Growth curves for subcutaneous UOK360 tumor-bearing mice treated with vehicle or erlotinib.

STR of original and patient tissue and preclinical models of FH-RCC1X and RMC32X.

10x images of FH-RCC1X and RMC32X tissues stained with Ki67, CD34 and CD31.

Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare and highly aggressive cancers. Although the combination of VEGF inhibition by bevacizumab and EGFR inhibition by erlotinib is clinically used for both diseases, the differential effect of each component has not been investigated. Transcriptomic profiling revealed that RMC and FH-deficient tumor tissues demonstrate increased EGFR but not VEGF expression compared with adjacent normal kidney. Subsequent in vitro studies revealed that RMC and FH-deficient cell lines are sensitive to erlotinib treatment, whereas clear-cell RCC cell lines are resistant. We developed patient-derived xenograft (PDX) models of tumors exposed to first-line therapies to represent treatment-experienced RMC and FH-deficient RCC models. These models were then used to determine tumor growth response to angiogenesis inhibition by bevacizumab alone or in combination with erlotinib. The FH-deficient RCC PDX model responded to either bevacizumab or erlotinib alone or in combination, whereas the RMC PDX model responded only to erlotinib, consistent with clinical and preclinical data suggesting that RMC is refractory to angiogenesis inhibition. Statistically higher expression of EGFR was observed in the RMC PDX model compared with the FH-deficient model, whereas higher phosphorylated tyrosine-416 SRC expression was observed in the FH-deficient PDX model compared with the RMC model. Our preclinical data suggest that EGFR signaling differentially modulates tumor growth in RMC and FH-deficient RCC and that angiogenesis inhibition is a valid target in FH-deficient RCC but not RMC.

PDF file 142K, Supplemental Materials and Methods, Supplemental Table S1-S2, and Supplemental Figure Legends Supplemental Table 1. Quantitative analysis of ENPP2 mRNA expression in tumor and normal renal endothelial cells. Endothelial cells from human RCC specimens (T) and their normal counterparts (N) were isolated from a single-cell suspension using anti-CD31-coated magnetic beads. Total RNA was prepared and real-time RT-PCR was performed for ENPP2 and GAPDH. Supplemental Table 2. The expression level of LPA1-4 is determined by real-time PCR. Part of results was confirmed by western blot. +++, average Ct < 31; +, average Ct ranges from 31 to 38; -, average Ct > 38 when average Ct of GAPDH ranges between 18 and 21, which is served as an internal control

PDF file 353K, Supplemental Figure 1. Identification of ATX/ENPP2 in sunitinib-treated RCC endothelium. (A) Gene expression analyses of known endothelial markers between anti-CD31-coated magnetic beads-bound renal endothelial population and unbound renal cells. (B) A cluster with the most significant difference in endothelial gene expression between sunitinib-treated and -untreated human RCC specimens. Supplemental Figure 2. LPA induces cell proliferation in RCC, but not in HUVECs. UMRC3 and HUVECs were cultured with medium only (CTL), 20 microM LPC, or 1 microM LPA. At the indicated times, cell proliferation was measured by MTT assay. Results are shown as percent of untreated control. Supplemental Figure 3. LPA stimulation of pro-angiogenic factor expression in RCC cells. RCC cell lines (UMRC3, 786-O, and Caki-1) and primary culture (HRC-223) were serum-starved for 24 hours and treated with 1 microM LPA for 3 hours. Total RNA was prepared and real-time PCR was performed for the indicated genes. Data are expressed as mean {plus-minus} S.D. of three replicates and are representative of two separate experiments. Supplemental Figure 4. Establishment of RCC xenograft models with the acquired resistance to sunitinib. (A) Animals were injected s.c. into the flanks with UMRC3 and treated daily with water (control) and 40 mg/kg sunitinib by oral gavage. Tumor measurement was performed twice per week. Upon the treatment, tumor growth showed an initial plateau during which the tumors stopped growing and then began to actively shrink. This period of active shrinkage is referred to as the sensitive phase. Tumors that shrank initially when the treatment began and showed a long-term trend toward continued growth later, were considered sunitinib-resistant. Arrowhead indicates the time point at which sunitinib treatment began. (B) Tumors under the sensitive and resistant phase of treatment were collected and subjected to immunohistochemistry by using monoclonal antibodies against CD34. (C) The microvascular density was calculated from three independent tumor sections per group and expressed as the mean plus-minus S.D. (Student's t-test; *, p<0.05 compared with untreated controls); scale bars, 50 microm. Supplemental Figure 5. Effects of Sunitinib on sprouting angiogenesis. (A-C) HUVECs were allowed to invade into 3D collagen matrices (2.5 mg/ml) containing 0.1 microM S1P in the absence (CTL) or presence of 40 ng/ml VEGF, IL-8, or bFGF combined with 0.1 microM or 1 microM Sunitinib. After 24 hours, cultures were fixed, stained, photographed (A) and quantified for invasion density (B), distance (C). For invasion density, data represent average numbers of invading cells per standardized field (n=3 fields). For invasion distance, data from 20 cells were averaged and are presented as means {plus-minus} SE. (**P<0.01 compared with vehicle control using Student t-test; nd; not determined)

