Tumor Blood Research Articles

Abstract C167: KTN0158, a humanized anti-KIT monoclonal antibody, demonstrates antitumor activity in dogs with mast cell tumors

Abstract Background: KTN0158 is a novel humanized anti-KIT IgG1 monoclonal antibody that binds canine, feline, non-human primate and human KIT with high affinity. KTN0158 potently inhibits wild-type and some oncogenic variants of KIT in vitro and modulates canine mast cell function and survival in vivo. A clinical trial was conducted in dogs with spontaneous mast cell tumors (MCT) where KIT is known to be widely expressed and where activating mutations in the form of internal tandem duplications (ITDs) in exon 11 are found in 30% of tumors. The purpose of this study was to evaluate the safety, biologic activity and pharmacokinetic/pharmacodynamic (PK/PD) profile associated with KTN0158 administration in dogs with MCT. Methods: Twelve dogs with measurable MCT were enrolled into this open-label phase 1 dose-escalating clinical trial. Three dose levels and 2 schedules were evaluated (10 or 30 mg/kg given on Day 0 only; 1 or 10 mg/kg given on Days 0 and 21). Serial tumor biopsies and blood samples for PK and PD analysis were collected pre- and post-treatment throughout the study. Dogs were assessed weekly with physical examination and standard laboratory tests (serum chemistries, hematology profiles, and urinalyses) for clinical toxicities and response. Adverse events (AEs) were recorded and graded according to published common terminology criteria for AEs in dogs. Determination of antitumor efficacy was based on objective tumor assessments made according to established RECIST criteria for solid tumors in dogs. Results: Of the 12 dogs entered in this study, 3 had tumors with exon 11 ITDs and 6 had evidence of metastasis to the local lymph node. All dogs treated with a single dose of KTN0158 at 10 or 30 mg/kg or two doses of KTN0158 at 1 or 10 mg/kg experienced clinical benefit. Partial responses were observed in 5 dogs (n = 1 KIT ITD) and stable disease was observed in 7 dogs (n = 2 KIT ITD). Upon study completion, 11 dogs underwent surgical removal of the MCT with or without extirpation of the draining lymph node. Histopathology failed to identify neoplastic mast cells in tumor samples from 2 dogs (patients 2 and 4) and 3 draining lymph nodes from 3 dogs (patients 2, 3 and 11) classified as metastatic at study entry. The most common adverse events associated with KTN0158 administration were reversible dose-dependent hematologic changes including anemia, neutropenia, and thrombocytopenia, generally occurring 7-14 days post-treatment. Doses of 30 mg/kg KTN0158 resulted in clinically significant toxicities not observed at other dosing levels, including evidence of edema/erythema, grade 4 neutropenia, and grade 3 thrombocytopenia. Evaluation of PK and PD biomarkers is ongoing. Conclusions: KTN0158 given at 1 and 10 mg/kg demonstrated an acceptable adverse event profile and single agent biologic activity in a relevant spontaneous large animal model of KIT driven malignancy (MCT). The objective responses observed in tumors with and without activating KIT mutation suggest that KTN0158 may provide clinical benefit to patients with GIST and other KIT-driven tumors and human clinical studies are planned. Citation Format: Cheryl London, Sarah Rippy, Heather Gardner, William Kisseberth, Gerald Post, Neal Janson, Linda Crew, Theresa LaVallee, Richard Gedrich. KTN0158, a humanized anti-KIT monoclonal antibody, demonstrates antitumor activity in dogs with mast cell tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C167.

ATPS-80ANTI-TUMORIGENESIS OF LENTI-VIRUS sh-HDGF IN RAT C6 GLIOMA MODEL

BACKGROUND: In our previous studies, we found Hepatoma derived growth factor (HDGF) is a mitogenic growth factor in the progression of human glioma. METHODS: In this paper, we study the gliomagenesis role of HDGF in syngenic glioma rat model by Lenti-virus sh-HDGF (LV-shHDGF) infection and tumors growth inhibition was analysis by MRI scanning and western blot analysis. This work was supported by NSC101-2314-B-075B-003-MY2 and VGHKS102-022. RESULTS: We found all glioma tumors growth after implanting but only HDGF knock down tumors had a significant reduction in the tumor size at the 4th-week MRI-scanning. After tumor volume was quantified on serial brain section, we found a 10-folds of reduction in the tumor size was observed in HDGF knock down tumors than scrambles (p < 0.005). By survival analysis, rats with gliomas had a less median survival of 25 days and the HDGF knock down group was still alive from the time point of implanting. According to the pathological analysis, the HDGF knock down tumors presented a significant cell apoptosis and reduction in the Ki67 mitotic index, CD31 angiogenesis index, and ANG-4 that exhibited markedly reduced tumor growth and blood vessel formation than controls. In addition, Western blot analysis showed exogenous HDGF increased the expression of PI3K, phospho-Akt, phospho-FAK, phospho-MDM2, PCNA and transcription factors of Ik-B, NF-kB, cFos, ANG-4, and HIF-a in a dose-dependent manner; however, these tumorigenesis moleculars were significantly decreased in HDGF knock cells. CONCLUSIONS: We concluded that HDGF is a mitogenic growth factor in the progression of glioma and it can be a useful therapeutic target for clinical management of glioma in the future.

