulated in Doxo-R, PTX-R, and post-NAC PDX samples compared to their respective controls. Among these three circRNAs, hsa_circ_001388 (also known as circNSD2) was the only transcript also predicted to be translated into a novel and uncharacterized protein given the presence of a high confidence translation initiation site and IRES sequence. Ongoing studies are aimed at determining the role of circNSD2 protein in breast cancer carcinogenesis, progression, and response to standard of care chemotherapeutics and the mechanistic process by which this protein functions. Additionally, the efficacy of specifically targeting this circRNA as a novel therapeutic approach is being explored. Conclusions: Increasing knowledge of the important functions of circRNAs underlying drug resistance will provide new opportunities for developing efficacious therapeutic strategies and prognostic/predictive biomarkers for TNBC. Citation Format: Xiyin Wang, Michael J. Emch, Xiaojia Tang, Jia Yu, Krishna R. Kalari, Liewei Wang, Matthew P. Goetz, John R. Hawse. The role of circular RNAs in triple negative breast cancer and chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1689.

AbstractObjectiveElevated serum chromogranin A (CGA) is associated with intrinsic or treatment‐related neuroendocrine differentiation (NED) in men with metastatic castration‐resistant prostate cancer (mCRPC). Fluctuations in serum CGA during treatment of mCRPC have had conflicting results. We analyzed the impact of (i) rising serum CGA and (ii) baseline CGA/PSA ratio during treatment to identify associations with abiraterone acetate (AA) therapy.MethodsBetween June 2013 and August 2015, 92 men with mCRPC were enrolled in a prospective trial with uniform serum CGA processing performed before initiating abiraterone acetate/prednisone (AA/P) and serially after 12 weeks of AA/P treatments. Serum CGA was measured using a homogenous automated immunofluorescent assay. Patients receiving proton pump inhibitors or with abnormal renal function were excluded due to possible false elevations of serum CGA (n = 21 excluded), therefore 71 patients were analyzed. All patients underwent a composite response assessment at 12‐weeks. Kaplan–Meier estimates and Cox Regression models were used to calculate the association with time‐to‐treatment failure analyses and overall survival.ResultsAn increase in chromogranin was associated with a lower risk of treatment failure (hazard ratio [HR]: 0.52, p = 0.0181). The median CGA/PSA ratio was 7.8 (2.6–16.0) and an elevated pretreatment CGA/PSA ratio above the median was associated with a lower risk of treatment failure (HR: 0.54 p value = 0.0185). An increase in CGA was not found to be associated with OS (HR: 0.71, 95% CI: 0.42–1.21, p = 0.207). An elevated baseline CGA/PSA ratio was not associated with OS (HR: 0.62, 95% CI: 0.37–1.03, p = 0.062). An increase in PSA after 12 weeks of treatment was associated with an increased risk of treatment failure (HR: 4.14, CI: 2.21–7.73, p = < 0.0001) and worse OS (HR: 2.93, CI: 1.57–4.45, p = < 0.0001).ConclusionsWe show that an increasing chromogranin on AA/P and an elevated baseline CGA/PSA in patients with mCRPC were associated with a favorable response to AA/P with no changes in survival. There may be limited clinical utility in serum CGA testing to evaluate for lethal NED as AA/P did not induce lethal NED in this cohort. This highlights that not all patients with an increasing CGA have a worse OS.

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 .