Background: Penile Cancer (PeCa) is a rare cancer with over 90% squamous cell carcinoma (SCC)histology. Risk factors include human papillomavirus (HPV) infection and phimosis. Molecular oncogenic pathways between HPV-Positive and HPV-Negative PeCa are difficult to validate because there are few preclinical models of PeCa and no models of HPV-Positive PeCa. Our goal was to establish PeCA patient-derived xenograft (PDX) models to elucidate the molecular signature of HPV-Positive and HPV-Negative PeCa for therapeutic benefit. Methods: PeCa specimens from penectomies and inguinal lymph node dissections (ILND) were collected from 2019-2021. Tumor specimens were dissected and tissue coated in Matrigel and transplanted subcutaneously into NOD SCID gamma (NSG™) mice. Tumor growth was monitored and was harvested at ≈ 1500m3 for subsequent propagation. Short tandem repeat fingerprinting (STR) analysis was performed on genomic DNA from PDX tissue and parental patient tumor tissue. Immunohistochemistry was performed on all PDX models and original patient primary tumors to validate the expression of p16 protein, a surrogate marker for HPV-Positive disease. p16 positivity was determined using a 0-3 scoring where a score of 3 was assessed as positive and a score of ≤2 as negative. For HPV genotyping: PCR was performed using DNA ELISA kit HPV, followed by RHA Kit HPV SPF10-LiPA25. Results: 8 PDXs were established from tissue from 7 patients. Age, race, and type of surgery were determined. SCC histology was confirmed in all the patients. Basaloid variant was present in 3 patients, and Verrucous variant was present in 1 patient. For pathological staging, 2 patients were staged as pT3N3Mx, 2 patients as pT2N1M0, 1 patient as pT3N0 and 3 patients as pT3Nx. One ILND had no evidence of disease. 2 PDX models (MDAPe3 and MDAPe7) were derived from two specimens from the same patient (1 pretreatment, 1 post-treatment). All models were matched to the patient's primary tumor by STR fingerprinting analysis. Squamous cell carcinoma histology was confirmed in all PDXs. 6 PDX models are currently in passage 3 and 6 PDXs were p16 positive. HPV genotyping in p16 positive PDXs detected high-risk HPV 16 and p16 negative xenografts were also HPV negative. Conclusion: In our study, we established PeCa PDX models that recapitulated the patients' histology. We report to our knowledge the first HPV-positive penile cancer xenografts. Plans for comprehensive characterization and future targeted therapy experiments are in development. Citation Format: Luis A. Segarra, Niki M. Zacharias, Alberto Pieretti, Angelita Alaniz, Tapati Maity, Sue Martinez, Priya Rao, Natalie Fowlkes, Jad Chahoud, Xin Lu, Magaly Martinez Ferrer, Christopher Wood, Curtis Pettaway. Establishment of patient-derived xenografts in penile cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3111.