ATPS-33EVIDENCE OF ABSENCE FOR CYTOMEGALOVIRUS IN PATIENTS WITH GLIOBLASTOMAS AND OTHER HIGH-GRADE GLIOMAS BY REAL-TIME PCR FOR DNA, IMMUNOHISTOCHEMISTRY FOR PROTEIN ANDIN-SITUHYBRIDIZATION FOR mRNA

BACKGROUND: Several groups have suggested a potential causative role of cytomegalovirus (CMV) in the development of high-grade gliomas (HGG), while others have not found any evidence of CMV in these cancers. We used 3 different molecular technologies to assay for the presence of CMV DNA, mRNA and protein in tumor tissue and blood samples of patients with HGG. METHODS: Part A: Fresh-frozen tissues from 27 glioblastomas (GBM) were analyzed for CMV by real-time PCR of US17. Part B: Prospective study of patients with newly-diagnosed HGG (grades III or IV) who underwent standard chemoradiation. Plasma and serum were collected at baseline, and plasma at follow-up visits. FFPE tumor tissue was analyzed for CMV proteins by genetically validated immunohistochemistry (IHC), in-situ hybridization assays targeting 1E1/2 and pp65, and by real-time PCR for US17 (tissue and plasma). RESULTS: Part A: CMV was detected in 0/27 fresh-frozen GBM tissue samples using real-time PCR. Part B: Eighteen patients with HGG (12 grade IV, 6 grade III) were enrolled. CMV tissue testing was negative in all samples by both probes for in-situ hybridization, both antibodies for IHC (n = 11 available samples), and CMV US17 real-time PCR (PCR successful in 8/14 samples). Serum CMV IgG avidity index was positive (0.55-0.82) in 8/15 cases, consistent with rates in the general population. CMV was detected in baseline plasma samples of 3/16 patients and in 1 single follow-up sample. All other plasma samples were negative for CMV (n = 34). CONCLUSION: CMV was not detected in any HGG tissue. Half of the patients with HGG did not have an exposure to CMV prior to their diagnosis, and viremia was rare at presentation and follow-up. An independent, unbiased investigation of CMV positivity rates using a central reference laboratory with standardized tissue samples and controls is warranted before further consideration of CMV-targeted therapy in HGG.

OP02ADOLESCENT/YOUNG ADULT MEDULLOBLASTOMA: TIME FOR A CHANGE IN MANAGEMENT STRATEGY

INTRODUCTION: About 20% of medulloblastoma cases arise between 15 and 40 years of age, representing only 1% of adult brain tumors. No prospective randomized trials of treatment for adults have ever been reported; however, retrospective reports in the modern era (Carrie 2007) suggest no outcome differences with lower craniospinal irradiation (CSI) doses ( 34Gy) alone (77 and 62% 5 and 10 year overall survival). These outcomes appear inferior to those currently reported in children with standard-risk medulloblastoma; however, these populations are now recognized to be dissimilar clinically, histologically and molecularly, with metastases, anaplastic/large cell histology and cmyc amplification (Group C) being very uncommon at diagnosis in adult patients (Kool 2012). Further, SHH tumors appear more prevalent in adults, except for the youngest children, and display distinct molecular differences. METHOD: We propose a multi-center prospective randomized trial of 36Gy CSI with tumor bed boost (18Gy) with or without an attenuated maintenance chemotherapy regimen for older adolescents/adults newly-diagnosed with non-metastatic medulloblastoma. Central review of imaging studies, histopathology and quality of life assessments will be mandated. Submission of paraffin-embedded tumor tissue and blood for sub-group classification, methylation profiling and whole exome sequencing, will be performed in order to create a correlational biobank to parallel the first prospective dataset in this population. CONCLUSION: This prospective, randomized clinical trial will set a new “benchmark” for diagnosis, prognostication and treatment of adults with medulloblastoma

PTPS-08HEMATOGENOUS DISSEMINATION OF MEDULLOBLASTOMA METASTASES TO THE LEPTOMENINGES

Metastatic medulloblastoma is a high-risk disease, because the majority of children dying of medulloblastoma expire from metastases rather than recurrence of the primary tumor. Current dogma dictates that medulloblastoma cells are shed from the primary tumor into the CSF, float through the spinal fluid and establish metastatic colonies on the leptomeningeal surface and spinal cord. Empirical evidence for this mechanism is lacking in the literature. Whole genome sequencing on matched trios of human primary tumor, metastases, and peripheral blood showed somatic mutations from the medulloblastoma at low clonal frequency in the blood samples — consistent with circulating tumor cells. In addition, we identified circulating tumor cells (CTC) in the blood of transgenic mice bearing metastatic medulloblastoma, and in mice intra-cranially implanted with patient derived MB. Surprisingly, when we implanted human or mouse medulloblastoma cells in the flank of immunocompromised mice, the MB cells colonized the leptomeningeal space causing death. As these MB xenografts are not in continuity with the CSF, they must have arisen through hematogenous dissemination. Importantly, flow cytometry shows that MB CTCs express CD34, the cell-surface marker used clinically in children to collect peripheral blood stem cells; suggesting that autologous bone marrow transplants from children with medulloblastoma are likely contaminated with CTCs. Finally we established a parabiosis model where two immunocompromised, sibling mice are surgically joined, and a common blood circulation is established. After parabiosis, one mouse is intra-cranially implanted with MB, and the mice are subsequently separated when the xenografted twin becomes symptomatic. The surviving twin goes on to die from leptomeningeal metastases. Our data challenge the existing paradigm of CSF mediated dissemination of medulloblastoma by demonstrating the capacity of medulloblastoma to spread through the blood system. Identification of a systemic route for MB metastases offers new avenues for the diagnosis and therapy of metastatic medulloblastoma.

