Tumor Blood Research Articles

Abstract 5606: Gallium maltolate inhibits brain tumor volume and blood volume in xenograft model.

Abstract Purpose: There are limited treatment options for glioblastomas (GBM). Tumor cells have a high requirement for iron; the latter is taken up by cells through transferrin receptor-mediated endocytosis of transferrin-iron. These receptors are highly expressed on GBM cells, which makes them an attractive target for transferrin receptor-directed therapies. Gallium is a group IIIa metal that can function as an iron mimetic by avidly binding to transferrin and incorporating into cells through the transferrin receptor. No studies have been performed to determine the efficacy of gallium-based therapies in brain tumors. Consequently, the goal of this study was to evaluate gallium maltolate in the treatment of a U87 xenograft brain tumor model. Methods: Athymc rats bearing U87 human grade IV astrocytoma cells were studied. Gallium maltolate (50 mg/kg/day, n=5) or saline (n=3) was given intravenously. via an alzet mini pump in the jugular vein. Magnetic resonance imaging (MRI) was performed on days 8 and 18 on a Bruker 9.4 T scanner. Enhancing tumor volumes were determined from the post-contrast T1w images, in all slices showing enhancing tumor. The spin and gradient echo relaxation rate changes were then determined giving estimates of microvascular and total blood volume. ((CBVmicro≈R2=R2MION-R*pre-MION CBVTotal≈ R2* = R2*MION-R2*pre-MION). Results: Gallium maltolate inhibited tumor growth (377132%), as measured by enhancing tumor volume, compared to saline controls (863481%). Treatment shows decrease of CBV micro and CBVTotal compared to the controls. The ratio of R2* /R2, which is a measure of mean vessel diameter, increased in saline treated controls but remained unchanged for the gallium maltolate treated rats. To our knowledge this is the first study performed that uses physiologic MRI measurements to investigate the effects of gallium maltolate on brain tumor xenografts. The differences shown are not statistically significant a result likely due to the small sample sizes, which is being remedied by ongoing additional studies. For the imaging studies included here tissue markers of proliferation (Ki67), hypoxia (HIF1) transferrin receptors, and vascular density (vWF) are being analyzed to provide additional information regarding mechanism of action. In general these results demonstrate, for the first time, that the novel gallium maltolate treatment holds promise for the treatment of malignant brain tumors. Citation Format: Kimberly R. Pechman, Andrew Lozen, Mona Al-Gizawiy, Kathleen Schmainda, Christopher R. Chitambar. Gallium maltolate inhibits brain tumor volume and blood volume in xenograft model. [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 5606. doi:10.1158/1538-7445.AM2013-5606

Blood oxygenation level dependent, blood volume, and blood flow responses to carbogen and hypoxic hypoxia in 9L rat gliomas as measured by MRI

To study vascular responsiveness to hypoxia and hypercarbia together with vessel size index (VSI) in a 9L rat glioma (n = 11) using multimodal MRI. VSI was determined using T2 and T2* MRI following AMI-227 contrast agent. Blood oxygenation level dependent (BOLD) signal response was determined using T2 EPI MRI, blood volume changes using AMI-227 and blood flow by means of continuous arterial spin labeling. VSI in the cortex, tumor rim, and core of 2.2 ± 1.0, 18.2 ± 5.4, and 23.9 ± 14.7 μm, respectively, showing a larger average vessel size in glioma than in the brain parenchyma. BOLD and blood volume signal changes to hypoxia and hypercapnia were much more profound in the tumor rim than the core. Hypoxia led to rim BOLD signal change that was larger in amplitude and it attained the low value much faster than either core or brain cortex. The vasculature in the rim appears more responsive to respiratory challenges in terms of volume adaptation than the core. Blood flow values within the gliomas were much lower than in the contralateral brain. Neither hypercarbia nor hypoxia had an effect on the tumor blood flow. Vascular responses of 9L gliomas to respiratory challenge, in particular hypoxia, are heterogeneous between the core and rim zones, potentially offering a means to classify and separate intratumor tissues with differing hemodynamic characteristics.

Vascular proliferation is associated with survival in pancreatic ductal adenocarcinoma

In pancreatic ductal adenocarcinoma (PDAC), the benefit of current chemotherapy and radiation therapy is very limited, even in radically resected patients. New treatment strategies, for example based on the inhibition of the tumour's blood supply, need to be explored. We have investigated angiogenesis markers and their associations with relapse and survival in 52 histologically confirmed cases of PDAC. Angiogenesis in the primary tumour was evaluated by microvessel density (MVD), vascular proliferation index (VPI) and the presence of glomeruloid microvascular proliferations (GMP). These features were analysed in the context of clinicopathological variables, KRAS mutation status, relapse location and survival. MVD (median 134 microvessels/mm(2) , range 88-177) and VPI (median 3.2%, range 1.6-4.9) were associated with larger tumour size and lymph node metastasis. MVD was also related to the occurrence of liver metastases. Both variables were associated with survival in univariate and multivariate analyses. GMPs were present in 32 (62%) of the cases. Patients who exhibited MVD and VPI values above median, and GMP positivity, had a median survival of only 4.2 months after surgery. In conclusion, the angiogenesis markers MVD and VPI have a significant impact on survival. By also including GMP, a subgroup of PDAC patients with particularly short survival could be identified.

