Multiple Solid Tumor Types Research Articles

Development and Validation of a Clinical Trial Patient Stratification Assay That Interrogates 27 Mutation Sites in MAPK Pathway Genes

Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

Abstract 3728: Anti-Notch1 antibody (OMP-52M51) impedes tumor growth and cancer stem cell frequency (CSC) in a chemo-refractory breast cancer xenograft model with an activating Notch1 mutation and screening for activated Notch1 across multiple solid tumor types.

Abstract The Notch pathway plays a central role in embryonic development, the regulation of stem and progenitor cells, and is implicated in many human cancers. Notch1 is also known to be activated by oncogenic mutations in tumors including T cell leukemia, chronic lymphocytic leukemia and non-small cell lung cancer. Here we report the discovery of similar mutations in breast cancer. We have developed a primary human xenograft model from a patient bearing an activating Notch1 mutation and have shown that blocking Notch signaling in this model with a novel anti-Notch1 antibody, OMP-52M51, impeded tumor growth and reduced cancer stem cell (CSC) frequency. OMP-52M51 is a humanized antibody that binds with high affinity and selectivity to human Notch1 and antagonizes Notch signaling. Additionally, we sought to determine the frequency of Notch pathway activation across a large panel of human tumors (n>600) by an immunohistochemistry assay that detects the activated form of Notch1 using a polyclonal antibody that specifically recognizes the Notch1 intracellular domain (ICD). Using this test and a rigorous H-score cut-off of 30, we found elevated Notch1 in 7-29% of the following cancers: chemo-resistant breast, gastric, cholangiocarcinoma, esophageal, hepatacellular carcinoma (HCC), and small cell lung cancer (SCLC). The class of patients that showed the highest frequency of elevated Notch1.ICD was chemo-resistant breast cancer (∼30%). This frequency was significantly higher than in unselected breast cancer patients, suggesting that Notch1 signaling plays a significant role in breast cancer chemoresistance. Interestingly, the human xenograft model which showed sensitivity to OMP-52M51 was derived from a patient that failed to respond to pre-operative chemotherapy and developed metastatic disease following surgery. Thus, chemo-resistant breast cancer as well as the other tumors with high Notch1.ICD may represent important classes of patients who could benefit from anti-Notch1 therapy in the clinic. Citation Format: Belinda Cancilla, Jennifer Cain, Min Wang, Lucia Beviglia, Jalpa Shah, Austin Gurney, John Lewicki, Laura Esserman, Tim Hoey, Ann M. Kapoun. Anti-Notch1 antibody (OMP-52M51) impedes tumor growth and cancer stem cell frequency (CSC) in a chemo-refractory breast cancer xenograft model with an activating Notch1 mutation and screening for activated Notch1 across multiple solid tumor types. [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 3728. doi:10.1158/1538-7445.AM2013-3728

Biological Characterization of TAK-901, an Investigational, Novel, Multitargeted Aurora B Kinase Inhibitor

Protein kinases Aurora A, B, and C play essential roles during mitosis and cell division, are frequently elevated in cancer, and represent attractive targets for therapeutic intervention. TAK-901 is an investigational, multitargeted Aurora B kinase inhibitor derived from a novel azacarboline kinase hinge-binder chemotype. TAK-901 exhibited time-dependent, tight-binding inhibition of Aurora B, but not Aurora A. Consistent with Aurora B inhibition, TAK-901 suppressed cellular histone H3 phosphorylation and induced polyploidy. In various human cancer cell lines, TAK-901 inhibited cell proliferation with effective concentration values from 40 to 500 nmol/L. Examination of a broad panel of kinases in biochemical assays revealed inhibition of multiple kinases. However, TAK-901 potently inhibited only a few kinases other than Aurora B in intact cells, including FLT3 and FGFR2. In rodent xenografts, TAK-901 exhibited potent activity against multiple human solid tumor types, and complete regression was observed in the ovarian cancer A2780 model. TAK-901 also displayed potent activity against several leukemia models. In vivo biomarker studies showed that TAK-901 induced pharmacodynamic responses consistent with Aurora B inhibition and correlating with retention of TAK-901 in tumor tissue. These preclinical data highlight the therapeutic potential of TAK-901, which has entered phase I clinical trials in patients within a diverse range of cancers.

507 - Phase I, Open-Label, Randomized, Crossover Study Evaluating the Effects of Linifanib on Qtc Intervals in Patients with Solid Tumors