IMCT-11NEXT GENERATION T CELL RECEPTOR SEQUENCING CAN IDENTIFY, QUANTIFY, AND TRACK TUMOR-SPECIFIC T CELL POPULATIONS BEFORE AND AFTER DENDRITIC CELL VACCINATION IN GLIOBLASTOMA MULTIFORME PATIENTS

While dendritic cell vaccination is a promising treatment for glioblastoma (GBM), sensitive methods of monitoring the immune response after treatment remain to be established. We sought to determine whether: 1) T cell receptor (TCR) repertoire overlap between peripheral blood and tumor and 2) tumor infiltrating lymphocyte (TIL) content, as assessed by next generation DNA sequencing, are predictors of immune response and survival. Genomic DNA from pre and post treatment tumor, as well as pre and post treatment peripheral blood, of 16 GBM patients enrolled in Phase I and II DC vaccination clinical trials was subjected to next generation sequencing through the TCRVβ region to quantify TIL content and assess the overlap between tumor and blood. Of the 5 patients for whom initial and relapsed tumors were available, two patients with high TCR sequence overlap between tumor and blood prior to treatment demonstrated the longest overall survival (OS) of 71.3 and 28.7 months. Two patients with poor OS (6.3 and 4.5 months) had low blood and tumor TCR overlap both before and after treatment. One multi-focal recurrent patient with intermediate OS (12.6 months) demonstrated an initially low overlap between pre-treatment tumor and blood; however, the TCR overlap increased 200-fold after treatment. Because we did not possess post-treatment, relapsed tumors for many patients, we tested whether the density of pre-treatment TILs was relevant. For the 16 patients from whom we had pre-treatment tumor tissue, the estimated TIL content significantly correlated with time to progression and OS (HR = 0.252, p = 0.0442; HR = 0.277, p = 0.0461), respectively. The estimated TIL content of pre-treatment tumor directly correlates with survival in GBM patients who received autologous tumor lysate-pulsed DC vaccination. Furthermore, the trends of tumor and peripheral blood TCR overlap suggest that this technology can be utilized to track tumor-specific T cell populations without prior knowledge of their antigen specificity.

GENO-09LANDSCAPE OF GENOMIC ALTERATIONS IN PITUITARY ADENOMAS

BACKGROUND: The genetic underpinnings of sporadic pituitary adenomas remain incompletely understood, despite their prevalence in adults. We describe a comprehensive genetic characterization of the largest mixed cohort of pituitary macroadenomas, including hormonally active and atypical adenomas. METHODS: 42 pituitary adenomas were selected to represent a spectrum of histopathologic subtypes, de novo and recurrent tumors, and MIB-1 proliferative indices to interrogate the heterogeneity intrinsic to pituitary tumors. Whole-exome sequencing was performed on tumor and paired blood to identify somatic copy-number alterations, mutations, insertions and deletions. RESULTS: We appreciated two classes of pituitary tumors based upon the fraction of the genome disrupted. The first class (71% of tumors) had somatic copy-number alterations involving less than 6% of the genome; the other class (29%) had significantly greater levels of genomic disruption, up to 99% of the genome involved. The widespread genomic disruption observed in the second class was largely accounted for by arm-level events, with minimal involvement of focal events, which is rare among tumor types. Levels of genomic disruption were associated with phenotype. Among the less disrupted group, 87% (26/30) were clinically non-functional adenomas, whereas the majority of the disrupted group were functional or atypical null-cell adenomas. The high and low genomic disruption classes did not differ in mean MIB-1 proliferative index. Frequent loss of chromosomes 11 and 1p were observed, as well as significant recurrent amplifications of chromosome 5, 7, 9, 12, and 14q. On the whole, the tumors harbored a low rate of mutations, similar to other benign brain tumors. CONCLUSIONS: Genomic characterization of a large series of sporadic pituitary adenomas reveals two distinct classes of tumor, based on genomic disruption from broad copy-number alterations, and associates with tumor subtype.

Correlation Between Tumor Size and Blood Volume in Lung Tumors: A Prospective Study on Dual-Energy Gemstone Spectral CT Imaging

Correlation Between Tumor Size and Blood Volume in Lung Tumors: A Prospective Study on Dual-Energy Gemstone Spectral CT Imaging

Tie-1: A potential target for anti-angiogenesis therapy.

