Giridhar, Karthik V.

Abstract Introduction: CDK 4/6 inhibitors (CDK4/6i) are FDA approved for treatment of hormone receptor (HR) positive, HER2 negative metastatic breast cancer (MBC). Recent work demonstrated that a rise in MCV was correlated with longer median progression free survival (Anampa et al, Haemoatologica 2018). We performed a single-institution retrospective analysis to evaluate whether palbociclib induced early changes in MCV serve as a pharmacodynamic biomarker to predict response to CDK4/6i. Methods: We identified a retrospective cohort of 81 patients with HR+, HER2- MBC treated with CDK4/6i as first line metastatic treatment at Mayo Clinic, Rochester. Hematologic indices were abstracted pretreatment and at the start of each cycle through the start of cycle 4 (C4). Wilcoxon signed test was used to examine the changes in MCV (ΔMCV) comparing pretreatment up to C4 and ΔMCV between cycles. Optimal cut points for ΔMCV were determined using Cutoff finder (Budczies et al., PLOS One 2012). Cox proportional hazard regression analysis was performed to evaluate associations of MCV and time to treatment failure (TTF), which was defined as the time from the start of the analyzed cycle to treatment discontinuation for any reason(in months). As eight comparisons were made, a Bonferroni correction established p-value <0.00625 as the adequate cutoff for statistical significance. Statistical analyses were performed using JMP (SAS Institute Inc, Cary, NC). Results: 60/81 patients had pretreatment and at least one subsequent lab data point that included MCV. The pretreatment mean hemoglobin (Hgb) was 12.8 [standard deviation (SD) 1.37 g/dL] and 6/60 (10.2%) patients with Hgb <11.0 g/dL. Mean pretreatment MCV was 89.0 femtoliters/cell [fl (SD 4.63)] and no macrocytosis (defined as MCV >100 fl) was observed. At C4, the mean Hgb was 12.1 g/dL (SD 1.11) with 4/49 (8.2%) with Hgb <11.0 g/dL. The C4 mean MCV was 96.6 fl (SD 5.23) and 8/29 (16.3%) developed macrocytosis. Wilcoxon signed analysis showed a significant increase in ΔMCV pretreatment to C2 [n=51, median 1.5 with interquartile range (IQR) -0.1-3.2, p<0.0001], C2 to C3 (n=44, median 2.9, IQR 1.4-5.2, p<0.0001), and C3 to C4 (n=41, median 3.4, IQR 1.9-4.9, p<0.0001). A rise in MCV≥8.15 from pretreatment to C4 was associated with a non-statistically significant improvement in TTF [n=49, hazard ratio (HR) 0.38, p = 0.06]. Between C2 and C3, a rise in MCV ≥2.9 was associated favorably with TTF (n=44, HR 0.36, p = 0.029). The estimated probability of remaining on treatment at 30 months was higher in those with C2-C3 ΔMCV ≥2.9 [44.1%; 95% Confidence Interval (CI) 17.3-68.2%] compared to those with a C2-C3 ΔMCV <2.9 (10.1%; 95%CI 1.5-28.7%). Discussion: We observed an increase in MCV in all patients receiving CDK4/6i by start of C4. The rise in MCV during CDK4/6i treatment may reflect drug induced cell-cycle arrest in hematopoietic cells. Evaluation in larger studies is needed to validate dynamic changes in MCV as a predictive biomarker of response to CDK4/6i therapy. Citation Format: Grace Mei Yee Choong, Roberto A Leon-Ferre, Ciara C O'Sullivan, Kathyrn J Ruddy, Tufia C Haddad, Timothy J Hobday, Prema P Peethambaram, Charles L Loprinizi, Minetta C Liu, Vera J Suman, Matthew P Goetz, Karthik V Giridhar. Evaluation of mean corpuscular volume (MCV) as a pharmacodynamic predictive biomarker in patients receiving CDK4/6 inhibitors for metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-42.

