Werner J. Struss

Treatment Outcomes and Tumor Loss of Heterozygosity in Germline DNA Repair–deficient Prostate Cancer

BACKGROUND Germline mutations in DNA repair genes were recently reported in 8-12% of patients with metastatic castration-resistant prostate cancer (mCRPC). It is unknown whether these mutations associate with differential response to androgen receptor (AR)-directed therapy. OBJECTIVE To determine the clinical response of mCRPC patients with germline DNA repair defects to AR-directed therapies and to establish whether biallelic DNA repair gene loss is detectable in matched circulating tumor DNA (ctDNA). DESIGN, SETTING, AND PARTICIPANTS We recruited 319 mCRPC patients and performed targeted germline sequencing of 22 DNA repair genes. In patients with deleterious germline mutations, plasma cell-free DNA was also sequenced. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Prostate-specific antigen response and progression were assessed in relation to initial androgen deprivation therapy (ADT) and subsequent therapy for mCRPC using Kaplan-Meier analysis. RESULTS AND LIMITATIONS Of the 319 patients, 24 (7.5%) had deleterious germline mutations, with BRCA2 (n=16) being the most frequent. Patients (n=22) with mutations in genes linked to homologous recombination were heterogeneous at initial presentation but, after starting ADT, progressed to mCRPC with a median time of 11.8 mo (95% confidence interval [CI] 5.1-18.4). The median time to prostate-specific antigen progression on first-line AR-targeted therapy in the mCRPC setting was 3.3 mo (95% CI 2.7-3.9). Ten out of 11 evaluable patients with germline BRCA2 mutations had somatic deletion of the intact allele in ctDNA. A limitation of this study is absence of a formal control cohort for comparison of clinical outcomes. CONCLUSIONS Patients with mCRPC who have germline DNA repair defects exhibit attenuated responses to AR-targeted therapy. Biallelic gene loss was robustly detected in ctDNA, suggesting that this patient subset could be prioritized for therapies exploiting defective DNA repair using a liquid biopsy. PATIENT SUMMARY Patients with metastatic prostate cancer and germline DNA repair defects exhibit a poor response to standard hormonal therapies, but may be prioritized for potentially more effective therapies using a blood test.

Circulating Tumor DNA Reveals Clinically Actionable Somatic Genome of Metastatic Bladder Cancer

Purpose: Targeted agents and immunotherapies promise to transform the treatment of metastatic bladder cancer, but therapy selection will depend on practical tumor molecular stratification. Circulating tumor DNA (ctDNA) is established in several solid malignancies as a minimally invasive tool to profile the tumor genome in real-time, but is critically underexplored in bladder cancer. Experimental Design: We applied a combination of whole-exome sequencing and targeted sequencing across 50 bladder cancer driver genes to plasma cell-free DNA (cfDNA) from 51 patients with aggressive bladder cancer, including 37 with metastatic disease. Results: The majority of patients with metastasis, but only 14% of patients with localized disease, had ctDNA proportions above 2% of total cfDNA (median 16.5%, range 3.9%–72.6%). Twelve percent of estimable samples had evidence of genome hypermutation. We reveal an aggressive mutational landscape in metastatic bladder cancer with 95% of patients harboring deleterious alterations to TP53, RB1, or MDM2, and 70% harboring a mutation or disrupting rearrangement affecting chromatin modifiers such as ARID1A. Targetable alterations in MAPK/ERK or PI3K/AKT/mTOR pathways were robustly detected, including amplification of ERBB2 (20% of patients) and activating hotspot mutations in PIK3CA (20%), with the latter mutually exclusive to truncating mutations in TSC1. A novel FGFR3 gene fusion was identified in consecutive samples from one patient. Conclusions: Our study demonstrates that ctDNA provides a practical and cost-effective snapshot of driver gene status in metastatic bladder cancer. The identification of a wide spectrum of clinically informative somatic alterations nominates ctDNA as a tool to dissect disease pathogenesis and guide therapy selection in patients with metastatic bladder cancer. Clin Cancer Res; 23(21); 6487–97. ©2017 AACR.

Magnetically-actuated drug delivery device (MADDD) for minimally invasive treatment of prostate cancer: An in vivo animal pilot study

The vast majority of prostate cancer presents clinically localized to the prostate without evidence of metastasis. Currently, there are several modalities available to treat this particular disease. Despite radical prostatectomy demonstrating a modest prostate cancer specific mortality benefit in the PIVOT trial, several novel modalities have emerged to treat localized prostate cancer in patients that are either not eligible for surgery or that prefer an alternative approach.