The tyrosine kinase system angiopoietin (Ang)/Tie interacts with vascular endothelial growth factor pathway and regulates vessel quiescence in adults as well as later steps of the angiogenic cascade related to vessel maturation. Since all Angs are able to bind to Tie-2 but none binds to Tie-1, the function of Tie-2 and its ligands have captured attention. However, emerging evidence indicates unique roles of the orphan receptor Tie-1 in angiogenesis under physiological and pathological conditions. It is required for maintaining vascular endothelial cell integrity and survival during murine embryo development and in adult and may be involved in modulating differentiation of hematopoietic cells in adult. Tie-1 exhibits poor tyrosine kinase activity and signals via forming heterodimers with Tie-2, inhibiting Tie-2 signaling mediated by Angs. This inhibition can be relieved by Tie-1 ectodomain cleavage mediated by tumor- and inflammatory-related factors, which causes destabilization of vessels and initiates vessel remodeling. Up-regulated Tie-1 expression has been found not only in some leukemia cells and tumor related endothelial cells but also in cytoplasm of carcinoma cells of a variety of human solid tumors, which is associated with tumor progression. In addition, it has pro-inflammatory functions in endothelial cells and is involved in some inflammatory diseases associated with angiogenesis. Recent research indicated that Tie-1 gene ablation exhibited significant effects on tumor blood- and lymph-angiogenesis and improved anti-Ang therapy, suggesting Tie-1 may be a potential target for tumor anti-angiogenesis treatment.

Abstract 1307: Assessment of gene expression in peripheral blood from patients with advanced melanoma using RNA-seq before and after treatment with anti-PD-1 therapy with pembrolizumab (MK-3475)

Abstract Pembrolizumab (MK-3475) is a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1) that is currently being studied in clinical trials across more than 30 types of cancer. Gene expression profiling was used to explore a less invasive method for gaining new insights into the pembrolizumab mechanism of action and to assess the potential for blood-based predictive biomarkers. RNA-seq data were obtained from the whole blood of 44 patients with advanced melanoma enrolled in the phase 1 KEYNOTE-001 clinical study before and after the first cycle of treatment with pembrolizumab. Objective response rates (ORR) were assessed per RECIST v1.1 by independent central review. The pembrolizumab dose and treatment schedule varied among the patients studied and included 10 mg/kg given once every 3 weeks (Q3W) (n = 21), 10 mg/kg Q2W (n = 12), and 2 mg/kg Q3W (n = 11). Among the 44 patients with melanoma included in the analysis, 75% received previous ipilimumab treatment. The ORR was 32%. Significant posttreatment changes in gene expression were confirmed for the target, PD-1, and its key ligand, programmed death ligand 1 (PD-L1). Changes in PD-1 and PD-L1 gene expression did not, however, show a significant association with ORR. Statistically significant posttreatment changes for an interferon-gamma (IFNG)-related 10-gene signature were observed (P &amp;lt; 0.002, upregulated in 70% of patients), and these changes were also associated with improved clinical outcome in terms of ORR (P = 0.042) and progression-free survival (P = 0.010). There appeared to be a possible association between IFNG gene signature posttreatment changes in blood in tumors with higher PD-L1 expression at baseline. There were no clear differences between posttreatment changes in the IFNG gene signature according to previous ipilimumab treatment. Baseline blood expression levels for PD-1, PD-L1, and the IFNG 10-gene signature were not found to be associated with clinical outcome. Additional post hoc analyses of baseline expression in combination with pathway analysis showed that the more highly ranked genes were linked to oxidative phosphorylation, suggesting that for patients whose tumors are not responding to pembrolizumab, their T cells may not be engaged with glycolysis, which is important for the transcriptional regulation of IFNG. The observation that nonresponders showed a lack of ability to induce activation of IFNG signaling suggests a possible link to defective T-cell effector function and may provide a means by which to monitor patient response to anti-PD-1 therapy. Citation Format: Mark D. Ayers, Michael Nebozhyn, Heather A. Hirsch, Razvan Cristescu, Erin E. Murphy, S. Peter Kang, Scot W. Ebbinghaus, Terrill K. McClanahan, Andrey Loboda, Jared K. Lunceford. Assessment of gene expression in peripheral blood from patients with advanced melanoma using RNA-seq before and after treatment with anti-PD-1 therapy with pembrolizumab (MK-3475). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1307. doi:10.1158/1538-7445.AM2015-1307

Abstract 4175: Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3)

Abstract VEGFR3 plays a key role in the regulation of lymphangiogenesis in adults, and is also important for tumor angiogenesis and metastasis. In order to characterize human cancer tissues for imunohistochemical (IHC) localization and prevalence of VEGFR3 protein, we developed a robust IHC assay in our lab using a mouse monoclonal primary antibody (Millipore). To optimize the various assay parameters, we utilized high-quality VEGFR3 positive (and internal negative) control tissues (Kaposi's sarcoma, angiosarcoma, lymph node). Reagent negative controls were satisfactory. Specificity of the primary anti-VEGFR3-antibody was supported by a discrete band at the expected molecular weight on western blots using a VEGFR3 transfected cell lysate and negative control cell lines. We also demonstrated lack of specific vascular VEGFR3 staining in pre-absorption experiments with VEGFR3 (but not VEGFR1 or 2) recombinant protein, supporting selectivity of the primary antibody for VEGFR3. Using the fully optimized assay, we stained a human multi-tumor screening tissue microarray (TMA) to demonstrate full range of specific vascular VEGFR3 staining in glioblastoma and carcinomas of the colon, breast, ovary, pancreas, lung, larynx, kidney, cervix, bladder, extrahepatic cholangiocarcinoma and malignant melanoma. Specific, unequivocal VEGFR3 immunoreactivity was interpreted qualitatively (VEGFR3 positive/negative) in tumor blood and lymphatic vessels. No tumor cell staining was seen. Some colon cancer tissues in multi-tumor TMA were VEGFR3+, while others were VEGFR3-. The observed variation in VEGFR3 expression and vascular distribution on screening TMA were further evaluated in two independent cohorts of well-characterized human colorectal cancer tissues (organ-specific TMAs) by a Board-certified, subspecialty GI pathologist (AN), with overall VEGFR3 prevalence rates of 62% (36 of 58 cases) and 56% (55 of 98 cases). Using CD34 and D2-40 IHC assays from a CLIA-certified lab, we confirmed IHC localization of VEGFR3 protein both in the stromal blood vessels and lymphatics within the invasive colorectal cancer stroma. In conclusion, following well-established IHC assay development and standardization protocols and efficient workflow paradigm, we have used a technically robust IHC assay to characterize routinely processed archival human cancer tissues and have demonstrated specific VEGFR3 expression patterns, tissue localization and prevalence in human colorectal cancer tissues. Based on its performance in our hands, this assay can be used to further evaluate patterns of VEGFR3 expression in areas of tumor angiogenesis in other human cancer tissues. Data-driven hypotheses generated from such investigations will be relevant to corroborate VEGF receptor biology and emerging clinical experience with anti-VEGF/anti-VEGFR therapies. Citation Format: Timothy R. Holzer, Drew M. Nedderman, Aejaz Nasir. Robust immunohistochemical assay to characterize human cancer tissues for prevalence of vascular endothelial growth factor receptor 3 (VEGFR3). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2015-4175