ABSTRACT Background Linifanib (ABT-869) is a novel, orally active and selective inhibitor of VEGF and PDGF family of receptor tyrosine kinases that has shown antitumor activity in multiple types of solid tumors. This study was conducted to evaluate the potential effects of linifanib on QTc prolongation in patients (pts) with advanced solid tumors. Methods Enrolled pts (N = 24; ≥18 years) had measurable disease refractory to standard therapies, ECOG PS 0-1, and adequate organ function. Pts received 2 sequences of regimens of 0.25 mg/kg orally administered linifanib up to a maximum dose of 17.5 mg. Pts received a morning dose on Days 1 and 7 under fasting and fed conditions in a crossover fashion. Serial triplicate ECG recordings were obtained on Day –1, and over 24 hours on Day 1 and 7; single recordings were obtained at screening and study completion or discontinuation. Plasma samples were collected for 72 hours on Day 1 and 7 for pharmacokinetic analysis. Effects of linifanib on cardiac repolarization were analyzed using a linear mixed effects model on time-matched baseline adjusted QTcF intervals. The primary end point was the time-matched difference for on-treatment QTcF from baseline (ΔQTcF). An intersection-union test was performed within the framework of the corresponding linear mixed effects model. The relationship between ΔQTcF and plasma linifanib concentration was explored using a linear mixed effects model. Results In the 24 pts evaluated, baseline QTcF ranged from 360.9 ms to 468.6 ms. After linifanib administration, the mean ΔQTcF ranged from –4.04 ms to 0.73 ms for the fasting regimen, and from –5.94 ms to –1.37 ms for the fed regimen. The upper 95% confidence bound was 4.30 ms (ie, 500 ms or change of >30 ms from baseline. Conclusion At the maximum tolerated dose of 0.25 mg/kg, linifanib had no effect on cardiac repolarization in pts with advanced solid tumors. Disclosure Y. Chiu: Full-time Abbott employee and stockholder. P.M. LoRusso: Patricia LoRusso's institution receives research funding from Abbott. J.L. Ricker: Full-time Abbott employee and stockholder. X. Li: Full-time Abbott employee and stockholder. R. Pradhan: Full-time Abbott employee and stockholder. D.M. Carlson: Full-time Abbott employee and stockholder.

203P - Placental Growth Factor (PIGF) as a Biomarker in Patients (PTS) With Solid Tumors Treated With Linifanib (ABT-869)

ABSTRACT Introduction Linifanib is a VEGF/PDGF receptor tyrosine kinase inhibitor (TKI). PlGF is a member of the VEGF family and is induced upon inhibition of VEGF signaling. We explored whether changes in PlGF levels during linifanib therapy could predict clinical outcome. Methods An exploratory, retrospective analysis was performed for pts enrolled internationally in phase (P) 1 (solid tumor) and hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) P2 linifanib monotherapy trials. Efficacy was assessed by RECIST and safety by NCI-CTCAE. Plasma PlGF levels were analyzed using the Architect system (Abbott) and relationships between PlGF levels, time-to-progression (TTP) or progression-free survival (PFS), and overall survival (OS) were determined. Results 130 pts were enrolled; 33 in P1; 44 HCC in P2 and 53 RCC in P2 studies. Across all studies, PlGF levels were induced in a dose and time- dependent manner by linifanib in pts with multiple solid tumor types. P1 pts were assessed for an association for PlGF with TTP. Of the pts assessed in P1 at day (D) 15, those with PlGF levels > 40 pg/ml had improved TTP (328 D; p = 0.01), compared to those with PlGF levels 66.3 pg/ml at D8-56 had improved PFS (p = 0.029) and OS (p = 0.02). In RCC, 5 pts with ΔPlGF below this threshold had median PFS of 100 D vs 207 D in the remaining pts (p = NS). Across the P2 studies, toxicity thresholds were determined using PlGF levels at D8 and toxicity events resulting in dose reductions or interruptions. The efficacy and toxicity thresholds were used to identify optimal ΔPlGF levels associated with improved OS in HCC (p = 0.01) and RCC pts (p = 0.001). Conclusions Optimal ΔPlGF plasma levels were identified and associated with improved outcome in HCC and RCC pts receiving linifanib. Monitoring of PlGF levels may allow clinicians to identify pts with increased sensitivity or toxicity risk on linifanib. Further evaluation in randomized linifanib studies is warranted, including dose adjustments based on ΔPlGF levels. Disclosure E.M. Mc Keegan: Full time Abbott employee who owns stock. A. Chakravartty: Full time Abbott employee who owns stock. P.J. Ansell: Full time Abbott employee who owns stock. S. Datwyler: Full time Abbott employee who owns stock. M.D. McKee: Full time Abbott employee who owns stock. J.L. Ricker: Full time Abbott employee who owns stock. D.M. Carlson: Full time Abbott employee who owns stock. All other authors have declared no conflicts of interest.

Application of Monoclonal Antibodies as Cancer Therapy in Solid Tumors

Monoclonal antibodies have become an important new class of therapeutic agents approved for use in solid tumors. They function through several different mechanisms including inhibition of tumor-related signal transduction, induction of apoptosis, inhibition of angiogenesis, enhancing host immune response against cancer and targeted delivery of cytotoxic agents to the tumor site. Several monoclonal antibodies have now received regulatory approval--trastuzumab, cetuximab, panitumumab, bevacizumab, catumaxomab, ipilimumab and denosumab--across multiple solid tumor types, including breast, colorectal, head and neck, non-small cell lung cancers and melanomas, amongst others. These agents are employed clinically in some neoadjuvant/adjuvant and radical treatment settings, as well as more extensively in the metastatic and palliative settings. Current research is focused on innovative compound design, novel targets, predictive biomarker discovery, enriched patient populations, and combination strategies in order to overcome resistance and prolong disease control. Here we provide an overview of monoclonal antibodies approved for use in clinical oncology and those currently in clinical development.