Background Male breast cancer (MBC) is a rare disease for which there is limited understanding of treatment patterns and prognostic factors. Methods Men with TNM stage I to stage III breast cancer diagnosed between 2004 and 2014 in the National Cancer Data Base were included. Trends in treatment modalities were described using the average annual percentage change (AAPC) and estimated using Joinpoint software for the analysis of trends. Kaplan‐Meier curves and the multivariate Cox proportional hazards regression model were used to compare survival between subgroups and to identify prognostic factors. Results A total of 10,873 MBC cases were included, with a median age at diagnosis of 64 years. Breast‐conserving surgery was performed in 24% of patients, and 70% of patients undergoing breast conservation received radiotherapy. Approximately 44% of patients received chemotherapy, and 62% of patients with estrogen receptor–positive disease received endocrine therapy. Oncotype DX was ordered in 35% of patients with lymph node–negative, estrogen receptor–positive/human epidermal growth factor receptor 2 (HER2)‐negative tumors. During the study period, there was a significant increase in the rates of total mastectomy, contralateral prophylactic mastectomy, radiotherapy after breast conservation, ordering of Oncotype DX, and the use of endocrine therapy ( P < .05). On multivariate analysis, factors found to be associated with worse overall survival were older age, black race, higher Charlson Comorbidity Index, high tumor grade and stage of disease, and undergoing total mastectomy. Residing in a higher income area; having progesterone receptor–positive tumors; and receipt of chemotherapy, radiotherapy, and endocrine therapy were associated with better overall survival. Conclusions Despite the lack of prospective randomized trials in patients with MBC, the results of the current study demonstrated that the treatment of this disease has evolved over the years. These findings further the understanding of the modern treatment and prognosis of MBC, and identify several areas for further research.

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.

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.

249 Background: Serum CGA has been identified as a candidate prognostic biomarker for mCRPC. In a two cohort study, we compared the prognostic value of serum CGA with a validated CTC assay. Methods: In the discovery cohort (DC), blood samples were collected from 256 men with mCRPC. In an independent validation cohort (VC), 92 men with mCRPC were enrolled in a biospecimen collection study. In both cohorts, men receiving proton pump inhibitors and those with non-castrate levels of testosterone ( > 50ng/dl) were excluded. Serum CGA was measured in a homogeneous automated immunofluorescent assay using time-resolved amplified cryptate emission. In the VC, CTC enumeration was performed using the FDA cleared CELLSEARCH CTC test prior to treatment with abiraterone acetate/ prednisone. Cox proportional hazard regression and Kaplan-Meier analysis were performed for associations with elevated CGA (above reference range), unfavorable (≥ 5) CTCs, and overall survival (OS). Results: In the DC, 200 men were eligible for analysis. The median age was 72 years (yrs), 81/200 pts had a Gleason score (GS) ≥ 8, 34/200 had an elevated CGA. At a median follow up of 2.2 yrs, 156/200 were deceased. In the subset of men with GS ≥ 8, elevated CGA was associated with shorter OS [hazard ratio (HR) 2.19, p = 0.017]. In the VC, 71 men were eligible for analysis. The median age was 71 yrs, 36/71 tumors were GS ≥ 8, 31/71 pts had an elevated CGA, 26/66 had unfavorable CTCs (≥ 5). At a median follow up of 1.8 yrs, 31/71 were deceased. Elevated CGA (HR 1.91, p = 0.043) and unfavorable CTC counts (HR 2.97, p = 0.0012) were adversely associated with OS. In the high GS group, both CTCs and CGA had the same area under the curve (AUC) of 0.72. Pts with elevated CTC and CGA had the poorest OS (HR 3.76, p = 0.008). Conclusions: Elevated serum CGA was negatively associated with OS in men with mCRPC. Serum CGA represents a prognostic biomarker that may complement CTC enumeration. Clinical trial information: NCT#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.