Purpose Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[F-18]fluoroglutamine ([F-18]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [F-18]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results In UMRC3 cells, [F-18]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60-90% in the presence of excess glutamine in the media and by 20-50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [F-18]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [F-18]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [F-18]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [F-18]FGln retention did not change significantly upon treatment with CB-839. Conclusions We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [F-18]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [F-18]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

Renal medullary carcinoma (RMC) and fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) are rare but highly aggressive malignancies that are often refractory to the therapies used for the more common RCCs. FH-deficient RCC most often occurs in individuals with hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC) and is characterized by loss of FH activity, a key metabolic enzyme. Bevacizumab plus erlotinib (B+E) is a preferred first-line therapy for FH-deficient RCC. RMC is characterized by loss of SMARCB1, which is involved in ATP-dependent chromatin remodeling. Platinum-based chemotherapy consisting of either cisplatin or carboplatin plus paclitaxel is the preferred first-line therapy for RMC. We developed patient-derived xenograft (PDX) models of tumors exposed to the commonly used first-line therapies to allow functional characterization of the metabolic hallmarks of treatment-experienced RMC and FH-deficient RCC. Methods: Resected tumor tissue was immediately implanted into NSG male and female mice. After tumor engraftment, animals were euthanized, and the tumor tissue implanted into another set of NSG mice. PDX tissues were confirmed to be genetically identical to the original patient tissue by short tandem repeat analysis. Histology was confirmed to be identical between original patient tumor and PDX by a trained clinical pathologist. Four tumors from each PDX model and four normal human kidney tissue samples were analyzed using reverse phase protein array (RPPA). Expression of proteins shown to be highly expressed by RPPA were further validated by Western blots. Results: We successfully generated one PDX model for RMC and one for FH-deficient RCC, respectively. The RMC model was generated from a 28-year old male with sickle cell trait, who had previously received cisplatin plus paclitaxel followed carboplatin plus paclitaxel. His lesion was characterized as ypT3apN1pMx and histology was consistent with RMC. The FH-deficient RCC model was generated from a 24-year-old female with HLRCC, who had been previously treated with B+E. Her lesion was characterized as pT3apN1pM1 and histology consistent with FH-deficient RCC. Germline testing revealed a R233H pathogenic mutation in the FH gene. RPPA revealed higher expression of hexokinase II (10x HLRCC, 16x RMC), lactate dehydrogenase A (27x HLRCC, 17x RMC), and glutaminase (4x HLRCC) in PDX tissue compared to normal kidney tissue. Western blots confirmed higher expression of glutaminase 1 in both HLRCC and RMC compared to normal kidney. Furthermore, we observed substantially higher hypoxia inducible factor 2α (HIF2α) protein expression in PDX tissue from FH-deficient RCC compared to RMC PDX tumors and normal kidney. Conclusions: Our study revealed distinct metabolic features in PDX models of FH-deficient RCC following treatment with B+E, and of platinum-experienced RMC, these differences may confer targetable vulnerabilities. Citation Format: Manuel Ozambela, Alberto Pieretti, Graciela M. Nogueras Gonzalez, Tapati Maity, Lei Wang, Carolyn De La Cerda, Breanna Alonzo, Luis Segarra, Angelita Alaniz, Priya Rao, Nizar Tannir, Jose A. Karam, Christopher G. Wood, Pavlos Msaouel, Niki M. Zacharias. Metabolic hallmarks of rare renal cell carcinoma patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3112.