Abstract 2847: PECAM-1 targeting extends overall survival in various advanced metastatic mouse tumor models

Abstract Objectives: Patients who die from cancer succumb to advanced metastatic progression (AMP). While targeted therapies are available that address tumor cell derived regulators of metastasis, few therapeutics are being developed to address the molecular mechanisms of AMP. Our group has previously shown that vascular endothelial cell (VEC) PECAM-1 is a critical molecular master switch which regulates AMP by controlling the release of VEC-derived, paracrine growth factors that drive its progression. Anti-PECAM-1 monoclonal antibody (mAb) effectively and safely treats even pre-terminal cancers in AMP-bearing mice. The current experiments were performed to assess whether, to what extent and how widely targeting PECAM-1 can improve overall survival in mice bearing advanced metastases. Methods: Groups of wildtype (WT) C57Bl/6 mice, together with age and gender matched PECAM-1 knockout (KO) mice were intravenously injected with 25,000 murine B16-F10 melanoma cells, 30,000 murine EL4 lymphoma cells or 1,000,000 murine MC38 colon cancer cells. We then injected matched groups of PECAM-1 KO vs WT mice with 1,000,000 B16-F10 cells, to model maximally aggressive AMP. All groups were assessed for overall survival, using IACUC approved guidelines. Results: In all cases, overall survival (OS) was significantly prolonged in PECAM-1 KO vs WT controls (Kaplan-Meier ranged from p = 0.018 to p = 0.0002). Targeting microenvironmental PECAM-1 was as effective in increasing OS in mice receiving the very high tumor burden of 1,000,000 B16-F10 cells as in mice receiving 25,000 cells, further confirming the efficacy of targeting PECAM-1 against the most advanced models of metastasis that can be achieved preclinically. Furthermore, anti-PECAM-1 mAb-based therapy of B16-F10 metastase was as effective as the PECAM-1 KO genotype in increasing OS. Conclusion: To date, many approved mAb- and small molecule-inhibitor based targeted-therapies have shown limited abilities to either prolong OS as single agents, or to effectively treat multiple different solid tumor types. These results suggest that anti-PECAM-1 mAb therapy, which specifically targets VEC and paracrine factors they produce, has the potential to significantly increase OS in the setting of lethal advanced metastatic progression, independent of tumor type. Anti-PECAM-1 mAb therapy is as effective as a PECAM-1 knockout in improving OS. Phase 1 clinical trials are planned within 2 years. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2847. doi:1538-7445.AM2012-2847

Abstract 3866: TWEAK receptor (Fn14) Is a novel target in melanoma: Characterization of unique targeted therapeutics

Abstract Previous studies have indicated that Fn14, the cell surface receptor for the cytokine TWEAK, is over-expressed in multiple human solid tumor types, including brain, breast, and lung cancers, and overexpression can be a negative prognostic indicator. We analyzed a series of melanoma cell lines and tumor tissue microarrays and detected Fn14 expression in ∼60% of the melanoma cell lines, including both B-Raf WT and B-Raf V600E lines. Fn14 expression was elevated in 178/190 (93.6%) of primary melanoma specimens and in 87/150 (58%) of melanoma metastases tested. Fn14 expression was not elevated in normal skin tissues. Initial development and characterization of an immunoconjugate designated ITEM4-rGel targeting Fn14 receptor has been published (Zhou et al., Mol. Cancer Ther. 10:1276 (2011)). We have now developed an Fn14-targeted immunotoxin more suitable for long-term clinical use. Specifically, we generated a humanized, dimeric single-chain version of ITEM-4 and fused this scFv to rGel. The resulting anti-Fn14 immunotoxin, designated hSGZ, bound to Fn14 with a Kd of ∼1.4 nM as determined by Biacore analysis. Confocal immunofluoresence studies showed that hSGZ specifically and rapidly (within 2 hrs) internalized into Fn14-expressing MDA-MB-435 melanoma cells. Cytotoxicity studies showed that hSGZ was highly cytotoxic to a panel of different melanoma cell lines (IC50 ranged from 0.1 pM to 1.1 nM) and was 2.2 to 2.8 ×105 fold more potent than free rGel. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. When hSGZ was combined with 5-FU, cisplatin, doxorubicin, etoposide or dacarbazine, we found an additive effect on melanoma cell growth inhibition. Mechanistic studies showed that hSGZ induced melanoma cell death consistent with a necrotic mechanism. Additionally, Fn14-targeted immunotoxins increased Fn14 expression and triggered cell signaling events similar to those induced by the TWEAK ligand. Finally, treatment of mice bearing human melanoma MDA-MB-435 and breast MDA-MB-231 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared to controls (P< 0.05). Fn14 appears to be an excellent new target for melanoma and the Fn14-targeting construct hSGZ appears to warrant further development as a novel therapeutic agent against Fn14-positive tumors. Melanoma lines appear to be the most sensitive tumor type tested but the reasons for this are unclear. Additional studies are in progress to investigate the biodistribution and pharmacokinetics of hSGZ in tumor-bearing mice. This work was conducted, in part, by the Clayton Foundation for Research (MGR); and supported by NIH grant NS055126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3866. doi:1538-7445.AM2012-3866

