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Background: Despite a growing understanding of the somatic landscape of breast tumors from BRCA1 and BRCA2 mutation carriers, less is known about breast tumors from carriers of germline mutations in other homologous recombination and DNA repair pathway genes such as ATM, CHEK2, and PALB2. Methods: We identified 44 clinically annotated breast cancer cases that included carriers of germline mutations in BRCA1 (n=9), BRCA2 (n=9), ATM (n=5), CHEK2 (n=7), and PALB2 (n=6) from the Hereditary Cancer Risk Program at BUMC. Sporadic breast cancers cases (n=8) were also collected. Genomic DNA and RNA were extracted from macro-dissected FFPE tumor sections, adjacent normal FFPE tissue, along with constitutional genomic DNA from blood. Expanded whole exome sequencing (WES) was performed on normal/tumor pairs and RNA-seq from tumors for each case. Bioinformatics analysis was performed using industry standard methods for somatic characterization. Results: All germline mutations were confirmed by WES. Somatic mutational analysis and copy number profiling from WES revealed the greatest similarities among BRCA1 and CHEK2 carriers. As expected, TP53 mutations were found in 8 of 9 BRCA1 carriers as all were triple negative subtype. We also detected somatic TP53 mutations in tumors from 4 of 7 CHEK2 carriers. Somatic TP53 mutations were found in only 1 of 7 BRCA2 tumors and 1 of 4 PALB2 tumors tested. Furthermore, BRCA1 and CHEK2 tumors showed trends of having higher mutation burden. Analysis of copy number BRCAness demonstrated stronger similarities between BRCA1, ATM, CHEK2, and PALB2 tumors. BRCA2 tumors were unique with fewer events and characterized by specific amplifications including 11q23 (CCND1) and 17q23 (BRIP1). Hierarchical clustering of RNA-seq data revealed strong clustering of BRCA1 tumors compared to all other tumors, predominantly attributed to breast cancer subtype. Furthermore, pathway analysis of genes that distinguish BRCA1 mutation positive versus non-BRCA mutated tumors showed strong correlation to pro-inflammatory and immune pathway signatures. Conclusions: Molecular analysis of 44 breast cancers from individuals with inherited predisposition to breast cancer via BRCA1, BRCA2, ATM, CHEK2, and PALB2 germline mutations demonstrated strongest somatic similarities between BRCA1 and CHEK2 tumors although all BRCA1 were TNBC and all CHEK2 tumors were ER positive. Marked differential gene expression differences in RNA expression patterns were observed in BRCA1 mutation carriers compared with all other groups analyzed. Our study is among the first to interrogate the profile of non-BRCA mutated hereditary breast cancers. Citation Format: Blum JL, Wong S, Pearson EJ, Nair A, Snipes GJ, Briones N, Baker A, Cropp CD, Carpten JD. Molecular analysis of breast cancers from individuals with hereditary cancer syndromes secondary to mutations in BRCA1, BRCA2, ATM, CHEK2, and PALB2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-02.