Purpose Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2 S ,4 R )-4-[ 18 F]fluoroglutamine ([ 18 F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. Procedures We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [ 18 F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. Results In UMRC3 cells, [ 18 F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60–90% in the presence of excess glutamine in the media and by 20–50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [ 18 F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [ 18 F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [ 18 F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [ 18 F]FGln retention did not change significantly upon treatment with CB-839. Conclusions We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [ 18 F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [ 18 F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

Von Hippel Lindau (VHL) inactivation, which is common in clear cell renal cell carcinoma (ccRCC), leads directly to the disruption of oxygen homoeostasis. VHL works through hypoxia-inducible factors (HIFs). Within this VHL-HIF system, prolyl hydroxylases (PHDs) are the intermediary proteins that initiate the degradation of HIFs. PHD isoform 3′s (PHD3) role in ccRCC growth in vivo is poorly understood. Using viral transduction, we knocked down the expression of PHD3 in the human ccRCC cell line UMRC3. Compared with control cells transduced with scrambled vector (UMRC3-SC cells), PHD3-knockdown cells (UMRC3-PHD3KD cells) showed increased cell invasion, tumor growth, and response to sunitinib. PHD3 knockdown reduced HIF2α expression and increased phosphorylated epidermal growth factor (EGFR) expression in untreated tumor models. However, following sunitinib treatment, expression of HIF2α and phosphorylated EGFR were equivalent in both PHD3 knockdown and control tumors. PHD3 knockdown changed the overall redox state of the cell as seen by the increased concentration of glutathione in PHD3 knockdown tumors relative to control tumors. UMRC3-PHD3KD cells had increased proliferation in cell culture when grown in the presence of hydrogen peroxide compared to UMRC3-SC control cells. Our findings illustrate (1) the variable effect of PHD3 on HIF2α expression, (2) an inverse relationship between PHD3 expression and tumor growth in ccRCC animal models, and (3) the role of PHD3 in maintaining the redox state of UMRC3 cells and their proliferative rate under oxidative stress.

Although genetic changes may be pivotal in the origin of cancer, cellular context is paramount. This is particularly relevant in a progenitor germ cell tumor and its differentiated mature teratoma counterpart when it concerns tumor heterogeneity and cancer dormancy in subsequent second malignancies (subsequent malignant neoplasms (SMNs)). From our tumor registry database, we identified 655 testicular germ cell tumor (TGCT) patients who developed SMNs between January 1990 and September 2018. Of the 113 solid organ SMNs, 42 had sufficient tumor tissue available for fluorescence in situ hybridization (FISH) analysis of isochromosome 12p [i(12p)]. We identified seven additional patients for targeted DNA and RNA sequencing of teratomas and adjacent somatic transformation. Finally, we established cell lines from freshly resected post-chemotherapy teratomas and evaluated the cells for stemness expression by flow cytometry and by the formation of teratomas in a xenograft model. In our cohort, SMNs comprising non-germ cell tumors occurred about 18 years after a diagnosis of TGCT. Of the 42 SMNs examined, 5 (12%) contained i(12p) and 16 (38%) had 12p gain. When comparing a teratoma and adjacent somatic transformation, targeted DNA and RNA sequencing demonstrated high concordance. Studies of post-chemotherapy teratoma-derived cell lines revealed cancer-initiating cells expressing multipotency as well as early differentiation markers. For the first time, we demonstrated the prevalence of i(12p) in SMNs and the presence of progenitor cells embedded within mature teratomas after chemotherapy. Our findings suggest a progenitor stem-like cell of origin in SMN and TGCT and highlight the importance of cellular context in this disease.