A new noninvasive blood-test for the early detection of colorectal cancer.

348 Background: Colorectal cancer (CRC) remains a major medical problem in the world. Its early detection impacts the prognosis and the cost of treatment. Fecal occult blood test is a current noninvasive screening method for CRC detection in asymptomatic average risk individuals. It detects less than 50% of cancers and yields a high number of “normal” colonoscopies. The dysregulation of DNA methylation has been recognized as playing a crucial role during CRC development. The aim of the study was to develop a CRC-specific methylated DNA test in serum. Methods: First, the GoldenGate Methylation microarray containing 1,505 CpG loci within 807 cancer-related genes was used to study methylation patterns in CRC patients. We performed such assays on 9 tissues (4 CRC, 4 normal autologous tissues, 1 polyp), 17 blood and urine pooled samples (7 CRC, 6 polyps, 4 normal) and 20 stool samples (7 CRC, 3 polyps, 10 normal). The clinical status of each sample was assessed by colonoscopy and/or pathology findings. Among methylated genes detected in tumour tissue, stools, and blood, NPY, PENK and WIF1 genes were selected taking into account their high degree of methylation. We then carried out a clinical validation by quantitative multiplex methylation-specific PCR (QM-MSP) in two phases: firstly on 30 tissues (15 CRC, 15 homologous normal tissues) and secondly on 193 serum samples (32 CRC, 161 normal). Results: For to obtain the best accuracy of QM-MSP test, we defined for both biological sources a cumulative methylation index (CMI) by adding of the methylation percentage of each gene. At the CMI of 20.0% on tissues, our QM-MSP test allows to diagnose the CRC with the highest accuracy (100% of specificity and 100% of sensitivity). From sera, the CMI has been standardized at 2.0% giving a sensitivity and specificity for detecting CRC of 94.4% and 59.4% respectively (NPV of 92.1% and PPV of 67.8%). Conclusions: We developed a QM-MSP-based analysis of NPY, PENK, and WIF1 methylated genes in serum. This test classified correctly and with a high accuracy the healthy individuals and colon cancer patients. We propose here new sensitive serum-based epigenetic biomarkers as an effective and simple new noninvasive test for CRC screening in the average and high risk populations.

Abstract IA14: Investigating the ICOS/ICOSL pathway as a target for combination therapy with anti-CTLA-4.

Abstract Biomarker studies used with immunotherapeutic strategies in the clinic have typically involved monitoring immunologic changes within the systemic circulation; however, recent data indicate that immunological changes within tumor tissues will be more likely to predict clinical responses. To obtain such data we conducted the first pre-surgical clinical trial with anti-CTLA-4 (ipilimumab) in a cohort of patients with localized bladder cancer. We are also conducting the first combination therapy pre-surgical trial with ipilimumab plus leuprolide acetate in patients with localized prostate cancer. Immunological data from these trials are obtained from both tumor tissues and blood samples. We found an increased frequency of CD4 and CD8 T cells expressing high levels of inducible costimulator (ICOS) and decreased frequency of FOXP3-expressing CD4 T cells within tumor tissues of treated patients. The CD4+ICOShi population contained effector T cells that produced IFN-γ and recognized the cancer-testis antigen NY-ESO-1 expressed on tumor cells. We therefore identified ICOS as marker of a subset of effector T cells that is increased in frequency after anti-CTLA-4 therapy. ICOS+ T cells are being explored as both a pharmacodynamic marker for treatment with anti-CTLA-4 as well as a novel target to improve the efficacy of anti-CTLA-4 therapy. These observations led us to test the possibility that engagement of ICOS could enhance the efficacy of anti-CTLA-4 therapy. To this end we transduced mouse B16F10 melanoma cells with a cDNA encoding ICOSL or a control construct. B16-ICOSL+ cells (IVAX) and control B16 cells were irradiated and used alone or in combination with anti-CTLA-4 to treat mice bearing established B16F10 tumors. We found that combination of the IVAX with anti-CTLA-4 was markedly more effective than the control vaccine plus anti-CTLA-4 or that of any single treatment alone. The increase in therapeutic efficacy was accompanied by a marked in increase in the density and functionality of CD4 and CD8 T cells within the tumor. These results suggest a novel strategy for manipulating the immune system to enhance anti-tumor responses: checkpoint blockade coupled with provision of agonist signals to enhance costimulation mediated by ICOS. Citation Format: Padmanee Sharma. Investigating the ICOS/ICOSL pathway as a target for combination therapy with anti-CTLA-4. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA14.