Introduction: The purpose of the present study is to examine whether tumor extracellular pH (pHe) is a sensitive and specific biomarker for measuring immediate, short term drug response to an mTOR inhibiting agent (RAD001, everolimus) by a novel, non-invasive MRI-based pH imaging method (acidoCEST). Methods: Two groups of SCID mice with Granta 519 human mantle cell lymphoma flank xenograft models were used in the study. First, to investigate if RAD001 has anti-tumor activity in the Granta 519 model, mice were injected IP with 5 mg/kg per day with RAD001 or vehicle (DMSO) (N=12 per group) every day until tumors reached 2000 mm3. Survival analysis was conducted using GraphPad software. In a separate correlative imaging study mice with tumors averaging 400 mm3 (N=8) were scanned with acidoCEST MRI pretreatment, one day post initial treatment, and one week after continuous treatment. To prepare for each acidoCEST MRI scan session, a mouse was anesthetized with 1.5% isoflurane, respiration rate and body temperature were monitored, and body temperature was maintained at 37.0᠑C with warm air. A bolus of 200 μL of 370 mgI/mL iopamidol was injected IV prior to scanning, followed by continuous infusion of iopamidol at 150 uL/hour during scanning. A CEST-FISP MRI protocol was performed with a Bruker Biospec 7T MRI scanner with a 72 mm volume coil using previously published procedures. The results were analyzed using Matlab, following previously published procedures. The average pHe was determined for each tumor. Pixels which registered a pHe of 6.00 to 6.99 were grouped together as the “acidic fraction,” and those pixels with pH ≥7 were collectively grouped together as the “neutral fraction.” The percent uptake of the contrast agent in the tumor area was measured to assess vascular perfusion in the tumor microenvironment. Results: RAD001 treatment results in a significant survival advantage in Granta 519 flank xenograft tumors. AcidoCEST imaging of tumors showed pretreatment an average pHe value of 6.82, demonstrating that the tumor model was moderately acidic. The standard deviation of pixelwise pHe values was 0.24, indicating moderate heterogeneity of pHe in the measured tumor area. Post initial treatment, the pHe had a statistically significant increase to 6.92 with a standard deviation of 0.14, indicating a decrease in average acidosis and a decrease in heterogeneity of acidosis. After continuous treatment, the pHe dropped to 6.74 with a standard deviation of 0.26, suggesting a return to a more normal metabolic rate. In keeping with the trend, the acidic fraction of the tumor decreased from 33.1% to 19.6%, before increasing to 49% during this study. The tumor growth study showed a temporary growth delay before returning to a more normal growth rate, which paralleled the acidoCEST MRI results. Conclusion: Tumor pHe measured with acidoCEST MRI can detect early response to RAD001. Additional pre-clinical studies are warranted to determine if this biomarker is a robust measure of early therapeutic response. In addition, translation of acidoCEST MRI to the clinic should be pursued to provide this biomarker for clinical studies. Citation Format: Paul J. Akhenblit, Christine M. Howison, Scott W. Malm, Liu Qi Chen, Amanda F. Baker, Mark D. Pagel. Monitoring early therapeutic response by measuring extracellular pH in a tumor model with acidoCEST MRI. [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 LB-161. doi:10.1158/1538-7445.AM2014-LB-161

Carfilzomib (CFZ), is an irreversible inhibitor of the chymotrypsin-like activity of the proteasome that was recently FDA approved for treatment of refractory and relapsed multiple myeloma. Early Phase 1B studies of CFZ showed signs of clinical activity in a number of solid tumors, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). We investigated the anti-tumor activity of CFZ monotherapy and in combination with cisplatin in NSCLC and SCLC cell lines and xenograft models. CFZ showed marked anti-proliferative activity in A549, H1993, H520, H460, H1299 NSCLC cell lines, with IC50 values after 96 hours of treatment ranging from 1.0 nM to 27 nM. CFZ + cisplatin resulted in antagonistic anti-proliferative activity in the NSCLC A549, H1993, and H520 cells, independent of sequence of drug administration. In vivo, CFZ monotherapy