Stanton, Melissa

572 Background: Renal medullary carcinoma (RMC) is an aggressive malignancy affecting predominantly young African Americans with sickle cell trait (SCT) or disease (SCD), while a pathologically similar collecting duct carcinoma (CDC) affects patients without sickle cell trait. Clinical responses to chemotherapy and IL-2 in RMC/CDC are poor and novel therapies are needed. Methods: 9 patients with RMC (ages 13-58 y. o., all male) and 15 patients (ages 26-74 y. o., M:F = 13:2) with collecting duct carcinoma (CDC) were studied. Expression of PD-L1 was evaluated with 2 monoclonal antibodies (SP142 and SP263) and tumor infiltrating lymphocytes (TIL) were evaluated for PD1 expression (MRQ-22 antibody) using immunohistochemistry (IHC). Additional studies included ALK protein expression (D5F3 antibody), gene translocation (break apart FISH), next generation sequencing (NGS), and microsatellite instability (MSI). Results: Cancer cell PD-L1 expression above the threshold ( ≥ 2+, ≥ 5%) was seen in 7/9 RMC and 5/13 CDC cases. Concordance between 2 PD-L1 antibodies was 94.4%. PD-1+ TIL were absent in 6/18 cases and variably present in 12/18 cases (from 1 to > 15 TIL/40x power field). No MSI was detected in any of the cases tested (0/6). No case expressed ALK protein, but one case of CDC showed ALK gene re-arrangement. Mutations were identified in SMARCB1, FH, TP53 (3x), ATM, BRCA2, CHEK2 (2x), NF2 (3x), SETD2, and CDKN2A. No mutations in VHL or KDR were detected. One patient with RMC (and SCT) achieved complete clinical remission after treatment with bevacizumab plus paclitaxel. Conclusions: RMC and CDC strongly express PD-L1 in the majority of cases (12/22), suggesting that these patients may benefit from targeting the PD-L1/PD1 interaction. The absence of MSI in these cancers indicates a different mechanism of PD-L1 upregulation from colorectal carcinomas. Consistent with our previous study that showed frequent activation of (pseudo)hypoxia-induced pathways in RMCs (Human Pathology 2011;42:1979), we describe a case of RMC successfully treated with anti-VEGF therapy.

570 Background: Vascular endothelial growth factor- (VEGF-) and mammalian target of rapamycin- (mTOR-) directed therapies represent a standard of care in metastatic renal cell carcinoma (mRCC). However, these agents are not employed based on an assessment of predictive biomarkers such as genomic alterations (GA). We sought to determine if an association exists between GA detected by comprehensive genomic profiling (CGP) and the response to VEGFR and mTOR pathway targeted therapies in a cohort of mRCC treated in a clinical practice setting. Methods: The results of CGP performed in the course of clinical care on 31 consecutive mRCC obtained from patients who had received VEGFR- and/or mTOR-inhibitors were reviewed. Duration of treatment (DOT), extent and duration of clinical response was obtained from review of medical records. All classes of genomic alterations - base substitutions, short insertions, deletions, gene fusions, rearrangements and copy number - were assessed via CGP. Descriptive statistics were used to determine the frequency of GAs in groups segregated by the DOT with VEGF-directed agents. Results: The most common GAs detected in this series were in VHL (70%), PBRM1 (48%), SETD2 (32%), TSC1 (29%), MLL (19%), TERT (16%), ARID1B (16%) and KDM5C (16%). Across 61 administrations of VEGF-directed therapy in 27 patients, exceptional responses (DOT > 21 months) were more frequent amongst patients with GAs in KDM5C, PBRM1, and VHL. Conversely, these patients also featured a lower frequency of GA associated with response to mTOR-directed therapy, such as TSC1. Conclusions: Multiple GAs are more prevalent in exceptional responders to VEGF-directed therapy. Prospective validation of these findings may lead to use of CGP to optimize therapeutic selection.