Incorporation of a selective sigma-2 receptor ligand enhances uptake of liposomes by multiple cancer cells

Background:The sigma-2 receptor is an attractive target for tumor imaging and targeted therapy because it is overexpressed in multiple types of solid tumors, including prostate cancer, breast cancer, and lung cancer. SV119 is a synthetic small molecule that binds to sigma-2 receptors with high affinity and specificity. This study investigates the utility of SV119 in mediating the selective targeting of liposomal vectors in various types of cancer cells.Methods:SV119 was covalently linked with polyethylene glycol-dioleyl amido aspartic acid conjugate (PEG-DOA) to generate a novel functional lipid, SV119-PEG-DOA. This lipid was utilized for the preparation of targeted liposomes to enhance their uptake by cancer cells. Liposomes with various SV119 densities (0, 1, 3, and 5 mole%) were prepared and their cellular uptake was investigated in several tumor cell lines. In addition, doxorubicin (DOX) was loaded into the targeted and unmodified liposomes, and the cytotoxic effect on the DU-145 cells was evaluated by MTT assay.Results:Liposomes with or without SV119-PEG-DOA both have a mean diameter of approximately 90 nm and a neutral charge. The incorporation of SV119-PEG-DOA significantly increased the cellular uptake of liposomes by the DU-145, PC-3, A549, 201T, and MCF-7 tumor cells, which was shown by fluorescence microscopy and the quantitative measurement of fluorescence intensity. In contrast, the incorporation of SV119 did not increase the uptake of liposomes by the normal BEAS-2B cells. In a time course study, the uptake of SV119 liposomes by DU-145 cells was also significantly higher at each time point compared to the unmodified liposomes. Furthermore, the DOX-loaded SV119 liposomes showed significantly higher cytotoxicity to DU-145 cells compared to the DOX-loaded unmodified liposomes.Conclusion:SV119 liposomes were developed for targeted drug delivery to cancer cells. The targeting efficiency and specificity of SV119 liposomes to cancer cells was demonstrated in vitro. The results of this study suggest that SV119-modified liposomes might be a promising drug carrier for tumor-targeted delivery.

A Combined Array-Based Comparative Genomic Hybridization and Functional Library Screening Approach Identifies mir-30d As an Oncomir in Cancer

Oncomirs are microRNAs (miRNA) that acts as oncogenes or tumor suppressor genes. Efficient identification of oncomirs remains a challenge. Here we report a novel, clinically guided genetic screening approach for the identification of oncomirs, identifying mir-30d through this strategy. mir-30d regulates tumor cell proliferation, apoptosis, senescence, and migration. The chromosomal locus harboring mir-30d was amplified in more than 30% of multiple types of human solid tumors (n = 1,283). Importantly, higher levels of mir-30d expression were associated significantly with poor clinical outcomes in ovarian cancer patients (n = 330, P = 0.0016). Mechanistic investigations suggested that mir-30d regulates a large number of cancer-associated genes, including the apoptotic caspase CASP3. The guided genetic screening approach validated by this study offers a powerful tool to identify oncomirs that may have utility as biomarkers or targets for drug development.