resulted in tumor growth inhibition in an A549 NSCLC murine xenograft model. In contrast, the SCLC cell lines SHP77 and H69 were more resistant to the anti-proliferative effects of CFZ, with IC50 values at 96 hours of 0.917 μM and >30 μM, respectively. In vitro, in the SHP-77 SCLC cell line CFZ + Cisplatin showed synergistic activity. In vivo, in a SHP-77 SCLC SCID murine xenograft model CFZ monotherapy resulted in an 8-day tumor growth delay to reach 1500 mm3. No additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin in this model. Western blot analysis from SHP-77 cell lines grown in vitro and treated with CFZ showed an increase in cleaved PARP and p53, supporting activation of apoptosis. We will report on inhibition of the chymotrypsin-like proteasome activity in CFZ-treated lung cancer cell lines. Together, these pre-clinical studies support further preclinical and clinical investigations of CFZ in NSCLC and SCLC, including novel CFZ + anti-cancer drug combinations. Citation Format: Amanda F. Baker, Barbara Sands, Lilliana Carbajal, Janet Anderl, Elena T. Chan, Christopher J. Kirk, Linda L. Garland. Carfilzomib demonstrates broad antitumor activity in preclinical lung cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2013-1022

Ovarian cancer is characterized by genomic and microenvironmental heterogeneity, both of which contribute to tumor progression, metastasis, and drug resistance. Hypoxia, or low oxygen tension, is one microenvironmental factor that influences genomic instability and promotes angiogenesis and metastasis. We investigated the gene expression levels of hypoxia-regulated genes in matched ovarian serious carcinoma primary and peritoneal metastasis from five patients. The hypoxia gene set used was based on a published metagene hypoxia signature established using breast and head and neck cancer samples by Buffa et al, 2010. To ensure that the genes selected were also hypoxia-regulated in ovarian tumor cells, we grew the Hey and Ovcar-3 ovarian cancer cell lines under normoxia, 1.0% and 0.2% oxygen for 4, 16, and 48 hours. RNA was extracted and cDNA prepared for quantitative real-time polymerase chain reaction (qRT-PCR). Each sample was analyzed in triplicate and normalized for loading using ribosomal protein, large, PO (RPLP0). Relative gene expression was calculated by comparing hypoxia treated samples to the normoxic control grown for the same length of time. The Hey cells were also grown in an intraperitoneal xenograft model and tumor tissue harvested following 22 days of growth. Primary tumor gene expression was normalized to the Hey cell line grown in normoxia. The metastatic tissue was also compared relative to each matched primary sample. From an initial list of 16 genes, we identified 9 genes that demonstrated robust increases in gene expression across treatment conditions in vitro in both OVCAR 3 and Hey cells: vascular endothelial growth factor (VEGF-A), solute carrier family 2 (SLC2A1; Glut-1), phosphoglycerate mutase 1 (PGAM1),lactate dehydrogenase A (LDHA), prolyl 4-hydroxylase, alpha polypeptide (P4HA1), adrenomedullin (ADM), v-yc myelocytomatosis viral related oncogene ( NDRG1; N-myc), aldolase A (ALDOA), carbonic anhydrase 9 (CA9). Xenograft tissue showed high overexpression of all 9 of these hypoxia regulated genes when normalized to the Hey normoxia cell line control grown in vitro, with CA9 levels being maximally elevated. On average, primary tumors showed highest expression levels of VEGF and CA9 when normalized to the in vitro Hey cell normoxia control. When the primary tumor samples were compared to matched metastatic tissue there was a strong correlation between expression patterns of VEGF-A and SLC2A1. Two of five matched samples showed over 3 fold elevation in VEGF and SL2A1 in the metastatic tissue relative to primary tumor. Our data suggests that there may be significant heterogeneity between the primary and metastatic tumors which could lead to sampling bias when investigating hypoxia regulated genes or gene products such as VEGF-A and SL2A1 as prognostic or therapeutic biomarkers. Citation Format: Amanda F. Baker, Scott Malm, Cindy Laughren, Setsuko K. Comparison of a hypoxia gene signature in matched ovarian primary tumors and peritoneal metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A6.