Abstract OT2-3-11: Tivozanib in combination with paclitaxel vs placebo with paclitaxel in patients with locally advanced or metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer with inferior survival outcomes. Although weekly paclitaxel (WP) is effective in the treatment (tx) of metastatic breast cancer (MBC), optimization of therapies for patients (pts) with TNBC is essential. Angiogenesis is a hallmark of advanced cancer, with subset analyses suggesting activity of angiogenesis inhibitors in TNBC. Tivozanib (TIVO) is a potent and selective inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3 with a promising role in metastatic renal cell carcinoma, and established safety in Phase I combination with WP in MBC. Purpose: This Phase II trial will assess the efficacy and safety of TIVO + WP in the first-line setting for pts with advanced or metastatic TNBC and evaluate the performance of candidate angiogenesis biomarkers. Objectives: The primary objective of this study is to compare progression-free survival (PFS) of pts treated with TIVO + WP vs pts treated with placebo (PB) + WP. Secondary objectives include objective response rate (ORR), overall survival (OS), safety and tolerability, quality of life, and correlative candidate biomarker endpoints. The pharmacokinetics of TIVO + WP also will be characterized. Study Design and Methods: This multicenter, randomized, PB-controlled, two-arm study will enroll pts with metastatic or unresectable TNBC (evaluable per RECIST) and no prior systemic therapy. Pts must have confirmed available archival tumor tissue. Pts will be stratified by ECOG performance score and number of metastatic sites, then randomized to receive either oral TIVO 1.5 mg once daily for 3 weeks (wks) on/1 wk off and intravenous WP 90 mg/m2 for 3 wks on/1 wk off, or PB + WP. One cycle will be 4 wks; tx will continue until disease progression or unacceptable toxicity. Archival tumor tissue and blood samples will be evaluated for response biomarkers, including a hypoxia sensitivity gene signature, a myeloid resistance gene signature, and angiogenic ligands. All pts will be followed for survival until death. Adverse events will be monitored throughout the study. Pharmacokinetic samples will be collected during cycles 1 and 2. PAM-50–defined intrinsic molecular subtype populations also will be evaluated retrospectively. Recruitment of 130 patients is planned, with an interim analysis after 80 pts to measure ORR (130 pts with a total of 82 investigator-assessed PFS events provides 80% power to detect statistically significant PFS differences between tx arms). Endpoint analyses will use the intent-to-treat population. The primary efficacy analysis will use investigator assessments of response and a two-sided 95% confidence interval for the hazard ratio produced using Cox proportional hazards regression models. OS will be compared using the log-rank test. Analyses of candidate biomarkers and determination of an optimal predictive cutoff for response also are planned. Trial enrollment will commence in fall 2012. Conclusion: This study will determine whether TIVO, a selective and potent VEGFR inhibitor, combined with WP improves clinical outcomes in pts with TNBC, and whether clinical activity is associated with candidate angiogenesis biomarkers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT2-3-11.

Abstract P4-04-02: Tumor-initiated peripheral myeloid cell expansion is reversed by radiation therapy

Abstract Myeloid lineage cells, particularly monocytes and macrophages, have an important role in the development and progression of cancer. Within established tumors, macrophages are critical for angiogenesis, invasion, metastasis, immunosuppression and response to therapy. In addition, both transplantable and transgenic mouse models of mammary cancer are associated with myeloid expansions detectible in the tumor, spleen and peripheral blood, and these myeloid cells are able to suppress T cell activation in vitro. Reducing the tumor burden through chemotherapy and resection has been shown to control the myeloid expansion. However, chemotherapies that reduce tumor burden also have direct effects on myeloid cells, and surgical trauma has been shown to mobilize myeloid cells. Radiation therapy is the third major option to treat cancer, but the consequence of radiation therapy to systemic myeloid populations has not been described. We applied tumor-specific radiation therapy to test the hypothesis that the frequency of myeloid lineage cells correlates with the primary tumor burden. We demonstrate in the MMTV-PyMT model of mammary cancer that myeloid expansion in the blood closely correlates with myeloid involvement in the tumor and with tumor progression. We demonstrate that radiation therapy of 4T1 mammary tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Despite these changes in the frequency of myeloid lineage cells, T cell numbers in the blood and spleen remains constant. However, radiation therapy improves the myeloid cell:CD8+ T cell ratio and results in increased proliferation of T cells in the spleen. Finally, we demonstrate that T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, while T cell responses to tumor-associated antigens are increased after radiation therapy of the tumor. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-02.