Abstract 1925: Streamlined analysis and interpretation of RNA editing variants from melanoma cancer samples

Abstract Of special interest in a clinical setting is the identification and interpretation of somatic variants from the DNA of a tumor/normal matched pair. Studies have shown that a large proportion of variants present in DNA are not present in the RNA and vice versa due to RNA-Editing and other unknown molecular processes1. In addition, recent publications show that RNA-editing plays an important role in cancer2,3. These findings can have strong implications for clinical diagnostics where the correct identification and interpretation of somatic variants from tumor/normal matched samples is essential. We took a published Whole Trancriptome/Whole Exome Sequencing dataset of tumor and peripheral blood from two patients with uveal melanomas5. We looked for somatic variants present in the RNA, but absent in the DNA using CLC Cancer Research Workbench. A strong mutation signature for C-T, A-G changes was found, which result from Adenosine to Inosine changes, a typical RNA-Editing event, which was recently published to be common in human and is mediated by ADAR proteins. By running Ingenuity Variant Analysis directly from CLC Cancer Research Workbench we found RNA-Editing found in these two patients is highly associated with cancer and more specifically with Melanoma directly. Some of the variants directly associated with Melanoma were classified as likely pathogenic and were not described in COSMIC, but present in Ingenuity's large Knowledge Base. 1) Widespread RNA and DNA Sequence Differences in the Human Transcriptome. Mingyao Li, Isabel X. Wang, Yun Li, Alan Bruzel, Allison L. Richards, Jonathan M. Toung, and Vivian G. Cheung. Science 1 July 2011: 333 (6038), 53-58. 2) RNA editing in RHOQ promotes invasion potential in colorectal cancer. Sae-Won Han et al. J Exp Med 2014 211:613-621. 3) A disrupted RNA editing balance mediated by ADARs (Adenosine DeAminases that act on RNA) in human hepatocellular carcinoma. Tim Hon Man Chan et al.Gut 2014;63:5 832-843 4) A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes.Genome Res. March 2014 24: 365-376 5) Harbour JW et al., “Frequent mutation of BAP1 in metastasizing uveal melanomas.”, Science, 2010 Nov 4;330(6009) Citation Format: Anika Joecker, Rupert Yip, Anne Arens, Bodil Oester, Douglas Basset, Bryant Macy. Streamlined analysis and interpretation of RNA editing variants from melanoma cancer samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2015-1925

Abstract 594: AGT mutations as a prognosis factor in patients with astrocytoma

Abstract Background: Astrocytomas are the most common and lethal brain tumors. The median survival of patients is 15 months. The study of genetic alterations may help to understand the mechanisms of development and behavior of the disease and thus understand the risk factors and prognostic factors. AGT gene and its peptide derivatives, are involved in cell proliferation and neoangiogenesis and resistance to apoptosis, hence the pathogenesis of astrocytomas. Objective: To identify somatic and germinal AGT gene variations in from patients with astrocytoma. Material and methods: 155 randomly selected files from patients diagnosed with astrocytoma between 2008-2014 were revised to determined clinical prognosis factor in Mexican population. In 25 patients, the molecular analysis of the whole codification region, 5´UTR and 3´UTR of the gene AGT was done in tumors biopsy and blood. Each amplified region had a depth of minimum 1500x. IonReporter software was utilize to analyze the genetic variants and using Integrative Genomic Viewer we confirmed that each variant had a Phred higher than 25. Genotypic frequencies were compared with HapMapMex. Results The clinical variants associated with prognosis were age (OR 0.01 IC 95% 1.02-1.07, p = 0.000), Grade IV (OR 17.11, IC 95% 1.89-132.13 p = 0.011), location of tumor (OR 1.17, IC 1.2-6.3, p = 0.016), total resection (OR = .186, IC 95% .098-.622, p = 0.003, Karnofsky (OR = .05, IC 95% 0.024-0.24, p = 0.003), motor deficiency (OR = 3.5, IC = 1.1-10.5, p = 0.025). In blood were found 11 genetic variants. rs4049, rs2148582, rs699, rs4762 and rs1926723 were associated with risk of tumor in the codominant model. rs5050 (- A20C) was statistically associated with the prognosis (OR 10.8, IC 1.16-100.51 p = 0.036). These variant it is part of a haplotype related to low expression of angiotensinogen. In biopsies of 2 patients missense mutations were found p.Arg477His in one patient and Gly226Ala -Pro165Ser in another one. Conclusion: In 8% of the tumors samples new somatic mutations were found. The genetic variant rs5050 of AGT is a prognosis biomarker in patients with astrocytoma. Citation Format: Talia Wegman-Ostrosky, Ernesto Soto-Reyes, Silvia Vidal-Millan, Sonia Mejia, José Sánchez-Corona, Luis A. Herrera. AGT mutations as a prognosis factor in patients with astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 594. doi:10.1158/1538-7445.AM2015-594