The vasculature of tumors is torturous and immature and in many cases lacks tone (the ability to self-regulate pressure). The administration of vasodilating agents induces a drop in systemic blood pressure which is not matched in the tumor, resulting in decreased intratumoral perfusion due to the “steal phenomenon”. This research uses this phenomenon to induce decreased tumor perfusion which results in increased tumor acidity and hypoxia. We hypothesize that this change in the tumor microenvironment can be used in combination with hypoxia- and acid-activated prodrugs (HAPs and AAPs) to enhance their efficacy, by increasing both the depth and distribution of hypoxia or acidosis within the tumor. Specifically, we have investigated the administration of hydralazine in several pancreatic xenograft tumors and used a needle microelectrode to measure decreases in pH as a result of decreased perfusion. We have also employed a computational math model to investigate the timing of the delivery of both hydralazine and HAPs to optimize the cell death. Preliminary computational results suggest that multiple rounds of vasodilators may be given with a single round of HAPs, depending on several factors including duration of vasodilation effect and half-life of the HAP. The pH investigation showed a substantial pH drop in one tumor type (MiaPaca2), but no change in two others (HS766t and SU8686), suggesting that the ‘steal’ phenomenon is not universal. Preliminary data suggest that combination therapy in the ‘steal’-sensitive models will increase efficacy. We are continuing research with the combination therapy, employing the timing strategies suggested by the computational models. We are also investigating different imaging modalities to monitor the effects of the steal phenomenon, including Ultrasound (for perfusion) and MRI (for bulk tumor changes and monitoring pH). In conclusion, the preliminary data supports the hypothesis that vasodilatation agents may be used to induce increases in acidosis and hypoxia within the microenvironment of some (but not all) tumors. These effects can be taken advantage of through combination with HAPs or AAPs to enhance antitumor effects. Further, computational math models can be employed to help determine the best timing for the combination therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2011-3520

Introduction: IL-6 has both diagnostic and prognostic significance in pancreatic cancer. We investigated the signaling pathways activated in response to IL-6 in pancreatic cell lines, with a focus on STAT-3 and Pim-1 kinase. Methods: IL-6 receptor (IL-6R) expression was evaluated by Western blot analysis across a panel of human pancreatic cell lines. Panc-1 and MiaPaCa2 cell lines were serum starved for 24 hrs and then treated with 100 ng/mL of IL-6 for various time courses from 0 to 6 hr. STAT-3 activation and Pim-1 kinase protein levels were assessed in the presence and absence of IL-6 by Western blot analysis. The ability of cucurbitacin B to block both STAT-3 and Pim-1 activation, and inhibit cell proliferation was investigated. Stably transfected Pim-1 kinase over-expressing cell lines were generated and characterized for their response to IL-6 in vitro, and their growth rate in vivo as flank tumors in scid mice. Results: IL-6R is broadly expressed across multiple cancer cell lines, with the highest expression observed in the Panc-1 cell line. IL-6 stimulation resulted in increased STAT-3 phosphorylation in Panc-1, and MiaPaCa2 tumor cell lines as well as the HP-DEV transformed pancreatic ductal cell line. Treatment with IL-6 resulted in increased Pim-1 protein expression in the Panc-1 but not the MiaPaCa2 parent cell lines. Induction of Pim-1 protein was also observed in both Panc-1 and MiaPaCa2 stably transfected over-expressing cell lines. Pre-treatment with the STAT-3 inhibitor cucurbitacin B resulted in inhibition of STAT-3 phosphorylation and decreased cell proliferation, but was not associated with suppression of Pim-1 protein expression in the Panc-1 cell lines. Over-expression of Pim-1 in the Panc-1 cell line was associated with no significant changes in basal apoptosis rate, cell growth rate in vitro or in flank xenografts, or IL-6 induced expression of VEGF. Conclusions: IL-6 stimulation activates both STAT-3 and Pim-1 kinase expression in pancreatic cell line models. Induction of Pim-1 kinase was cell line specific, suggesting that the molecular context is essential for this pathway to be activated. Suppression of STAT-3 with a pharmacological inhibitor resulted in exaggerated Pim-1 expression in over-expressing cell lines implying Pim-1 up-regulation may be one of the compensatory and/or stress pathways activated when STAT-3 is inhibited. Stable over-expressing Pim-1 kinase Panc-1 and MiaPaCa2 cells did not exhibit significant changes in growth or apoptosis, unlike previously reported studies in the literature with other pancreatic cancer cell line models. This may be due to the multiple pathways that are dysregulated in these cell lines. Both STAT-3 and Pim-1 kinase have previously been identified as drug targets in multiple malignancies, including leukemia, lymphoma, prostate, and pancreatic cancer. Our study suggests that both pathways may be activated in patients with elevated IL-6 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2011-2930