Michelle Muldong

Tumor infiltrating B-cells are increased in prostate cancer tissue

BackgroundThe presence of increased B-cell tumor infiltrating lymphocytes (TILs) was seen in mouse prostate cancer (PCa) but has not been fully documented in human PCa. We, therefore, investigated the density of infiltrating B cells within human PCa utilizing a quantitative computational method.MethodsArchived radical prostatectomy specimens from 53 patients with known clinical outcome and D’Amico risk category were obtained and immunohistochemically (IHC) stained for the B cell marker, CD20. Slides were reviewed by a genitourinary pathologist who manually delineated the tumoral regions of PCa. Slides were digitally scanned and a computer algorithm quantified the area of CD20 stained B-cells as a measure of B cell density within the outlined regions of prostate cancer (intra-tumoral region), versus extra-tumoral prostate tissue. Correlations were analyzed between B-cell density and demographic and clinical variables, including D’Amico risk groups and disease recurrence.ResultsFor the entire cohort, the mean intra-tumoral B cell density was higher (3.22 SE = 0.29) than in the extra-tumoral region of each prostatectomy section (2.24, SE = 0.19) (paired t test; P < 0.001). When analyzed according to D’Amico risk group, the intra-tumoral B cell infiltration in low risk (0.0377 vs. 0.0246; p = 0.151) and intermediate risk (0.0260 vs. 0.0214; p = 0.579) patient prostatectomy specimens did not show significantly more B-cells within the PCa tumor. However, patient specimens from the high-risk group (0.0301 vs. 0.0197; p < 0.001) and from those who eventually had PCa recurrence or progression (0.0343 vs. 0.0246; p = 0.019) did show significantly more intra-tumoral CD20+ B-cell staining. Extent of B-cell infiltration in the prostatectomy specimens did not correlate with any other clinical parameters.ConclusionsOur study shows that higher B-cell infiltration was present within the intra-tumoral PCa regions compared to the extra-tumoral benign prostate tissue regions in prostatectomy sections. For this study we developed a new method to measure B-cells using computer-assisted digitized image analysis. Accurate, consistent quantitation of B-cells in prostatectomy specimens is essential for future clinical trials evaluating the effect of B cell ablating antibodies. The interaction of B-cells and PCa may serve as the basis for new therapeutic targets.

PCSD1, a new patient-derived model of bone metastatic prostate cancer, is castrate-resistant in the bone-niche.

IntroductionProstate cancer bone metastasis occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with therapy resistance and rapid decline. Androgen deprivation therapy (ADT) is standard of care for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to ADT. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. Here we report the castrate resistant growth in the bone niche of PCSD1, a patient-derived intra-femoral xenograft model of prostate bone metastatic cancer treated with the anti-androgen, bicalutamide.MethodsPCSD1 bone-niche model was derived from a human prostate cancer femoral metastasis resected during hemiarthroplasty and serially transplanted into Rag2−/−;γc−/− mice intra-femorally (IF) or sub-cutaneously (SC). At 5 weeks post-transplantation mice received bicalutamide or vehicle control for 18 days. Tumor growth of PCSD1 was measured with calipers. PSA expression in PCSD1 xenograft tumors was determined using quantitative RT-PCR and immunohistochemistry. Expression of AR and PSMA, were also determined with qPCR.ResultsPCSD1 xenograft tumor growth capacity was 24 fold greater in the bone (intra-femoral, IF) than in the soft tissue (sub-cutaneous, SC) microenvironment. Treatment with the anti-androgen, bicalutamide, inhibited tumor growth in the sub-cutaneous transplantation site. However, bicalutamide was ineffective in suppressing PCSD1 tumor growth in the bone-niche. Nevertheless, bicalutamide treatment of intra-femoral tumors significantly reduced PSA expression (p < =0.008) and increased AR (p < =0.032) relative to control.ConclusionsPCSD1 tumors were castrate resistant when growing in the bone-niche compared to soft tissue. Bicalutamide had little effect on reducing tumor burden in the bone yet still decreased tumor PSA expression and increased AR expression, thus, this model closely recapitulated castrate-resistant, human prostate cancer bone metastatic disease. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study bone metastatic disease and for pre-clinical drug development of novel therapies for inhibiting therapy resistant prostate cancer growth in the bone-niche.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer

Objective Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. Methods Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2−/−γc−/− male mice. Results Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. Conclusion PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Abstract B23: Novel epidermal growth factor receptor inhibitors cross the blood-brain barrier and inhibit the growth of metastatic non-small cell lung cancer