P2Y12 Is Involved in Cancer Metastasis,

Abstract 3356The platelet ADP receptor P2Y12 plays a prominent role in amplifying platelet activation, aggregation and thrombus formation, and the P2Y12 inhibitor Clopidogrel is widely used clinically to treat coronary artery, peripheral vascular and cerebrovascular diseases. Recently, several publications analyzed the TRITON-TIMI 38 clinical trial (test the efficacy and safety of Prasugrel, a newly FDA approved thienopyridine P2Y12 inhibitor) revealing an increase in multiple types of solid tumors with Prasugrel use, casting doubt on the safety of anti-platelet therapy targeting P2Y12. In this study, we used animal models to investigate the safety of targeting P2Y12, and to elucidate the probable mechanism for the effects of P2Y12 antagonists on metastasis. Tumor growth and metastasis are basic features of malignant cancer. Pulmonary metastasis in the experimental, and spontaneous mouse metastasis models were used to identify the role of P2Y12, and the effects of its inhibitors in tumor growth and metastasis. In the experimental pulmonary metastasis group, one week before tail vein injection of 2×105 melanoma B16 cells, wild type (WT) and P2y12–/– mice were treated every other day with PBS, Clopidogrel or Prasugrel by oral gavage and continue in whole experiment process. Tumor nodules were counted, and the average nodule sizes measured at 20 days after B16 cell injection. P2y12–/– mice had fewer lung metastatic loci than the lungs of the WT controls. The experimental and control lungs had similar size nodules. But Clopidogrel dose dependently (1.5mg/kg and 30mg/kg) caused an increase of metastatic loci and loci size in both WT and P2y12–/– mice. Prasugrel (0.2mg/kg and 4mg/kg) did not affect the number or size of metastatic loci produced in either strain. These results demonstrated that p2y12 deficiency negatively affected cancer metastasis.In contrast, Clopidogrel, but not Prasugrel, promotes metastasis and cancer growth. Importantly, Clopidogrel induced metastasis promotion and growth was not P2Y12 dependent. A plausible explanation of this difference between the two drugs may reside in the fact that both are pro-drugs of thienopyridine family. Presumably, activation of the pro-drug clopidogrel, but not Prasugrel produces one or more metabolites able to promote cancer growth and metastasis in vivo.This supposition was tested, in part by asking if the effects of Clopidogrel are limited to the B16 model of metastasis. The Lewis lung carcinoma (LLC) spontaneous pulmonary metastasis model was used for this purpose. LLC cells (2.0×106) were implanted intradermally in each mouse. Surgery was done 14 days after cell implantation to completely remove the primary tumors (about 1.0g or 1cm3 in size). One month later, lungs were removed, rinsed with PBS, and pulmonary metastatic nodules counted and measured. P2Y12 deficiency decreased the spontaneous formation of pulmonary metastatic loci, but had no effect on the primary tumor. Also, Clopidogrel, dose dependently promoted lung metastasis.Next, the mechanism of the effect of P2Y12 absence on tumor metastasis was investigated. In vitro aggregation experiments revealed that B16 melanoma cells directly interact with p2y12+/+ platelets and cause platelet-cancer cell aggregation. In contrast, P2Y12 deficiency results in significantly less or no cancer cell-platelet aggregation. Interestingly, LLC cells do not cause platelet aggregation. Immunohistochemistry and immunofluorescence analyses of lung samples from the spontaneous metastasis model indicated an obvious attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for metastatic niche formation. Moreover, this effect is transplantable, as wild-type mice reconstituted with p2y12–/– bone marrow have a similar phenotype as P2y12–/– mice. These results imply that the mechanism of P2Y12 function on cancer metastasis is complicated, probably mediated through regulation of both cancer cell-platelet direct interaction and metastatic niche formation. Further work is needed to resolve this issue. In summary, it is clear that P2Y12 is a safe drug target for anti-thrombotic therapy. But, metabolic derivatives of the pro-drug Clopidogrel apparently promote cancer metastasis and growth; whereas the newly approved pro-drug Prasugrel lacks that effect in animal models, and hopefully in humans. Disclosures:No relevant conflicts of interest to declare.

SF1126, a Pan-PI3K Inhibitor Has Superior Preclinical Activity to CAL-101 a PI3K Delta-Specific Inhibitor in Aggressive B-Cell Non-Hodgkin's Lymphoma

Abstract 2720The PI3K pathway is constitutively active in B-cell non-Hodgkin lymphomas (B-NHL). PI3K pathway targeted therapies have focused on inhibiting mTORC1 (rapalogs) with a ∼20–48% response rate due to inactivation of mTORC1 resulting in G1 cell-cycle arrest or apoptosis. A mechanism of resistance to rapalogs is that mTORC2 is unaffected resulting in undesirable Akt activation. Strategies to block Akt up-regulation require novel agents that simultaneously block PI3K, mTORC1 and mTORC2. SF1126 is a novel pan-PI3K/mTORC1/mTORC2 inhibitor conjugated to an integrin targeted peptide RGD with potent anti-tumor activity in multiple solid tumor types. Here, we demonstrated SF1126 had potent anti-B-NHL activity and is superior to CAL-101 a PI3K delta-isoform specific inhibitor in a panel of aggressive B-NHL cell lines. Cells treated with SF1126 exhibited >90% decrease in pAkt and pGSK-3β confirming the mechanism of action of a pan-PI3K inhibitor. Moreover, SF1126 induced apoptosis in a dose and time dependent manner confirmed by flow cytometry, PARP cleavage and with an IC50 < 4μM. In contrast, CAL-101 was less active compared to SF1126 in inducing apoptosis (12% versus 25% in SUDHL-4 and 15% versus 23% in TMD-8) and cell proliferation (5.62μM versus 3.28μM SUDHL-4 and 5.31μM versus 1.47μM in TMD-8). SF1126 induced G1 cell cycle arrest at 2μM which contributes to suppression of cell proliferation. The cell cycle protein cyclin D1 is downstream of mTORC1, and the over-expression of cyclin D1 is a hallmark of mantle cell NHL (MCL). Consistent with this, cyclin D1 was significantly decreased by SF1126 compared to CAL-101. Lastly, the addition of Rituximab to SF1126 or CAL101 increased the apoptosis over single agent therapy in B-NHL cell lines. In conclusion, we demonstrate that SF1126 potently inhibits the constitutively activated PI3K/mTORC/Akt pathway in aggressive B-cell NHL cell lines with consequent suppressive effects on cell cycle progression, cell proliferation and induction of apoptosis. These findings provide a rationale for SF1126 in combination with rituximab as a novel therapeutic strategy for aggressive B-NHL and warrant early phase clinical trial evaluation [Funded by the Lymphoma SPORE 1 P50 CA 130805 01A1]. Disclosures:Garlich:Semafore Pharmaceuticals: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Methylation Status of the Collagen Prolyl-3 Hydroxylases (C-P3H) and C-P4H Genes in Multiple Myeloma: P3H2 Is Selectively Methylated