410 Background: TGCT is a prototype stem-cell malignancy recapitulating the ontogeny of solid tumors. We studied the roles of differentiation versus dedifferentiation in a bona fide well-differentiated tumor, namely mature teratomas, associated with or without somatic transformation. Methods: Between 2001 and 2013, we identified 7 out of 13 cases in which teratoma and somatic transformation were present within the same residual tumor after chemotherapy and resected at our institution with sufficient quantity and quality for DNA and RNA analysis. We performed primary cell culture, flow cytometry, and xenografts to evaluate stem-ness biomarkers and tumor phenotypes of freshly resected mature teratomas. Results: We detected very few mutations within a preselected gene pool (T200) in all tumor samples. The "within patient mutation agreement" from a mutation matrix of 57 genes, in which at least one mutation occurred between teratoma and somatic transformation, was 86.2 to 94.8%. There was disparity in gene expression, including miR7-3HG, ARHGEF35, and DLX6, between the 7 matched tumor pairs (log2 fold change of about 2). Prospective primary cell culture studies using cell surface stem-ness markers (SSEA3, TRA1-60, Cripto-1, CD90, CD133, CD44) indicate presence of stem cells embedded within residual mature teratomas (n = 3) after chemotherapy. Conclusions: Molecular profiling confirms a common clonal origin between teratoma and somatic transformation but refutes the idea of dedifferentiation of teratoma to somatic transformation. Presence of stem cells embedded within mature teratomas and capable of differentiation into separate ontogenetic lineages could account for the origin of somatic transformation in TGCT.[Table: see text]

592 Background: Zinc finger protein 395 (ZNF395) is frequently altered in several tumor types. However, the role of ZNF395 remains poorly studied in patients with clear cell renal cell carcinoma (RCC). In this study, we investigated the in vitro and in vivo role of ZNF395 in ccRCC. Methods: cBioPortal For Cancer Genomics was used to correlate the expression of ZNF395 with RCC patient clinical, pathological and molecular profiles. ZNF395 protein and mRNA levels were studied in several RCC cell lines in vitro. Subsequently, ZNF395 knockdown was performed in 786-O and UMRC3 RCC cells and overexpression was done in Caki-1 and 769-P RCC cells. We then evaluated ZNF395 modulation in these cell lines by in vitro MTT, migration and invasion assays. Finally, we studied the effect of ZNF395 knockout and overexpression in vivo using SCID xenograft models. Results: Patients with higher expression of ZNF395 experienced longer disease-free survival and overall survival. Using in vitro models, we confirmed that knockdown of ZNF395 decreased ZNF395 expression, and increased proliferation, migration and invasiveness of 786-O and UMRC3, while overexpression of ZNF395 increased ZNF395 expression, and reduced proliferation, migration and invasiveness of Caki-1 and 769-P. Using in vivo mouse models, knockdown of ZNF395 expression in 786-O promoted tumor growth while its overexpression in Caki-1 resulted in tumor growth inhibition. We are currently performing experiments to understand the process by which ZNF395 regulates ccRCC pathogenesis. Conclusions: Our data support the role of ZNF395 as an important tumor suppressor gene in the pathogenesis of RCC.

4516 Background: Previous studies have shown minimal impact of TKIs on primary renal tumor downsizing. Axitinib is a VEGFR TKI that has been recently approved for use in patients with metastatic clear cell renal cell carcinoma (RCC). In this prospective phase II trial, we sought to investigate the safety and role of axitinib in downsizing tumors in patients with non-metastatic renal cell carcinoma, prior to undergoing surgical resection. Methods: Patients with locally advanced (clinical stage T2-T3b N0 M0) biopsy-proven clear cell RCC were eligible for this phase II clinical trial. The primary outcome was objective response rate (using RECIST) following the administration of axitinib for 12 weeks prior to undergoing radical nephrectomy. Secondary outcomes included safety, tolerability, and feasibility of administration of axitinib in this patient population. Patients were given axitinib 5mg PO BID, and dose titration was allowed. Axitinib was continued until 36 hours prior to surgery. A dedicated radiologist independently reviewed all CT scans to evaluate for response using RECIST. Results: The study goal of enrolling 24 patients has been recently reached. At present, nineteen patients have completed the studies required for assessment of the primary outcome and are hereby reported. Fifteen patients were males, and four were females. Median age was 61 years (range 42-83 years). All patients had biopsy-proven clear cell RCC. All 19 patients continued axitinib for 12 weeks, and underwent surgery as planned without delay. Adverse events of any grade were: arthralgia in 6, hypothyroidism in 14, fatigue in 15, and hypertension in 16 patients. No wound complications occurred after surgery. Nine patients (47%) experienced a partial response by RECIST, and 10 patients had stable disease. There was no progression of disease while on axitinib. Conclusions: Axitinib is well tolerated in the neoadjuvant setting in patients with planned surgery for locally advanced non-metastatic clear cell RCC. The drug showed tumor downsizing activity when given for 12 weeks prior to surgery. Adverse events of any grade were common and easily manageable with routine care. Clinical trial information: NCT01263769.