Abstract SS-01: Metformin effects on breast preneoplasia

Abstract Background: Metformin has been associated with a significant reduction in cancer incidence and mortality in diabetic patients relative to other antidiabetic drugs, including positive results specifically in breast cancer. In a recent window-of-opportunity trial in 200 non-diabetic women with breast cancer, we showed a heterogeneous effect of metformin on breast cancer proliferation (Ki-67 labeling index) depending on insulin resistance, with a trend to a decreased proliferation in women with insulin resistance (HOMA>2.8) and an opposite trend in women with normal insulin sensitivity (Bonanni et al. JCO 30:2593, 2012). Here we performed an exploratory study to determine whether metformin has antiproliferative effects on adjacent lobular or ductal intraepithelial neoplasia (LIN or DIN) and on distant ductal hyperplasia and whether these effects are different according to specific host and tumor characteristics. Previous studies showed that Ki-67 LI in atypical lesions predicts subsequent breast cancer risk. Methods: Baseline core biopsies of tumor tissue and blood samples were obtained at study entry and before surgery for pre/post-treatment comparisons. Patients were randomly assigned to metformin, 850 mg or placebo once daily on day 1-3 followed by two 850 mg tablets from day 4 to 28. At the time of surgical removal of the tumor, three to five specimens of adjacent (≥1 cm from the tumor) and distant (>1 cm from the tumor) grossly normal tissue (i.e., the surgical margins of quadrantectomy or lumpectomy, or the grossly free quadrants from mastectomy specimens) were obtained to assess systematically the prevalence of LIN or DIN and ductal hyperplasia. Results: Overall, the prevalence of LIN, DIN and ductal hyperplasia was 4.5% (9/200), 66.5% (133/200) and 35% (69/200), respectively. The Ki-67 LI was positively associated with DIN grade (p-trend<0.001). The median (and IQ range) Ki-67 LI distribution of LIN+DIN was 12% (8-20) and 10% (6-22) on metformin and placebo, respectively (p=0.6), nor was there a significant difference of Ki-67 LI in any subgroup between treatment arms. However, compared with placebo, metformin exhibited different effects on Ki-67 according to DIN grade (delta=+4.4% in DIN1 vs -27.9% in DIN3, p-interaction=0.09), cancer HER2 status (median 12% vs. 10% in HER2-ve and 28% vs. 35% in HER2+ve, p-interaction=0.03), abdominal circumference (p=0.08), and BMI (p=0.17). Conclusions: Our findings illustrate the notion that the window of opportunity pre-surgical model provides insight into a drug's preventive potential by targeting tumor adjacent dysplastic (or intraepithelial neoplastic) cells and distant ductal hyperplastic cells. The model unravels the existence of the field cancerization effect in apparently normal breast tissue. Similar to our results on breast cancer tissue, metformin had no overall significant effect on breast preneoplasia proliferation but exhibited heterogeneous effects depending upon specific host and preneoplasia characteristics. Further studies are necessary to better understand the clinical implications of these findings. (Supported by the Italian Association for Cancer Research and Italian Ministry of Health 2009-RF-1532226). Citation Format: Andrea DeCensi, Valentina Aristarco, Matteo Lazzeroni, Clara Varricchio, Bernardo Bonanni, Matteo Puntoni, Giancarlo Pruneri, Massimiliano Cazzaniga, Andrea Vingiani, Davide Serrano, Aliana Guerrieri-Gonzaga, Oreste Gentilini, Harriet Johansson. Metformin effects on breast preneoplasia. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr SS-01.

Moving next-generation sequencing into the clinical realm: Detection of somatic mutations in cancer by targeted amplicon sequencing.

60 Background: Molecular targeted therapies are increasingly important in treating cancer patients; robust analytically validated clinical assays are required for patient selection in early-stage clinical trials. The goal of Molecular Characterization Laboratory (MoCha) is to develop clinical diagnostic assays using next generation sequencing methods to support clinical studies in DCTD (CTEP). Methods: We developed a custom assay for somatic mutation detection using Fluidigm access array technology for amplicon generation followed by sequencing with the Illumina Miseq. A panel of 62 amplicons covering 6 Kb genomic regions was designed to detect 92 DNA loci, including common therapeutically actionable targets, in 37 genes. Analytical studies were performed using genomic DNA samples from fresh or formalin fixed cancer cell-lines and a normal hapmap individual (CEPH). We subsequently applied this assay to characterize DNA samples from both tumor tissues and blood specimens from ovarian cancer patients. Results: The assay detected known variants in both frozen and fixed DNA samples reproducibly with high sensitivity and specificity (<2%). Using a series of positive control plasmid spikes mixed into a normal reference CEPH DNA at pre-defined copy number ratios, we verified the assay is sensitive to detect variants at 5% allelic frequency with a minimum 400 X coverage. We identified somatic mutations in TP53 and PIK3CA in a few patients, and a germ-line variant D1583N in ATM genes occurring in one-third of tested patients. Conclusions: We developed and validated a next generation sequencing assay suitable for patient selection for clinical trials. Plans are to correlate sequencing and clinical results when clinical data are available.

Preparation and antitumor activity of bFGF-mediated active targeting doxorubicin microbubbles

Characterization and antitumor activity of basic fibroblast growth factor-mediated active targeting doxorubicin microbubbles (bFGF-DOX-MB) were investigated. Pluronic F68 with chemical conjugation of doxorubicin (DOX-P) and peptide KRTGQYKLC-conjugated DSPE–PEG2000 were prepared. bFGF-DOX-MB had a normal distribution of particle size, with average particle size of 2.7 μm. Using A549 mouse model, bFGF-DOX-MB combined ultrasound showed the best inhibition effect on tumor volume growth among all the test groups. Similar conclusion was obtained from experimental measurements of tumor weight change and blood cell count. From the results, chemotherapeutic drug inhibition on tumor growth could be enhanced by local ultrasound combined with active targeting bFGF-DOX-MB, which might provide a potential application for ultrasound-mediated chemotherapy.