Abstract 4748: Revealing the molecular portrait of triple negative breast tumors from an understudied population through omics analysis of formalin-fixed and paraffin-embedded tissues

Abstract Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal receptor 2 (HER2), is an aggressive form of breast cancer (BC) that is more prevalent in certain populations, in particular in low and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. We performed an integrative genomic analysis on FFPE samples from TNBC patients to identify molecular programs associated with this disease in an understudied population. We implemented whole exome sequencing of 12 tumor tissues and blood pairs, complemented with miRNA and mRNA profiling of the tumor tissues, using FFPE archived pathological samples from Mexican women. Sequencing analyses found TP53 and RB1 genes as the most frequently mutated. Transcriptional programs were characterized by the overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), the repression of cell cycle control pathways (TP53, RB1), the deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the utility of archival clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing the development of treatment strategies and of retrospective studies on the exploration of the geographic diversity of BC. Citation Format: Felipe Vaca-Paniagua, Rosa María Alvarez-Gomez, Carlos Perez-Plasencia, Hector Aquiles Maldonado-Martínez, Veronica Fragoso-Ontiveros, Federico Lasa-Gonsebatt, Luis Alonso Herrera, David Cantú, Enrique Bargallo-Rocha, Alejandro Mohar, Geoffroy Durand, Nathalie Forey, Catherine Voegele, Maxime Vallee, Florence Le Calvez-Kelm, James McKay, Maude Ardin, Stephanie Villar, Jiri Zavadil, Magali Olivier. Revealing the molecular portrait of triple negative breast tumors from an understudied population through omics analysis of formalin-fixed and paraffin-embedded tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2015-4748

Abstract 4917: Identification of genetic heterogeneity by whole exome sequencing using cell free DNA from blood samples

Abstract Objectives: Circulating cell-free DNA (cfDNA) is expected to be useful for diagnosis of genetic alterations in cancer. However, it has been still difficult to comprehensively analyze somatic mutations using cfDNA, because of the low quantity in blood. We examined the feasibility of whole exome sequencing using cfDNA and assessed the ratio of mutation reads in cfDNA for somatic mutations comparing with the corresponding tumor DNA in ovarian cancer specimens. In addition, we assessed whether cell free DNA might reflect the tumor heterogeneity in ovarian cancer. Methods: The pair of fresh frozen tumor samples and blood samples was obtained from two cases of ovarian serous adenocarcinoma under informed consent and approval of institutional review board. The tumor DNA and cfDNA were extracted from the specimens and whole-exome sequencing was performed using 1ug of the tumor DNA or 25ng of cfDNA. The list of the mutated genes was compared between cell free DNA and the corresponding tumor DNA. Validation of the mutations and calculation of the tumor content ratio in cfDNA was assessed using target sequencing. Result: We successfully detected the somatic mutations from cfDNA. The somatic mutations detected in cfDNA covered from 21% (49/233) to 55% (141/258) of the mutations detected in the corresponding tumor DNA. TP53 was included in the mutated genes, and the base substitution (chr17:7578257 C&amp;gt;A) was identified in 5 of 121 reads (4%) in cfDNA. We validated the tumor content ratio of cfDNA is 8% by target sequencing. One of the two cases showed the correlation between the mutant allele frequency in the tumor DNA and the detection ratio of the mutations in cfDNA (p = 0.004, logistic regression analysis). However, the correlation was not observed in another case (p = 0.3923, logistic regression analysis). Of note, we detected various types of mutations in cfDNA alone. One of such genes is RAPGEF2. The base substitution (ch4:160253031 C&amp;gt;T) was detected only in cfDNA (11/53, 21%), but not in tumor DNA (0/120, 0%). Conclusion: Our data suggest that whole exome sequencing of cfDNA is feasible. The mutations detected only in cfDNA might reflect the tumor heterogeneity, and cfDNA might provide us novel biomarkers for diagnosis of recurrence and/or metastasis. Further study is warranted to elucidate the method to utilize the genetic information from cfDNA. Note: This abstract was not presented at the meeting. Citation Format: Kayo Asada, Katsutoshi Oda, Kengo Gotoh, Reiko Kurikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Yuji Ikeda, Yuriko Uehara, Kenbun Sone, Osamu Hiraike-Wada, Kei Kawana, Tetsu Yano, Yutaka Osuga, Tomoyuki Fujii, Hiroyuki Aburatani. Identification of genetic heterogeneity by whole exome sequencing using cell free DNA from blood samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4917. doi:10.1158/1538-7445.AM2015-4917