You have accessJournal of UrologyKidney Cancer: Advanced1 Apr 2014MP57-13 PHASE II TRIAL OF NEOADJUVANT AXITINIB IN PATIENTS WITH LOCALLY ADVANCED NON-METASTATIC CLEAR CELL RENAL CELL CARCINOMA Jose Karam, Catherine Devine, Diana Urbauer, Marisa Lozano, Tapati Maity, Kamran Ahrar, Pheroze Tamboli, Nizar Tannir, and Christopher Wood Jose KaramJose Karam , Catherine DevineCatherine Devine , Diana UrbauerDiana Urbauer , Marisa LozanoMarisa Lozano , Tapati MaityTapati Maity , Kamran AhrarKamran Ahrar , Pheroze TamboliPheroze Tamboli , Nizar TannirNizar Tannir , and Christopher WoodChristopher Wood View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1788AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Previous studies have shown minimal impact of tyrosine kinase inhibitors on primary renal tumors. In this prospective phase II trial, we sought to investigate the safety and role of the tyrosine kinase inhibitor axitinib in downsizing tumors in patients with non-metastatic clear cell renal cell carcinoma (ccRCC), prior to undergoing surgical resection. Methods Patients with locally advanced (clinical stage T2-T3b N0 M0) biopsy-proven ccRCC were eligible for this study. Patients were given axitinib 5mg by mouth twice daily for 12 weeks, and dose titration was allowed. Axitinib was continued until 36 hours prior to surgery. The primary outcome was objective response rate (using RECIST) following the administration of axitinib for 12 weeks prior to undergoing radical or partial nephrectomy. Secondary outcomes included safety, tolerability, feasibility of administration of axitinib and quality of life in this patient population. A dedicated radiologist independently reviewed all CT scans to evaluate for response using RECIST. Trial was registered with clinicaltrials.gov as NCT01263769 and approved by IRB. Results Twenty-four patients were treated between May 2011 and April 2013. All patients had biopsy-proven clear cell RCC. Twenty-three patients continued axitinib for 12 weeks, and underwent surgery as planned without delay. One patient stopped treatment before 12 weeks due to adverse events (AEs) and was taken to surgery early. Median reduction of primary renal tumor size was 28.3% (range 5.3-42.9%). Eleven patients (45.8%) experienced a partial response by RECIST, and 13 patients had stable disease. There was no progression of disease while on axitinib. The most common AEs were hypertension, fatigue, oral mucositis, hypothyroidism, and hand-foot syndrome. No grade 4 AEs were observed. Intraoperatively, no complications or unusual bleeding were encountered. Postoperatively, 2 grade 3 and 13 grade 2 complications were noted, while no grade 4 or 5 complications occurred. FKSI did change over time (p < 0.0001), with quality of life worsening in comparison to the screening assessment by week 7 (p = 0.0004). However, by week 19, quality of life was not found to be statistically different from screening (p = 0.3344). Conclusions Axitinib was clinically active and reasonably well tolerated in the neoadjuvant setting in patients with locally advanced non-metastatic ccRCC. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e646 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Jose Karam More articles by this author Catherine Devine More articles by this author Diana Urbauer More articles by this author Marisa Lozano More articles by this author Tapati Maity More articles by this author Kamran Ahrar More articles by this author Pheroze Tamboli More articles by this author Nizar Tannir More articles by this author Christopher Wood More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract Purpose: Sunitinib is currently considered as the standard treatment for advanced renal cell carcinoma (RCC). We aimed to better understand the mechanisms of sunitinib action in kidney cancer treatment and in the development of acquired resistance. Experimental Design: Gene expression profiles of RCC tumor endothelium in sunitinib-treated and -untreated patients were analyzed and verified by quantitative PCR and immunohistochemistry. The functional role of the target gene identified was investigated in RCC cell lines and primary cultures in vitro and in preclinical animal models in vivo. Results: Altered expression of autotaxin, an extracellular lysophospholipase D, was detected in sunitinib-treated tumor vasculature of human RCC and in the tumor endothelial cells of RCC xenograft models when adapting to sunitinib. ATX and its catalytic product, lysophosphatidic acid (LPA), regulated the signaling pathways and cell motility of RCC in vitro. However, no marked in vitro effect of ATX-LPA signaling on endothelial cells was observed. Functional blockage of LPA receptor 1 (LPA1) using an LPA1 antagonist, Ki16425, or gene silencing of LPA1 in RCC cells attenuated LPA-mediated intracellular signaling and invasion responses in vitro. Ki16425 treatment also dampened RCC tumorigenesis in vivo. In addition, coadministration of Ki16425 with sunitinib prolonged the sensitivity of RCC to sunitinib in xenograft models, suggesting that ATX-LPA signaling in part mediates the acquired resistance against sunitinib in RCC. Conclusions: Our results reveal that endothelial ATX acts through LPA signaling to promote renal tumorigenesis and is functionally involved in the acquired resistance of RCC to sunitinib. Clin Cancer Res; 19(23); 6461–72. ©2013 AACR.