Biallelic tumour suppressor loss and DNA repair defects in de novo small-cell prostate carcinoma: Molecular dissection of de novo small-cell prostate carcinoma

Small‐cell prostate carcinoma (SCPC) is an aggressive malignancy that is managed similarly to small‐cell lung cancer. SCPC can evolve from prostate adenocarcinoma in response to androgen deprivation therapy, but, in rare cases, is present at initial cancer diagnosis. The molecular aetiology of de novo SCPC is incompletely understood, owing to the scarcity of tumour tissue and the short life‐expectancy of patients. Through a retrospective search of our regional oncology pharmacy database, we identified 18 patients diagnosed with de novo SCPC between 2004 and 2017. Ten patients had pure SCPC pathology, and the remainder had some admixed adenocarcinoma foci, but all were treated with first‐line platinum‐based chemotherapy. The median overall survival was 28 months. We performed targeted DNA sequencing, whole exome sequencing and mRNA profiling on formalin‐fixed paraffin‐embedded archival tumour tissue. We observed frequent biallelic deletion and/or mutation of the tumour suppressor genes TP53, RB1, and PTEN, similarly to what was found in treatment‐related SCPC. Indeed, at the RNA level, pure de novo SCPC closely resembled treatment‐related SCPC. However, five patients had biallelic loss of DNA repair genes, including BRCA1, BRCA2, ATM, and MSH2/6, potentially underlying the high genomic instability of this rare disease variant. Two patients with pure de novo SCPC harboured ETS gene rearrangements involving androgen‐driven promoters, consistent with the evolution of de novo SCPC from an androgen‐driven ancestor. Overall, our results reveal a highly aggressive molecular landscape that underlies this unusual pathological variant, and suggest opportunities for targeted therapy strategies in a disease with few treatment options. Copyright

The role of netrin-1 in metastatic renal cell carcinoma treated with sunitinib

Introduction Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies like sunitinib, target the formation of new vessels allowing nutrient deprivation and cell death. However, recent studies have shown that patients develop resistance after approximately 1 year of treatment and show disease progression while on therapy. Therefore, we propose to identify the protein(s) responsible for increased migration with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease. Material and Methods Human renal cancer cell lines (Caki-1, Caki-2, ACHN) were treated with increasing doses of sunitinib to develop a sunitinib-conditioned renal cell carcinoma cell line. mRNA microarray and qPCR were performed to compare the differences in gene expression between Caki-1 sunitinib-conditioned and non-conditioned cells. NTN1 was assessed in our in vivo sunitinib-conditioned mouse model using immunostaining. xCELLigence and scratch assays were used to evaluate migration and MTS was used to evaluate cell viability. Results Human renal cell carcinoma sunitinib-conditioned cell lines showed upregulation of netrin-1 in microarray and q-PCR. Increased migration was demonstrated in Caki-1 sunitinib-conditioned cells when compared to the non-treated ones as well as, increased endothelial cell migration. Silencing of netrin-1 in sunitinib-conditioned Caki-1 cells did not demonstrate a significant reduction in cell migration. Conclusion Netrin-1 is highly upregulated in renal cell carcinoma treated with sunitinib, but has no influence on cell viability or cell migration in metastatic RCC.

MP60-07 THE CALCIUM-SENSING RECEPTOR (CASR) IS RESPONSIBLE FOR THE DEVELOPMENT OF BONE METASTASIS IN RENAL CANCER

INTRODUCTION AND OBJECTIVES: We previously reported that high MET and matriptase expression in RCC cells in bone metastasis indicates their importance in bone metastasis (Mukai et al. Hum Cell, 2015). MET is a high-affinity receptor tyrosine kinase of hepatocyte growth factor (HGF). HGF is secreted as an inactive singlechain precursor, which requires proteolytic activation for conversion to an active form. Matriptase is the most efficient known cellular activator of pro-HGF. Furthermore, activation of matriptase is regulated by HGF activator inhibitor (HAI). In this study, we employed a previously reported mouse model of bone metastasis (Strube et al. Clin Exp Metastasis, 2010) to clarify the significance of the matriptase-induced HGF/MET signaling axis in RCC bone metastasis. METHODS: Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of female nude mice (5 weeks old). After 6 weeks, we confirmed the formation of bone metastasis by whole-body bioluminescent imaging, and extracted specimens. Expression of matriptase, MET and HAI was analyzed by PCR, immunohistochemistry (IHC) and immunoblots. Phosphorylation of MET was also investigated. Based on the result, we produced HAI-2 (specific inhibitor of matriptase) stable knock down (KD) 786-O cells, and analyzed the difference of expression in each molecule, cell-migration assay and invasion assay. RESULTS: Expression of matriptase was increased significantly in bone metastasis compared with parent cell line, and we confirmed increased phosphorylation of MET in bone metastasis. On the other hand, decreased expression of HAI-2 was observed in bone metastasis. Interestingly, increased matriptase expression was observed by HAI-2 KD in 786-O cells. In addition, invasive activity was increased significantly by knock down of HAI-2. CONCLUSIONS: These results have suggested that matriptase contributes to the HGF-dependent MET activation in the pericellular microenvironment of bone metastasis in RCC. In addition, upregulation of matriptase and downregulation of HAI-2 may have important roles in their progression.