Pre-operative embolisation of choroid plexus tumours in children. Part II. Observations on the effects on CSF production

Choroid plexus tumours are one of the few causes of hydrocephalus secondary to increased CSF production. Operative treatment aided by pre-op embolisation is being used in our institution as a primary option of treatment. Our aim was firstly to quantify the effects of embolisation on CSF production and secondly to assess whether the use of pre-operative embolisation would lead to reduction of CSF production thus reducing the need for CSF diversion procedures in the perioperative and long term. From 1996 till 2009, 30 patients (mean age, 2.25years) underwent surgical treatment for 24 choroid plexus papillomas and 6 choroid plexus carcinomas. Thirteen underwent pre-operative super-selective embolisation of the feeding vessels with Histoacryl glue. The need for CSF diversion-external ventricular drain (EVD)/shunt-was recorded together with the daily CSF production between the two groups (embolised: EMB+ vs. not embolised: EMB-) RESULTS: The embolisation was successful in 13 of 15 (86.6%) patients. The average post-op daily CSF production between the EMB+ and EMB- groups was (67 vs. 135ml/day; p = 0.005). EVD days in situ post-operatively was 7.9 vs. 12.1 (p = 0.033). However, the need for permanent CSF diversion was similar in both groups (five vs. six). We have established the safety of pre-operative embolisation as an adjunct to operative treatment of choroid plexus tumours. As we expected, this technique, by removing the tumour's blood supply, reduces the rate of CSF production. This has had a positive impact on the post-operative management of these patients. We cannot say the same for the need of permanent CSF diversion in our study.

Deficiency of C–C chemokine receptor 5 suppresses tumor development via inactivation of NF–ĸB and inhibition of monocyte chemoattractant protein-1 in urethane-induced lung tumor model

To evaluate the significance of C-C chemokine receptor type 5 (CCR5) in lung tumor development, we compared carcinogen-induced tumor growth in CCR5 knockout (CCR5(-/-)) mice and wild-type (CCR5(+/+)) mice. CCR5(-/-) mice showed reduced urethane (1g/kg)-induced tumor incidence when compared with those of CCR5(+/+) mice. We investigated the activation of nuclear factor-kappaB/STAT3 since these are implicated transcription factors in the regulation of genes involving tumor growth. Significant inhibition of DNA-binding activity of nuclear factor-kappaB and STAT3, and the translocation of p50 and p65 into the nucleus and the phosphorylation of IĸB were found in the lungs of CCR5(-/-) mice compared with the lungs of CCR5(+/+) mice. Expression of apoptotic protein such as cleaved caspase-3, cleaved PARP and Bax was elevated, whereas the expression levels of survival protein such as Bcl-2 and cIAP1 was decreased in the lungs of CCR5(-/-) mice. Interestingly, we found that the level of monocyte chemoattractant protein-1 (MCP-1), a tumor growth-promoting cytokine, was significantly reduced in the lung tumor tissue and blood of CCR5(-/-) mice compared with the level in CCR5(+/+) mice. In addition, CCR5 small interfering RNA (siRNA) and inhibitor of MCP-1 blocked lung cancer cell growth, which was abolished by the addition of MCP-1 protein in cultured lung cancer cells. Moreover, inactivation of CD8(+) cytotoxic T cell and dendritic cells was significantly increased in the blood, lung tumors and spleens of CCR5(-/-) mice compared with that of CCR5(+/+) mice. Therefore, these results showed that CCR5 deficiency suppressed lung tumor development through the inhibition of nuclear factor-kappaB/STAT3 pathways and the downregulation of MCP-1 in the carcinogen-induced lung tumor model.

P4.01 Delivery of High Quality Molecular Panel Tests for 9,000 Cancer Patients Routinely Consented for Use of Molecular and Clinical Data

ABSTRACT Background Molecular analysis of tumours may be used to identify patients predicted to benefit from targeted therapies. The Cancer Research UK Stratified Medicine programme is piloting plans to apply testing broadly across the UK healthcare system, linking molecular phenotype to clinical outcomes enabling further research and cohort identification of patients. Methods The Stratified Medicine Programme has been developed in partnership with Astra Zeneca, Pfizer, the UK Department of Health and academic researchers. Phase One is a two year feasibility study to demonstrate the collection and molecular analysis of consented sections of surplus diagnostic tumour tissue and blood samples from 9,000 patients across a network of 20 hospitals.Samples are tested for mutations in genes of potential clinical interest (KRAS, BRAF, NRAS, PIK3CA, TP53, PTEN, TMPRSS-ERG, EGFR, EML4-ALK and KIT). Different technologies are used across the three testing laboratories but all tests are technically validated with external quality assurance monitoring and targets for completion in clinically relevant timescales. Data (diagnostic including molecular information, treatment and clinical outcome) is collected through existing hospital systems, and transferred to a central data repository hosted within the Eastern Cancer Registry Information Centre. Results The study opened in September 2011 with 2160 patients consented to participate by April 2012, 607 sets of molecular results returned and planned accrual rates for Year One met (300 samples/month). Test requests and reports are exchanged electronically with standardisation of reporting being developed. Partnerships with two clinical trials for pre-screening of cohorts have been agreed.Programme accrual by tumour type. Conclusions We will report the emerging results, observations of processes and potential issues identified in delivering a scalable model for routine, high quality, prospective molecular characterisation of tumours for cancer patients, with consent for the collection, storage and research use of population-scale genetic and outcomes data. Table 1 . Programme accrual by tumour type Year one targets Patients consented Breast 641 455 Colorectal 644 263 Lung 641 610 Melanoma 213 150 Ovary 200 132 Prostate 660 529