Abstract 4172: Differential angiogenesis-related cytokines release in tumor interstitial fluid and plasma in ER-positive and triple-negative breast cancers overexpressing VEGF

Abstract Interstitial fluid (IF) is a key component of the tumor microenvironment (TME) that has been largely overshadowed by proteomic and genomic analysis of the cancer cell and stromal compartments of the TME. Early studies of tumor IF by Gullino and colleagues showed elevated protein and lactic acid levels and the absence of glucose. Modern analytical methods allow for comprehensive characterization of IF proteins to better understand the TME. We modified Gullino's model and created a collection chamber adapted to collect IF from mouse tumors. We used ER-positive MCF-7 and triple negative MDA-MB-231 breast cancer cells in SCID mice. Additionally, we created VEGF-overexpressing cells from each cell line to evaluate the effect of VEGF overexpression on angiogenic cytokines in tumor IF. The chamber was implanted with small tumor pieces in the subcutaneous space until the tumor encompassed the chamber (4 to 12 weeks depending on cell types). The tumor was then removed and the tumor IF (approximate volume 40μl) and blood plasma were collected. IF and plasma samples were analyzed using a multiple cytokine immunoassay (Proteome Profiler Human Angiogenesis Kit, R &amp; D Systems Inc., Minneapolis MN). Among the 55 human angiogenesis-related cytokines tested, only 4 were detected in MCF-7 tumors IF, and 24 were detected in MDA-MB-231 IF sample. As anticipated, high VEGF levels were detected in MCF-7 overexpressing cells. Additionally, we observed a tendency to an elevated level of TIMP metallopeptidase inhibitor 1 (TIMP-1). All 4 cytokines detected in MCF-7 IF were also detected in MDA-MB-231 IF. VEGF overexpression had minimal effect on the tested cytokines. However, decreased levels of angiogenin and endostatin were observed. In plasma of tumor-bearing mice, 5 cytokines were detected. For instance, Serpin E1 (PAI-1) and TIMP-1 were detected in the plasma of both MCF-7 and MDA-MB-231, at lower levels than in IF. However, urokinase plasminogen activator was present only in mice with triple negative MDA-MB-231 tumors. We anticipate that a better understanding of the TME, via the characterization of the IF, will help identify new targets for diagnosis and therapy of cancer. We intend to examine correlations between the present data and human databanks to confirm the relevance of our model systems. Citation Format: Louis Dore-Savard, Esak Lee, Aleksander S. Popel, Zaver M. Bhujwalla. Differential angiogenesis-related cytokines release in tumor interstitial fluid and plasma in ER-positive and triple-negative breast cancers overexpressing VEGF. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4172. doi:10.1158/1538-7445.AM2015-4172

Effect of iron nanoparticles on tumor cells and immunocytes.

e22094 Background: The aim of the study was to analyze the effectof iron nanoparticles on functional state of tumor and mononuclear blood cells of patients with malignant tumors in vitro. Methods: ...

Abstract OT1-1-01: LORELEI: A Phase II randomized, double-blind study of neoadjuvant letrozole plus taselisib (GDC-0032) versus letrozole plus placebo in postmenopausal women with ER-positive/HER2-negative, early stage breast cancer

Abstract Background: Taselisib (GDC-0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3-kinase (PI3K) alpha, gamma, and delta isoforms, with 30-fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that taselisib has enhanced activity against PIK3CA mutant cancer cell lines. Clinical data have also demonstrated confirmed partial responses in patients with PIK3CA mutant breast cancer treated with single-agent taselisib. Preclinical and clinical data also show enhanced antitumor activity when taselisib is combined with either letrozole or fulvestrant. Study design: LORELEI is a phase II, two-arm, randomized, double-blind, multicenter, neoadjuvant study of letrozole and taselisib versus letrozole and placebo in postmenopausal women with newly diagnosed ER+/HER2-, untreated, stage I-III operable breast cancer. Other relevant eligibility criteria include tumor size ≥ 2 cm, unilateral disease, ECOG PS ≤1, and available and evaluable tumor tissue for central review of PIK3CA mutation analysis. Patients will be randomized (1:1) to receive continuous daily letrozole (2.5 mg) with either placebo or taselisib (4mg on a 5 days on/ 2 days off schedule) for 16 weeks. Study treatment is followed by surgery. Adjuvant treatment will be given as per physician’s discretion. Stratification at randomization is based on tumor size and nodal status. Endpoints: The co-primary endpoints are overall objective response rate (ORR) by centrally assessed breast magnetic resonance imaging (MRI) via modified RECIST criteria and pathologic complete response (pCR) rate in breast and axilla at time of surgery in all enrolled patients and PIK3CA mutant (MT) patients. Secondary endpoints include ORR by centrally assessed MRI and pCR rate in PIK3CA wild-type (WT) patients. Other secondary endpoints performed in all enrolled patients and separately as per PIK3CA mutation status include: assessment of ORR using breast ultrasound, clinical breast exam (i.e. palpation) and mammography; changes in Ki67 levels from baseline to week 3, baseline to surgery and week 3 to surgery; centrally assessed preoperative endocrine prognostic index (PEPI) score; changes in enhancing tumor volume from baseline to surgery as measured by breast MRI via central assessment. Exploratory analyses include expression of biomarkers predictive of response to letrozole plus taselisib from tumor tissue or blood. Statistical methods: The sample size was calculated to detect an absolute percentage increase of 24% in ORR via MRI (40% in the letrozole-placebo arm vs. 64% in the letrozole-taselisib arm in the PIK3CA MT cohort) with 80% power at 16% two-sided significance level. The sample size will also detect an absolute percentage increase of 18% in pCR rate (1% in the letrozole-placebo arm vs 19% in the letrozole-taselisib arm in the PIK3CA MT cohort) with 80% power at 4% two-sided significance level. Target accrual: Approximately 330 pts at 110 global sites across Europe, North and South America, and Asia-Pacific. Reference Study ID Numbers: GO28888/BIG-3-13/SOLTI 1205/ABCSG 38. Citation Format: Cristina Saura, Evandro de Azambuja, Peter Dubsky, Mafalda Oliveira, Kamal S Saini, Christian Fes, Ray S Lin, Timothy R Wilson, Jill Fredickson, Hema Parmar, Jerry Y Hsu, Martine Piccard, Michael Gnant, Jose Baselga. LORELEI: A Phase II randomized, double-blind study of neoadjuvant letrozole plus taselisib (GDC-0032) versus letrozole plus placebo in postmenopausal women with ER-positive/HER2-negative, early stage breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-01.