Using PDX for Biomarker Development

Our rapidly expanding understanding of the molecular landscape of all cancers is driving not only drug discovery but also biomarker development. Especially next-generation sequencing of RNA and DNA and also other high-throughput technologies have provided the necessary tools to determine which genes and pathways are driving tumor growth and progression and, as a result, which genes and pathways need to be targeted for therapeutic intervention. The alterations in the genes and pathways themselves become predictive markers that can be used to identify which patients will benefit from targeted interventions. In this context, patient-derived xenografts have become a critical tool in biomarker development.

Abstract 3080: Y-box binding protein 1 is crucial in acquiring drug resistance in advanced renal cell carcinoma

Objective: Investigating the role of Y-box binding protein 1 (YB1) in drug resistant advanced kidney cancer. Background: Renal cell carcinoma (RCC) is the 6th most common malignancy with approximately 1,800 deaths in 2015 and 2.3% annual increase in Canada. Despite the partial or total surgical removal of kidney in patients with localized RCC, metastatic patients are treated with tyrosine kinase inhibitors (TKIs) in a purely palliative approach. However, TKI-resistance (Sunitinib) is developed after a median time of 10-14 months. Therefore, identifying the factor(s) responsible for TKI-resistance development and disease advancement in RCC is imperative. It is now widely recognized that evolutionarily conserved Y-box binding protein 1 (YB1) is essential for cell growth and survival. Upregulation of YB1 in numerous cancer types was found to be positively correlated with tumor growth, metastasis and drug-resistance development. YB1 is also involved in intercellular communication through its secretion in the tumor microenvironment by cell-surface transporters, ABC-transporters. Summary of the data: Endothelial cells (HUVEC) were co-cultured with Caki-1DC (Sunitinib-resistant, developed in our lab) and Caki-1WT (Sunitinib-sensitive) RCC cell-lines, and increased migration of HUVEC was observed with Caki-1DC compared to Caki-1WT. A drastic increase in YB1 and its downstream target ABCB1 in Caki-1DC compared to Caki-1WT cells was also detected. Consistent with previous reports, we observed granular structures in the Caki-1DC cells that support potential secretion of YB1 into the tumor microenvironment. Moreover, blocking ABCB1 reverted the Caki-1DC cells to being drug-sensitive. Experimental procedures: Caki-1WT and Caki-1DC were co-cultured with HUVEC cells followed by scratch assay to test for HUVEC cell migration and associated secretory factors. Western blot and qPCR were used to determine the protein and mRNA levels respectively between the two cell-lines. Immuno-histochemical staining was carried out on the RCC tumors from Sunitinib-sensitive and resistant mouse models (developed in our animal facility). Granular structures were observed using immunofluorescence staining against YB1. Presto-blue was used for cell biomass assay following different drug treatments. Summary: The molecular function of YB1 in RCC and its potential in targeted therapy is not well understood. Therefore, understanding the function of YB1 in metastatic RCC and in drug-resistance development is of vital importance. Our data suggests that inhibition of YB1 may slow disease progression and, possibly, revert the drug resistance mechanism. The results from this study have the potential to introduce YB1 inhibitors in conventional RCC chemotherapy, alone or in combination, to improve survival in advanced kidney cancer patients. Citation Format: Ninadh M. D9Costa, Peter Raven, Zheng Tan, Werner Struss, Sebastian Frees, Claudia Chavez-Munoz, Alan I. So. Y-box binding protein 1 is crucial in acquiring drug resistance in advanced renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3080. doi:10.1158/1538-7445.AM2017-3080