Spontaneous tumor rupture and surgical prognosis of patients with hepatocellular carcinoma

Background.Spontaneous rupture is an uncommon but the most fatal complication of hepatocellular carcinoma (HCC) and is recognized as a risk factor for tumor recurrence. The present study is to investigate the short- and long-term survival of the patients with HCC rupture and evaluate the influence of tumor rupture on patient's survival after hepatic resection. Patients and methods.The clinical data of 101 patients with HCC rupture from 2000 to 2010 were reviewed retrospectively. The management of tumor rupture and clinicopathological parameters affecting 30-day mortality of the patients were recorded and evaluated. Long-term survival of the 41 patients undergoing hepatic resection was compared with 446 patients with non-ruptured HCC at the same time period. Results. The 30-day mortality rate of the 101 patients with HCC rupture was 35.6% and median survival was 79 days. The independent risk factors affecting 30-day mortality were tumor size and blood transfusion quantity. Compared with 446 non-ruptured HCC patients, 41 patients with ruptured HCC undergoing hepatic resection had a similar overall and disease-free survival to 446 without rupture (Log-rank test, p = 0.704 and 0.084, respectively). Multivariate analysis revealed that age, gender, and tumor size were independently significant factors in differentiating tumor rupture from non-rupture. Conclusion.Early mortality of spontaneous rupture of HCC was dependent on preoperational liver function, tumor status, and severity of bleeding. Prolonged survival can be achieved in patients with ruptured HCC after hepatic resection as those without the complication.

SDHA and SDHB mutations in KIT/PDGFRA WT gastrointestinal stromal tumors.

10087 Background: KIT/PDGFRA wild-type (WT) GISTs harbour mutations on SDHB and SDHC and, more recently, we described mutations on SDHA using massively parallel sequencing approach. We sequenced SDHA and SDHB genes in a larger series in order to validate the data. Methods: SDHA gene (1-15 exons) and SDHB gene (1-8 exons) (even not all exons in all samples) were sequenced on tumor (T) and/or peripheral blood (PB) of WT GIST patients by Sanger Sequencing method. DNA was extracted from tumor specimens by the QIAmp DNA Mini kit (Qiagen, Milan, Italy) and amplified with specific primer pairs designed to amplify exons but not SDHA pseudo-genes located on chromosomes 3 and 5. Then, PCR products were purified with the Qiaquick PCR purification kit (Qiagen, Milan, Italy) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). Sanger sequencing was performed on ABI 3730 Genetic Analyzer (Applied Biosystems). Results: SDHA gene exons were sequenced on a total of 27 WT GIST patients, in particular on T, PB and both from 12, 6 and 9 patients respectively. SDHB gene exons were sequenced on a total of 18 out of 27 patients, in particular on T, PB and both from 7, 8 and 3 patients respectively. 8 SDHA mutations were found in 5 samples (18.5%). Besides those previously identified, 5 new SDHA mutations were found in other 3 samples: one sample harboured R171C and R589Q heterozygous missense mutation in exons 5 and 13 respectively. The other one harboured G419R and E564K heterozygous missense mutations in exons 9 and 13 respectively. The third sample harboured a delCAG immediately upstream of exon 5, in heterozygosis on PB and in homozygosis on T. A SDHB heterozygous mutation (301delT) in exon 4 was found on 1 PB sample. Conclusions: the presence of SDHA mutations has been confirmed in a subgroup of WT GIST patients. All subunits of SDH complex should be sequenced on WT GIST patients in order to explore the frequency and any linkage between each other and the pathogenetic and clinical significance.