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011247 ASSOCIATION OF P95HER2 WITH TARGETED THERAPY RESISTANCE IN TYPE 2 PAPILLARY RENAL CELL CARCINOMA Stephen H. Culp, Xiu-Ying Zhang, Tapati Maity, and Christopher G. Wood Stephen H. CulpStephen H. Culp Houston, TX More articles by this author , Xiu-Ying ZhangXiu-Ying Zhang Houston, TX More articles by this author , Tapati MaityTapati Maity Houston, TX More articles by this author , and Christopher G. WoodChristopher G. Wood Houston, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.316AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Papillary Renal Cell Carcinoma (pRCC) is the second most common of all renal parenchymal tumors and is divided into two subtypes based on histologic and cytogenetic profiles. Although type 1 pRCC is more common, type 2 pRCC tumors tend to be higher grade and metastasize more often resulting in poorer patient prognosis. Although targeted therapy (TT) has improved progression-free survival in patients with conventional RCC, anecdotal reports show that TT response in advanced pRCC is poor. Our objective was to identify alternative receptor tyrosine kinase (RTK) pathways that may contribute to aggressiveness and TT resistance in type 2 pRCC. METHODS We previously established the MSC9 xenograft tumor model, representing advanced type 2 pRCC, and have shown that these tumors respond initially in vivo to TT (sunitinib and RAD001) with uniform development of TT resistance. To ascertain differences between TT-responsive and resistant tumors, athymic nude mice were implanted with bilateral subcutaneous tumors. The first tumor was harvested at day 14 after treatment initiation, corresponding to TT response, and the second tumor was harvested once there was evidence of TT resistance based on volume change in tumor growth. Western immunoblotting analysis was used to identify changes in protein expression and phosphorylation. RESULTS We identified changes in the HER2 RTK pathway with TT resistance. Specifically, expression and phosphorylation of full-length p185HER2 protein decreased in TT-responsive tumors and returned to baseline in TT-resistant tumors. More importantly, MSC9 tumors expressed, at baseline, truncated p95HER2 protein, a product not seen in primary kidney tumor or normal renal tissue. Both expression and phosphorylation of p95HER2 protein decreased significantly in TT-responsive tumors but returned to baseline with TT resistance. Finally, protein levels of the metallopeptidases ADAM17 and ADAM10 (involved in the cleavage of p185HER2 protein) were elevated at baseline in MSC9 tumors, decreased with TT response, and returned to baseline with TT resistance. CONCLUSIONS Our results demonstrate that the HER2 pathway and, more importantly, the presence of truncated p95HER2, likely contribute to baseline aggressiveness and TT resistance in type 2 pRCC tumors. In addition, production of truncated p95HER2 likely results from changes in ADAM10 and ADAM17 expression. Further research is needed to better define these alterations in the HER2 pathway and identify potential therapeutic targets for patients with advanced type 2 pRCC. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e100 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stephen H. Culp Houston, TX More articles by this author Xiu-Ying Zhang Houston, TX More articles by this author Tapati Maity Houston, TX More articles by this author Christopher G. Wood Houston, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 201080 ACTION OF TARGETED THERAPY ON PAPILLARY RENAL CELL CARCINOMA TUMOR MODELS Stephen H. Culp, Xiu-Ying Zhang, Tapati Maity, and Christopher G. Wood Stephen H. CulpStephen H. Culp More articles by this author , Xiu-Ying ZhangXiu-Ying Zhang More articles by this author , Tapati MaityTapati Maity More articles by this author , and Christopher G. WoodChristopher G. Wood More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.128AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Targeted systemic agents are the standard of care for metastatic renal cell carcinoma (RCC). Most studies, however, have evaluated the action of vascular endothelial growth factor receptor (VEGFR) or mammalian target of rapamycin (mTOR) inhibitors in patients with clear cell RCC (ccRCC) since these molecules target pathways altered by the mutation or deletion of the von-hippel landau protein. Few studies exist regarding the efficacy of these agents in advanced papillary RCC (pRCC). Our objective was to evaluate the action of sunitinib (a VEGFR inhibitor) and/or RAD001 (an mTOR inhibitor) on the in vitro and in vivo behavior of established types 1 and 2 pRCC tumor models. METHODS We have established tumor models from primary tumors representing types 1 (M48) and 2 (MSC9) pRCC that grow both in vitro and in vivo. For in vitro studies, cells were treated with increasing dosages of sunitinib (0.1 to 4uM) and/or RAD001 (1 to 100nM) for up to 7 days and growth and cell density determined by trypan blue exclusion and Sulforhodamine B In Vitro Toxicology assays, respectively. Wound healing assays were used to assess effects on cell migration. To determine the effect of sunitinib and/or RAD001 on in vivo growth, tumor xenografts were implanted bilaterally into the flanks of athymic nude mice. Mice were randomized into 1 of 4 groups and treated daily by gavage: Control (dH20 only), sunitinib (40mg/kg), RAD001 (5mg/kg), or a combination of sunitinib (20mg/kg) and RAD001 (2.5mg/kg). A single tumor was harvested on day 14 after treatment initiation and the second tumor harvested once there was evident resistance to therapy. RESULTS Sunitinib and RAD001 inhibited in vitro growth of both M48 and MSC9 cells in a dose-dependent manner with the optimum dose being 2uM for sunitinib and 10nM for RAD001. Sunitinib and RAD001 together resulted in an additive effect on growth inhibition. Cell migration was decreased in both cell lines by RAD001 or sunitinib. In vivo growth of M48 and MSC9 xenograft tumors was initially inhibited by sunitinib and/or RAD001. However, in vivo resistance to both agents uniformly occurred after approximately 25 days. CONCLUSIONS Sunitinib and RAD001 inhibit both the in vitro and in vivo growth of types 1 and 2 pRCC tumor models. Effectiveness of these agents in pRCC may be due in part to alterations in hypoxic pathways similar to what is observed in ccRCC. As with ccRCC, resistance to therapy uniformly occurred in both tumor models. Further research is needed to better define the mechanisms of resistance and proper sequence of therapy in advanced pRCC. Houston, TX© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e33-e34 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.Metrics Author Information Stephen H. Culp More articles by this author Xiu-Ying Zhang More articles by this author Tapati Maity More articles by this author Christopher G. Wood More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...