Deaths from solid tumors are often not due to the primary lesion but to metastatic disease at distal sites such as the lung, liver, and brain. Patients with non-small cell lung cancer (NSCLC) experience brain metastases, a poor prognostic marker, at an incidence rate of 30-50%. A significant proportion of the metastatic tumors express activating mutations of the EGFR, including exon 19 deletions as well as point mutations within the enzyme active site (L858R), which confer increased sensitivity to EGFR inhibitors. However, current small-molecule therapeutics poorly inhibit the growth of NSCLC brain metastases due to difficulty crossing the blood-brain barrier and harmful off-target effects. Thus, NSCLC patients with brain metastases have access to very few emerging treatment options. We have synthesized a novel class of compounds that irreversibly inhibit the epidermal growth factor receptor (EGFR) in the nanomolar range in vitro, and demonstrate a high degree of selectivity for EGFR mutants found frequently in NSCLC, in particular exon 19 deletions and L858R mutations, while sparing wild-type (WT) EGFR. Two compounds, LL-001 and LL-019, from Capella Therapeutics, Inc., inhibited L858R EGFR-mediated autophosphorylation and phosphorylation of downstream targets Akt and ERK MAP kinase at a concentration of approximately 100nM and blocked the kinase activity of an EGFR mutant, T790M, which confers resistance to first-generation EGFR inhibitors. In contrast, these compounds only weakly inhibited WT EGFR and failed to inhibit either the insulin receptor or insulin-like receptor-I, suggesting that these inhibitors would produce minimal off-target side effects in treated patients. Both compounds induced full remission of subcutaneous tumors with the human NSCLC cell line H1975 (T790M/L858R+). Most notably, pharmacokinetic studies showed that these compounds, when administered at a concentration of 150mg/kg in mice, showed no signs of toxicity and were found in the brain at concentrations ranging from 12-171 times higher than the GI50 for inhibition of EGFR+ NSCLC cell line proliferation in vitro. To determine whether these compounds could treat NSCLC brain metastases, we developed intracranial and intracardiac injection orthotopic xenograft models of brain metastatic NSCLC. LL-001 and LL-019 could induce remission of brain tumors in mice injected intracranially with the human NSCLC cell line HCC827-luciferase, which expresses an exon 19 deletion mutant of EGFR. Likewise, both compounds inhibited the growth of the human NSCLC cell line PC9M in an intracardiac model of brain metastatic NSCLC, and greatly extended the survival of mice compared to those receiving vehicle alone. Our results indicate that LL-001 and LL-019 can cross the blood-brain barrier at levels sufficient to inhibit the growth of brain metastatic lung cancer, with minimal deleterious off-target effects. These compounds show promise for the treatment of critically underserved NSCLC patients with brain metastases. Citation Format: Michelle Muldong, Christina Jamieson, Yun Oliver Long, Ida Deichaite, David W. Anderson, Alan Lewis, Nicholas A. Cacalano. Novel epidermal growth factor receptor inhibitors cross the blood-brain barrier and inhibit the growth of metastatic non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B23.

Abstract B49: Novel epidermal growth factor receptor inhibitors cross the blood-brain barrier and inhibit the growth of metastatic non-small cell lung cancer

Deaths from solid tumors are often not due to the primary lesion but to metastatic disease at distal sites such as the lung, liver, and brain. Patients with non-small cell lung cancer (NSCLC) experience brain metastases, a poor prognostic marker, at an incidence rate of 30-50%. A significant proportion of the metastatic tumors express activating mutations of the EGFR, including exon 19 deletions as well as point mutations within the enzyme active site (L858R), which confer increased sensitivity to EGFR inhibitors such as erlotinib, gefitinib, and AZD9291. Despite successful use of small-molecule kinase inhibitors for the treatment of EGFR+ primary lung tumors, current therapeutics poorly inhibit the growth of NSCLC brain metastases due to difficulty crossing the blood-brain barrier. For this reason, NSCLC patients with brain metastases are often excluded from clinical trials with novel therapies and thus have access to very few emerging treatment options. We have synthesized a novel class of compounds that inhibit the epidermal growth factor receptor (EGFR) in the nanomolar range in vitro, and demonstrate a high degree of selectivity for EGFR family members as well as EGFR mutants found frequently in NSCLC, including exon 19 deletions and L858R mutations, while sparing wild-type (WT) EGFR. Two compounds, LL-001 and LL-019, from Capella Therapeutics, Inc., inhibited EGFR-mediated autophosphorylation and phosphorylation of downstream targets Akt and ERK MAP kinase at a concentration of approximately 100nM and blocked the kinase activity of an EGFR mutant, T790M, which confers resistance to front-line EGFR inhibitors such as Tarceva (erlotinib). Both compounds induced full remission of subcutaneous tumors using the human NSCLC cell line H1975 (T790M/L858R+). Most notably, pharmacokinetic studies showed that these compounds, when administered at a concentration of 150mg/kg in mice, showed no signs of toxicity and were found in the brain at concentrations ranging from 12-171 times higher than the GI50 for inhibition of EGFR+ NSCLC cell line proliferation in vitro. In order to determine whether these compounds could effectively treat NSCLC brain metastases, we developed intracranial and intracardiac injection orthotopic xenograft models for the study of brain metastatic NSCLC. We found that LL-001 and LL-019 could induce remission of brain tumors in mice injected intracranially with the human NSCLC cell line HCC827-luciferase, which expresses an exon 19 deletion mutant of EGFR. Likewise, both compounds inhibited the growth of the human NSCLC cell line PC9M in an intracardiac model of brain-metastatic NSCLC, and greatly extended the survival of mice compared to those receiving vehicle alone. Our results indicate that LL-001 and LL-019 can cross the blood-brain barrier at levels sufficient to inhibit the growth of brain-metastatic lung cancer and show promise for the treatment of a critically underserved NSCLC patient population with brain metastases. Citation Format: Michelle Muldong, Christina AM Jamieson, Ida Deichaite, Alan Lewis, David W. Anderson, Yun Oliver Long, Nicholas A. Cacalano. Novel epidermal growth factor receptor inhibitors cross the blood-brain barrier and inhibit the growth of metastatic non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B49.