Abstract Monoclonal antibodies that target the programmed death-1 (PD-1)–programmed death ligand-1 (PD-L1) axis have antitumor activity against multiple cancers. The presence of sarcomatoid differentiation in renal cell carcinoma (RCC) is associated with resistance to targeted therapy and poor responses to IL2 immunotherapy. Given the aggressive nature of RCC with sarcomatoid differentiation and the exclusion of sarcomatoid histology from metastatic RCC clinical trials, less is understood regarding selection of therapies. Here, we characterized the PD-1/PD-L1 axis in RCC with sarcomatoid differentiation. We directly compared two PD-L1 antibodies and found concordance of PD-L1 positivity in 89% of tested RCCs with sarcomatoid differentiation. Coexpression of PD-L1 on neoplastic cells and the presence of PD-1–positive tumor-infiltrating lymphocytes were identified in 50% (13 of 26) of RCCs with sarcomatoid differentiation. In contrast, only 1 of 29 clear cell RCCs (3%) had concurrent expression of PD-L1 and PD-1 (P = 0.002). Our study suggests that RCC with sarcomatoid differentiation may express PD-1/PD-L1 at a higher percentage than RCC without sarcomatoid differentiation, and patients with these tumors may be good candidates for treatment with anti–PD-1/PD-L1 therapies. Cancer Immunol Res; 3(12); 1303–7. ©2015 AACR.

Abstract Background: Renal cell carcinoma (RCC) is the most common type of kidney cancer responsible for ∼80% of kidney cancer cases. Circulating tumor cells (CTCs) are emerging as an important prognostic and predictive biomarker in several types of metastatic cancers. However, methods for CTC detection that target only EpCAM and/or cytokeratin (CK) to enrich CTCs, may fail to recognize the CTCs in clear cell RCC (ccRCC) because ccRCC lacks expression of EpCAM. The objective of this study was to develop a new technology capable of detecting CTCs including those with a mesenchymal phenotype in metastatic RCC. Methods: Blood samples from patients affected by metastatic RCC were provided by Mayo Clinic Arizona and analyzed by Creatv MicroTech. CellSieveTM microfilters, which have 7 µm diameter pores in a uniform array, with 160,000 pores in a 9 mm diameter area, were used for separation of CTCs in the blood specimen. 7.5 mL of whole blood was diluted in the prefixation buffer and filtered through the microfilter. Clinical immunohistochemical markers for RCC such as CD10 and vimentin were used to identify candidate CTCs. The cells collected on the filter were fixed, permeabilized, and stained with DAPI and fluorescent antibodies specific to CD10, vimentin, and CD45. The assay protocol was developed using three RCC cell lines, 786-O, Caki-1, and Caki-2, and validated using blood samples from patients with metastatic RCC. Results: The capture efficiencies for 786-O, Caki-1, and Caki-2 cell lines were determined to be 98%, 98% and 97%, respectively. On-filter antibody staining revealed heterogeneous expressions of vimentin and CD10 in RCC cells. Patient samples were collected in duplicate CellSaveTM tubes and processed one day later. A CD10+/vimentin+/CD45- cell population was detected in blood samples with enumeration ranging from 4 to 86 cells/7.5 mL of blood. We found that the RCC CTC profile was distinct from other types of cancers. The typical CTCs display abnormal morphology, including large nuclei (typically 15-30 µm in size), irregular cell size and shape, and high nucleus-to-cytoplasm ratio. Although CD10 and vimentin antibodies have cross-reactivity with white blood cells (WBC), the CTCs could be distinguished from WBC based on morphology, cell size and CD45- staining. Conclusions: We demonstrated that the CellSieveTM microfiltration assay is a very simple and efficient method to isolate a CD10+/vimentin+/CD45- cell population from patients affected by metastatic ccRCC. Future studies include fluorescence in situ hybridization to confirm loss of 3p, a molecular event that occurs in >90% of ccRCC. We are also analyzing blood samples from patients treated with tyrosine kinase inhibitors to determine if cell enumeration is predictive of response to treatment. This technology might greatly facilitate detection of EpCAM negative ccRCC CTCs. The clinical significance of these unusual, large, rod-shaped, naked nuclei require further molecular characterization. Citation Format: Peixuan Zhu, Thai Ho, Erik P. Castle, Richard W. Joseph, Melissa L. Stanton, Shuhong Li, Daniel Adams, Olga V. Makarova, Platte T. Amstutz, Cha-Mei Tang. Isolation and identification of circulating tumor cells in renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3081. doi:10.1158/1538-7445.AM2014-3081