Phase II trial of temsirolimus (TEM) plus sorafenib (SOR) in hepatocellular carcinoma (HCC).

TPS501 Background: The multikinase inhibitor SOR prolongs survival in patients with HCC not amenable to curative therapies. In HCC preclinical models, the combination of SOR with an inhibitor of the mammalian target of rapamycin (mTOR) pathway is synergistic, though single-agent mTOR inhibition did not improve survival in HCC patients after failure of SOR in a phase III trial. We previously completed a phase I study of the mTOR inhibitor TEM combined with SOR in 25 HCC patients which identified the maximum tolerated dose as TEM 10 mg IV weekly and SOR 200 mg PO BID. This two-center, phase II study was developed to examine the efficacy of the combination and to explore candidate biomarkers. The study was approved and funded by the National Comprehensive Cancer Network (NCCN) Oncology Research Program from general research support, with activation October 2012. Methods: The study is registered on ClinicalTrials.gov (NCT01687673). Design: Single-arm, one stage phase II trial. Primary endpoint: Time to progression (TTP) by RECIST 1.1. Other endpoints: Progression-free survival, response rate, overall survival, proportion with alpha fetoprotein decline ≥ 50%, toxicity, hepatitis B virus reactivation rate, and exploratory biomarkers including mTOR pathway protein expression in tumor, circulating tumor cells, and blood and tumor micro-RNA profiles. Sample size: 25 evaluable patients are required to detect a difference between the null hypothesis of median TTP &lt; 3 months versus alternate hypothesis of median TTP ≥ 6 months (a clinically-meaningful outcome in advanced HCC), with 1-sided significance level of 10% and power 88% under the exact test. Main eligibility criteria: HCC not amenable to curative therapies, histologically-confirmed, ≥ 1 untreated, radiographically-measurable site of disease. No prior systemic therapy. ECOG ≤ 1. Child-Pugh score ≤ 7 with bilirubin ≤ 2 mg/dL. Treatment and procedures: TEM 10 mg IVweekly plus SOR 200 mg PO BID in 28-day cycles, with collection of archival tumor samples and blood samples at baseline, on treatment, and at progression. Accrual:Sixteen of 25 planned evaluable patients have enrolled. An interim analysis for safety after 30% enrollment met pre-specified target to continue. Clinical trial information: NCT01687673.

Preoperative ultrasound features as prognostic factors for patients with hepatocellular carcinoma.

Ultrasound is the most common imaging tool used to scan the tumours of hepatic carcinoma patients. However, very few studies have been performed to evaluate ultrasound imaging features for predicting tumour prognosis. Therefore, the goal of the current study was to evaluate preoperative ultrasound characteristics as prognostic factors that could affect survival rate after liver resection for hepatocellular carcinoma (HCC). A total of 104 HCC patients who underwent resection were retrospectively reviewed with regard to their clinical data, preoperative ultrasound characteristics, and survival rate. Preoperative ultrasound parameters included cirrhosis, tumour site, size, echo pattern, portal vein thrombosis, intra-tumour blood flow signal, peak systolic velocity (V max), and resistance index (RI). The Kaplan-Meier method was used to calculate survival. Pre-resection prognostic factors were assessed using univariate log-rank test and a multivariate Cox proportional hazards model. The median survival was 37months. The 1-, 3-, and 5-year disease-free survival (DFS) rates were 78.85, 53.85, and 26.92%, respectively, and the overall survival (OS) rates at 1, 3, and 5years were 85.58, 69.23, and 46.15%, respectively. On univariate analysis, shorter survival was associated with mixed echo pattern, larger tumour size, portal vein thrombus, affluent flow signal, and higher V max. Application of the Cox multivariate proportional hazards model indicated that tumour size and blood flow signal in the tumours were independent prognostic factors. The overall survival for HCC patients undergoing hepatic resection can be stratified on a sonographic basis of tumour size and intra-nodular vasculature. These prognostic factors may be useful to determine appropriate treatment for HCC patients.