Blood and tumor inflammation markers in non-small cell lung cancer (NSCLC): Blood leukocytosis as an independent risk factor in early stage.

e21110 Background: Cancer inflammation is associated with impaired survival in a range of cancers. We reviewed blood and intratumoral inflammatory markers in NSCLC. Methods: At the Departmentof Thoracic Surgery, Skejby Hospital, Aarhus, Denmark, consecutive patients with resected NSCLC from 2000 to 2008 were reviewed, and 906 patients with complete clinical data were identified. A subset of 341 consecutive patients, resected between 2003 and 2006, also had intratumoral CD66b+ neutrophils and CD163+ macrophages measured by immunohistochemistry and evaluated by stereological assessment. Results: A total of 526, 197, and 183 patients had stage I, II, and III, respectively. Multivariate analysis stratified for tumor stage revealed elevated blood leukocytes above upper limit of normal as a significant prognostic factor for recurrence-free survival (RFS)(hazard ratio [HR] 1.9; 95% CI 1.4-2.6; p<0.0001), cancer specific survival (CSS)(HR 1.9; 95% CI 1.4-2.7; p<0.0001), and overall survival (OS)(HR 1.5; 95% CI 1.1-1.9; p<0.006) in stage I NSCLC, but not in stage II and III. No prognostic impact of intratumoral neutrophils or macrophages was seen on CSS, RFS, or OS, neither in the entire cohort, nor limited to stage I patients with elevated blood leukocytes or with normal counts. Controlling intratumoral neutrophils and macrophages for localization restricted to tumor tissue, stromal tissue, or blood vessels, respectively, were also with no statistically significant difference. Conclusions: Blood leukocytosis is an independent prognostic factor for short recurrence free survival, cancer specific survival, and overall survival in stage I NSCLC, but not in stage II and III. However, intratumoral neutrophils or macrophages did not impact prognosis. Further studies are needed to elucidate the role of cancer inflammation in NSCLC.

MAVERICC: A randomized phase II study of mFOLFOX6-bevacizumab (BV) versus FOLFIRI-BV with prospective biomarker stratification in previously untreated metastatic colorectal cancer (mCRC).

TPS3635^ Background: The identification of prognostic and predictive biomarkers could significantly improve the risk-benefit ratio and cost-effectiveness of 1st-line mCRC regimens. This is the first prospective study of tumoral ERCC1 (chemo-resistance marker to platinum compounds) and plasma VEGF-A as potential biomarkers for oxaliplatin- and BV-containing regimens, respectively, in an effort to further define the optimal chemotherapy backbone with biologic therapies, including BV, for mCRC. Methods: In this randomized, open-label, global, phase II study, patients (N=360) with histologically or cytologically confirmed CRC and ≥1 measurable metastatic lesion are stratified at screening by tumoral ERCC1 mRNA expression (high vs low, cutoff of 1.7 [ERCC1/β-actin mRNA]). Eligibility criteria include completion of adjuvant therapy >12 months before screening and an ECOG performance status ≤1. Blood samples are collected to quantify plasma VEGF-A levels. Patients within each ERCC1 stratification group are randomized 1:1 to mFOLFOX6-BV or FOLFIRI-BV administered in 2-week cycles. BV will be given at a dose of 5 mg/kg IV q2w. Patients will remain on study treatment until disease progression (PD) or unacceptable toxicity. If oxaliplatin or irinotecan need to be discontinued, BV and 5-fluorouracil or capecitabine are to be continued until PD. The primary objectives are: 1) to assess ERCC1 and VEGF-A as biomarkers of progression-free survival (PFS) for oxaliplatin- and BV-containing regimens in 1st-line mCRC, and 2) within ERCC1 high patients, to test whether FOLFIRI-BV is associated with a prolonged 1st-line PFS compared to mFOLFOX6-BV. Secondary objectives include assessing the impact of these markers on overall survival, objective response, hepatic metastases resection, and safety. Exploratory endpoints include correlative analyses with additional tumor tissue, blood, and SNP markers. The first patient was enrolled in August 2011. An interim biomarker distribution assessment of the first 100 patients is planned, and the evaluation of the primary endpoints is estimated for early 2015. Clinicaltrials.gov: NCT01374425.

Sequential first-line treatment for metastatic colorectal cancer with capecitabine, irinotecan, and bevacizumab: Capecitabine plus bevacizumab (Cape-Bev) versus capecitabine, irinotecan plus bevacizumab (CAPIRI-Bev): The AIO KRK 0110/ML22011 randomized, phase III trial.

TPS3639 Background: Several randomized trials have indicated that combination chemotherapy applied in metastatic colorectal cancer (mCRC) does not significantly improve overall survival when compared to the sequential use of cytotoxic agents (CAIRO, MRC Focus, FFCD 2000-05). The present study investigates the question in bevacizumab-based first-line treatment including escalation- and de-escalation strategies. Methods: The AIO KRK 0110/ML22011 trial (ClinicalTrials.gov NCT01249638) is a two-arm, multicenter, open-label randomized phase III trial comparing the efficacy and safety of Cape-Bev versus CAPIRI-Bev in the first-line treatment of metastatic colorectal cancer. Patients with unresectable metastatic colorectal cancer, ECOG PS 0-1, will be assigned in a 1:1 ratio to receive either capecitabine 1250 mg/m2 bid for 14d (d1-14) plus bevacizumab 7.5 mg/kg (d1) q3w (Arm A) or capecitabine 800 mg/m2 BID for 14d (d1-14), irinotecan 200 mg/m2 (d1) and bevacizumab 7.5 mg/kg (d1) q3w (Arm B). Patients included into this trial are required to consent to the analysis of tumor tissue and blood for translational investigations. In Arm A, treatment escalation from Cape-Bev to CAPIRI-Bev is recommended in case of progressive disease (PD). In Arm B, de-escalation from CAPIRI-Bev to Cape-Bev is possible after 6 months of treatment or in case of irinotecan-associated toxicity. Re-escalation to CAPIRI-Bev after PD is possible. The primary endpoint is time to failure of strategy (TFS). Secondary endpoints are ORR, OS, PFS, safety and quality of life. Conclusion: The AIO KRK 0110 trial is designed for patients with disseminated, but asymptomatic mCRC who are not potential candidates for surgical resection of metastasis. Two bevacizumab-based strategies are compared: one starting as single-agent chemotherapy (Cape-Bev) allowing escalation to CAPIRI-Bev and another starting with CAPIRI-Bev and allowing de-escalation to Cape-Bev and subsequent re-escalation if necessary. By January 2012, 79 of planned 516 patients have been enrolled.