Abstract 4639 Background–Aim:Prolyl hydroxylation is an important and the most common post-translational modification that affects the structure and function of collagen. Enzymes that participate in this process are the collagen prolyl-3 hydroxylases (C-P3H) and the C-P4H. We have previously showed that P3H3 is methylated in multiple solid tumour types, whereas methylation in P3H2 appears to be specific for breast cancer and multiple myeloma (MM). In this study we assessed the CpG methylation status of the P3H genes (P3H1, P3H2, P3H3) in a larger cohort of MM patients and for the first time the CpG methylation status of the P4H genes (P4HA1, P4HA2, P4HA3) and investigated for clinical relevance. Patients and Methods:Bone marrow aspirate samples from 47 MM patients (28 males, median age 66 years) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the CpG methylation in P3H1, P3H2, P3H3, P4HA1, P4HA2 and P4HA3 CpG islands. Genomic DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. For the study of the P3H2 CpG methylation status we used 2 sets of independent primers, which map to different areas of P3H2 CpG island. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, b2 microglobulin, serum albumin, hypercalcemia, ISS stage, extramedullary disease, bone disease, anemia and renal failure (eGFR< 50 ml/min). Results:None of the six genes under study was methylated in the control bone marrow samples. Methylation in the CpG island of P3H2 was detected in 44 % of patients (95% CI 27.8–62.8%) No methylation was detected in the CpG islands of P3H1, P3H3, P4HA1, P4HA2 and P4HA3. Trends noted were that patients with methylated P3H2 were more likely to have ISS stage 3 (OR 2.2, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size. Conclusions:P3H2 is methylated at high frequency in MM, whereas P3H1, P3H3, P4HA1, P4HA2, P4HA3 were unmethylated, implying a striking specificity for methylation in P3H2 in MM. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of P3H2 methylation in MM. Disclosures:No relevant conflicts of interest to declare.

Positron emission tomography imaging of CD105 expression with 89Zr-Df-TRC105

High tumor microvessel density correlates with a poor prognosis in multiple solid tumor types. The clinical gold standard for assessing microvessel density is CD105 immunohistochemistry on paraffin-embedded tumor specimens. The goal of this study was to develop an (89)Zr-based PET tracer for noninvasive imaging of CD105 expression. TRC105, a chimeric anti-CD105 monoclonal antibody, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS) and labeled with (89)Zr. FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and Df-TRC105. PET imaging, biodistribution, blocking, and ex-vivo histology studies were performed on 4T1 murine breast tumor-bearing mice to evaluate the pharmacokinetics and tumor-targeting of (89)Zr-Df-TRC105. Another chimeric antibody, cetuximab, was used as an isotype-matched control. FACS analysis of HUVECs revealed no difference in CD105 binding affinity between TRC105 and Df-TRC105, which was further validated by fluorescence microscopy. (89)Zr labeling was achieved with high yield and specific activity. Serial PET imaging revealed that the 4T1 tumor uptake of (89)Zr-Df-TRC105 was 6.1 ± 1.2, 14.3 ± 1.2, 12.4 ± 1.5, 7.1 ± 0.9, and 5.2 ± 0.3 %ID/g at 5, 24, 48, 72, and 96 h after injection, respectively (n = 4), higher than all organs starting from 24 h after injection, which provided excellent tumor contrast. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiments, control studies with (89)Zr-Df-cetuximab, and ex-vivo histology all confirmed the in vivo target specificity of (89)Zr-Df-TRC105. We report here the first successful PET imaging of CD105 expression with (89)Zr as the radiolabel. Rapid, persistent, CD105-specific uptake of (89)Zr-Df-TRC105 in the 4T1 tumor was observed.

Mining the adenovirus virome for oncolytics against multiple solid tumor types.

Although there are 55 serotypes of adenovirus that infect humans, Ad5 is the most widely studied due to the availability of commercial kits for its genetic manipulation. In fact, engineered adenovirus serotype 5 is currently being used in all of the 87 global clinical trials utilizing adenovirus for the treatment of cancer. Unfortunately, Ad5 is one of the most seroprevalent serotypes, meaning that this virus has to confront additional immunological barriers to be effective in Ad5-immune patients. In this work, we compare Ad5 to 13 other adenoviral serotypes from species B, C, D, and E for oncolytic potential in both immunodeficient mouse and immunocompetent hamster models. Our results indicate that species D adenoviruses are not effective oncolytics against most solid tumors. Conversely, lower seroprevalence Ad6 and Ad11 hade anti-cancer activity comparable to Ad5. This work strongly supports the consideration of Ad6-based oncolytic therapies for the treatment of breast, ovarian, kidney, and liver tumors.

Roles of bone marrow cells in skeletal metastases: no longer bystanders.