MP87-10 GENOMIC ANALYSIS OF A LONGITUDINAL SERIES OF SURGICAL PROSTATE CANCER BONE METASTASES AND XENOGRAFTS FROM THE SAME PATIENT REVEALED SELECTION OF A PROGRESSIVELY THERAPY RESISTANT METASTATIC CLONE

METHODS: We used a panel of cell lines i.e 786-0, A498 (VHL -) and Caki-1, Caki-2, ACHN (VHL +) for CCC and LNCaP, VCap, PC3, DU145, 22RV1 for PCa. Tumor/normal corresponding tissues pairs from 42 and 41 CCC and PCa patients, respectively, were also used. DNPH1 isoforms expression was measured by RTqPCR and Western blot. Since no specific chemical inhibitors are available, the role of DNPH1 isoforms in vitro and in vivo was evaluated using siRNAs and expressing vectors (wild-type or inactive, dead active site). In vivo, we used the tumor xenografted nude mice model to assess the role and the underlying mechanisms of DNPH1 in tumor growth. RESULTS: We show that CCC and PCa express only isoforms 1 and 2. These are 174 and 148 aminoacids long, respectively, and are identical till aminoacid 126. DNPH1 expression was deregulated in 71% of CCC cases but upregulated in 75% of PCa cases, regardless of the stage. Both isoforms behave similarly but isoform 1 represented 80% of total DNPH1 expression. In CCC cell lines, the transfection with wildtype DNPH1 expressing vectors decreased cell growth up to 60% by stimulating cell proliferation and inhibiting apoptosis, while the transfection with the inactive vector had no effect. In PCa cells, siRNAs targeting DNPH1 isoforms 1 and 2 decreased cell growth dosedependently by up to 50% through inhibition of cell proliferation and induction of apoptosis. We are currently evaluating the effect of both isoforms in nude mice xenografted with CCC cell lines transfected with the DNPH1 expressing vector and in PCa-bearing nude mice treated with in vivo siRNAs. First results corroborate in vitro observations. CONCLUSIONS: Our results show the opposite tumor suppressor/oncogene properties of DNPH1 in CCC and PCa and should allow to design new therapeutic options for these refractory diseases.

Abstract A43: Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer

Prostate cancer metastasis to bone occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with disease progression, therapy resistance, poor prognosis, and rapid decline. Androgen ablation therapy is standard of care for advanced prostate cancer, however, the role of androgens in bone metastatic prostate cancer is not understood. The effects of anti-androgens as seen on bone scans can also be inconsistent with the biochemical PSA response. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. It is essential to understand the unique interaction of prostate cancer with the bone environment and to develop novel therapies that target these pathways. Here we report the development of novel patient-derived intra-femoral xenograft models of prostate bone metastatic cancer. METHODS: Surgical prostate cancer bone metastasis specimens were transplanted by direct injection into the femurs of Rag2-/-γc-/- mice or sub-cutaneously into the right flank. Patient-derived xenograft (PDX) tumors that grew out were analyzed for prostate cancer biomarker expression using quantitative RT-PCR and immunohistochemistry. Bone lesion formation was measured using micro-computed tomography (μCT). RESULTS: Prostate cancer surgical bone metastasis specimens have been collected from which we have established new serially transplantable, prostate cancer bone metastasis xenograft models – PCSD1, PCSD4 and PCSD5. PCSD1 (Prostate Cancer San Diego 1) was molecularly characterized as advanced, luminal epithelial-type prostate cancer. PCSD1 intra-femoral xenografts formed mixed osteoblastic/osteolytic lesions that closely mimicked those of the patient. Treatment with the anti-androgen, bicalutamide, did not inhibit intra-femoral PCSD1 xenograft growth although there was a decrease in PSA as seen in some patients treated with anti-androgen who had discordant PSA and bone scan tests. CONCLUSION: PCSD1, PCSD4 and PCSD5 are new patient-derived prostate cancer bone metastasis-derived xenograft models. PCSD1 xenograft model closely recapitulates the mixed osteolytic/osteoblastic bone metastatic lesions seen in patients, and we are using it to develop novel therapies for inhibiting prostate cancer growth in the bone-niche. Citation Format: Christina Jamieson, Christina Wu, Amy Strasner, Jason R. Woo, Michelle Muldong, Young B. Jeong, Michael A. Liss, Omer Raheem, Tomonori Yamaguchi, Heather Leu, Deborah Marshall, Sheldon Morris, Nicholas A. Cacalano, Koichi Masuda, Catriona H.M. Jamieson, Anna A. Kulidjian, Christopher J. Kane. Novel prostate cancer patient-derived xenograft models of bone metastatic castrate-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A43.