Abstract 2333: Serotonin: A known neurotransmitter functions as an angiokine to support cancer progression

Abstract Platelet aggregation leads to significant serotonin release from its major storage and results in increasing of serotonin levels at injury site and thrombotic tumor environment. Many studies have shown that platelet activation plays a crucial role in tumor progression; however, the role of serotonin in angiogenesis and tumor progression has not been well studied. In this study, we have tested the hypothesis that 5-HT promotes angiogenesis, and antagonizing 5-HT activity has anti-angiogenic benefit in controlling tumor growth. Real time PCR and western blot studies showed that endothelial cell expressed 5-HT receptor 1B (HTR1B) in higher level than other 5-HT receptors. Endothelial cell proliferation and tube formation were significantly affected by blocking HTR1B and the cAMP and IP1 assays have revealed the HTR1B as the inhibitory type of GPCR. Stimulation of endothelial cell with 5-HT or HTR1B agonist has led to activation of two individual signaling pathways: ERK and Akt / mTOR. In further studies, p70S6K was recognized as the merging point of these signaling. These kinases have been also activated by known angiogenic factors (VEGF and FGF) but the mechanism of activation was different from serotonin and it was through their tyrosine kinase receptors. In contrast, pretreatment of endothelial cell with a selective HTR1B antagonist have led to blockade of the 5-HT induced kinases’ activation. We also demonstrated angiogenic promoting activity of 5-HT with a mouse Matrigel plug (in vivo angiogenesis model system) and antiangiogenic potential of antagonists against HTR1B with an ex vivo tumor angiogenesis model (xenograft CAM assay) and xenograft mice model of human ovary cancer (SKOV-3). Selective HTR1B antagonist displayed the synergistic effect (combination index analysis) in blocking tumor-induced new blood vessel formation when it was applied in combination with Sutent (VEGFR2 antagonist). HTR1B antagonist also showed preclinical efficacy in the xenograft model of human ovary cancer by reducing over 50% in tumor size and blood vessel density marker.The results of this study enhanced our understanding of the serotonin-signaling pathway in human endothelial cells during angiogenesis. This research also revealed the potential of 5-HT signaling as the new target for antiangiogenic development. 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 2333. doi:1538-7445.AM2012-2333

Validating the woodchuck model for studying antiangiogenic therapies for human hepatocellular carcinoma (HCC).

239 Background: Anti-angiogenic agents have become the backbone of advanced HCC treatment and provide clinical benefit despite infrequent radiographic response. Animal models that mimic human HCC and treatment response are greatly needed. The laboratory woodchuck (Maramota monax) is a unique animal model that replicates the complex human liver milieu of HCC in the context of chronic hepatitis B viral infection and was selected for this translational study. Methods: Humans: Patients with inoperable HCC were consented and prospectively treated with sunitinib 37.5mg PO qd for one week prior to liver directed therapy(chemoembolization). Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was done pre-sunitinib and after one week as part of the trial. Woodchucks: Laboratory bred eastern woodchucks were inoculated at birth with known titers of sera from a chronic infectious pool of chronic woodchuck hepatitis virus (WHV), confirmed by ultrasound exam at 12-18 months to have developed HCC. Methods for assessment of woodchuck HCC blood flow using DCE-MRI were optimized. Woodchucks received sunitinib 12mg/kg/day poQD and identical image acquisition and analysis techniques were used for both species and identical parameter sets were obtained for cross comparison done by Dr. Ashton in a blinded fashion. Results: A total of seven patients and eight woodchucks with HCC were included (2 scans per patient/woodchuck, pre and post sunitinib). The “Exact Wilcoxon rank-sum test” was used for statistical analysis. Comparison between median Ktrans, AUCBN(90), necrotic and total volumes of the tumors in both groups revealed all parameters to be similar and only total tumor volume reached statistical significance (P=0.002). Conclusions: Our data indicate similarities between human and woodchucks in the tumor histology, blood flow and baseline and post antiangiogenic therapy functional imaging assessment by DCE-MRI. The current sample size of n=7+8 can detect large differences of roughly 1.5 standard deviation units between humans and woodchucks for our measures at alpha=0.05 and power=0.80. Further studies that explore the translational potential of this unique animal model are warranted.