Bone serves one of the most congenial metastatic microenvironments for multiple types of solid tumors, but its role in this process remains under-explored. Among many cell populations constituting the bone and bone marrow microenvironment, osteoblasts (originated from mesenchymal stem cells) and osteoclasts (originated from hematopoietic stem cells) have been the main research focus for pro-tumorigenic roles. Recently, increasing evidence further elucidates that hematopoietic lineage cells as well as stromal cells in the bone marrow mediate distinct but critical functions in tumor growth, metastasis, angiogenesis and apoptosis in the bone microenvironment. This review article summarizes the key evidence describing differential roles of bone marrow cells, including hematopoietic stem cells (HSCs), megakaryocytes, macrophages and myeloid-derived suppressor cells in the development of metastatic bone lesions. HSCs promote tumor growth by switching on angiogenesis, but at the same time compete with metastatic tumor cells for occupancy of osteoblastic niche. Megakaryocytes negatively regulate the extravasating tumor cells by inducing apoptosis and suppressing proliferation. Macrophages and myeloid cells have pro-tumorigenic roles in general, suggesting a similar effect in the bone marrow. Hematopoietic and stromal cell populations in the bone marrow, previously considered as simple by-standers in the context of tumor metastasis, have distinct and active roles in promoting or suppressing tumor growth and metastasis in bone. Further investigation on the extended roles of bone marrow cells will help formulate better approaches to treatment through improved understanding of the metastatic bone microenvironment.

Abstract 1766: Development of single chain immunotoxins targeting the fibroblast growth factor-inducible 14 (Fn14) receptor on solid tumor cells

Abstract Previous studies have indicated that Fn14, the cell surface receptor for the cytokine TWEAK, is over-expressed in multiple human solid tumor types, including brain, breast, pancreatic, esophageal, colon and lung cancers. Using a melanoma tumor array, we recently found that Fn14 expression is elevated in 177/190 (93%) primary melanoma specimens and in 87/150 (58%) melanoma metastases specimens. This suggests that Fn14 may be a novel therapeutic target in this disease setting as well. We developed an immunoconjugate designated ITEM4-rGel composed of a high-affinity anti-Fn14 monoclonal antibody (ITEM-4) conjugated to recombinant gelonin (rGel), a highly cytotoxic, ribosome-inactivating n-glycosidase. To develop Fn14-targeted immunotoxins more suitable for clinical use, a humanized single-chain version of ITEM-4 (designated hscFvIT4) specific for Fn14 was successfully produced and characterized. BiaCore analysis of ITEM-4, ITEM4-rGel and hscFvIT4 binding to the Fn14 extracellular domain indicated that these proteins bound to Fn14 with similar affinity, with Kds of ∼ 1.1 nM, ∼ 0.7 nM, and ∼ 6.2 nM, respectively. Using hscFvIT4 and rGel toxin, we next engineered a bivalent immunotoxin (hscFvIT4/rGel/29) by adding a COOH-terminal dimerization domain. Cytotoxicity studies showed that ITEM4-rGel and its humanized counterpart, hscFvIT4/rGel/29, were highly cytotoxic to Fn14-expressing cells (IC50 ranged from 0.8 pM-25 nM) and were 50 to 0.5 x106 fold more potent than free rGel. Minimum contact time studies showed that as little as 8 hr exposure achieved maximal cytotoxic effect. Both ITEM4-rGel and hscFvIT4/rGel/29 were shown to specifically bind to Fn14-expressing tumor cells as analyzed by ELISA. Confocal immunofluoresence studies showed that ITEM4-rGel and hscFvIT4/rGel/29 specifically and rapidly (within 2 hrs) internalized into MDA-MB-435 melanoma cells but not into Fn14-deficient mouse embryonic fibroblasts. Mechanistic studies showed that ITEM4-rGel induced apoptosis in antigen-positive T24 human bladder carcinoma cells as measured by Annexin V staining. Treatment of MDA-MB-435 cells with hscFvIT4/rGel/29 induced the non-canonical NF-κB pathway and down-regulated survivin expression. Preliminary mouse xenograft tumor model studies are ongoing. We propose that the hscFvIT4/rGel/29 construct may warrant further development as a novel therapeutic agent against a broad range of Fn14-positive solid tumor types. Research conducted, in part, by the Clayton Foundation for Research (MGR), and supported by NIH grant NS55126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW). 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 1766. doi:10.1158/1538-7445.AM2011-1766

Abstract 1770: Enavatuzumab (PDL192), a humanized monoclonal antibody to TweakR, displays broad anticancer activity which is dependent on NFκB signaling

Abstract TWEAK (TNFSF12) is a multifunctional cytokine that binds to the cell surface receptor TweakR (Fn14, TNFRSR12A). TweakR is overexpressed in many solid tumor types, including cancers of breast, pancreatic, and ovarian origin. The initial functional description of TWEAK described it as a weak inducer of apoptosis in certain cancer cell lines. TWEAK is also known to function in other diverse biological processes such as inflammation, tissue repair, angiogenesis, cell migration, and growth inhibition. The growth inhibitory activity of TWEAK observed in some cancer cell lines and the overexpression of its receptor in cancer led to investigation of targeting TweakR as a therapeutic modality. Enavatuzumab (PDL192), a humanized IgG1 mAb to TweakR, exhibits some of the same functional activities of TWEAK and is currently in clinical investigation for the treatment of solid tumors due to its significant in vitro and in vivo growth inhibitory activity in models of multiple solid tumor types. The purpose of the current study was to provide insight into the mechanism of action of enavatuzumab and to further our understanding of the biology of TweakR in tumor cell lines. A panel of 106 cancer cell lines was tested for the ability of enavatuzumab to inhibit in vitro cell growth to identify characteristics of response. Of the 106 cell lines tested, 33 cell lines had &amp;gt;25% growth inhibition in response to the drug. Using luciferase transcriptional reporter constructs, it was revealed that the NFκB pathway was activated in response to enavatuzumab treatment in responder cells. Both classical (p50/p65) and non-classical NFκB (p52/RelB) pathways were subsequently shown to be induced by enavatuzumab treatment using Western blot and functional ELISA in responder cell lines. Using Affymetrix™ microarrays, we observed that cell lines sensitive to enavatuzumab had a higher number of transcriptional changes compared with resistant cells and included many genes known to be regulated by NFκB. To confirm a role for NFκB in enavatuzumab-mediated signaling, we inhibited NFκB pathway induction using siRNAs to the individual NFκB subunits p50, p65, p52, and RelB as well as the upstream kinases IKKα and IKKβ. The siRNAs were able to block enavatuzumab's growth inhibitory activity in multiple sensitive cell lines. Further confirmation of the importance of NFκB was obtained using a small molecule inhibitor of IKKα/β (IKK16) which significantly reduced the drug-induced growth inhibition. The finding that NFκB drives the growth inhibitory activity of enavatuzumab is a compelling function for a family of transcription factors that are more frequently associated with cancer growth and survival than inhibition of cell proliferation. 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 1770. doi:10.1158/1538-7445.AM2011-1770

Gadd45 Stress Sensors in Malignancy and Leukemia

Gadd45 proteins, including Gadd45a, Gadd45b, and Gadd45g, have been implicated in stress signaling in response to physiological and environmental stress, including oncogenic stress, which can result in cell cycle arrest, DNA repair, cell survival, senescence, and apoptosis. The function of Gadd45 as a stress sensor is mediated via a complex interplay of physical interactions with other cellular proteins implicated in cell cycle regulation and the response of cells to stress, notably PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. Altered expression of Gadd45 has been observed in multiple types of solid tumors as well as in hematopoietic malignancies. Using genetically engineered mouse models and bone-marrow transplantation, evidence has been obtained indicating that Gadd45 proteins can function to either promote or suppress tumor development and leukemia; this is dependent on the molecular nature of the activated oncogene and the cell type, via engagement of different signaling pathways.

The Prolyl-3-Hydroxylase Leprecan Like 1 (Leprel1) Is a Selective Target for Epigenetic Silencing In Multiple Myeloma.

AbstractAbstract 3645 Background–Aim:Gene silencing is a major mechanism of tumour suppressor gene inactivation in neoplasia, including multiple myeloma (MM). Prolyl hydroxylation is an important post-translational modification that affects the structure and function of collagen. The human genome contains 3 prolyl 3 hydroxylases (P3H) which are closely related in structure and function: Leprecan (P3H1), Leprecan Like 1 (Leprel1, P3H2) and Leprecan Like 2 (Leprel2, P3H3). Leprel2 is methylated in multiple solid tumour types, whereas methylation in Leprel1 appears specific for breast cancer. Leprecan is not detectably methylated in any solid tumour we have analysed. Here, we examine the CpG methylation status of the P3H genes in patients with MM. Patients and Methods:Bone marrow aspirate samples from 38 MM patients (22 males, 16 females) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study methylation in the Leprel 1, Leprel2 and Leprecan CpG islands. Genomic DNA was isolated and bisulphite modification performed using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. We used 2 sets of independent primers, which map to different areas of the leprel 1(P3H2) CpG island. Pyrosequencing is ongoing to confirm the sensitivity and specificity of MSP for analysis of P3H gene methylation in our study population. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, ISS stage, extramedullary disease, bone disease, anemia, renal failure, multiple treatments and relapsed/refractory disease. Results:None of the three genes under study was methylated in the control bone marrow samples, but methylation in the CpG island of Leprel 1 was detected in 20 cases (52%). No methylation was detected in the CpG islands of Leprel2 or Leprecan. Trends noted were that patients with methylated leprel 1 were more likely to have ISS stage 3 (OR 1.5, p=0.6), to have received multiple treatments (OR=1.7, p=0.4) and to have received radiotherapy for either bone lytic lesions or extramedullary disease (OR=5.6, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size. Conclusions:This is the first demonstration of methylation of genes encoding collagen, the prolyl hydroxylases in MM, suggesting that this may be a novel class of myeloma suppressors. Interestingly, Leprel1 was methylated at high frequency, whereas Leprecan and Leprel2 were unmethylated, implying a striking specificity in MM for methylation in Leprel1. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of leprel1 methylation in MM. Disclosures:No relevant conflicts of interest to declare.