Citations

Abstract Background: Candidate gene and candidate pathway-based pharmacogenomic studies have been used to investigate the inherited variability of prognosis of lung cancer treated with platinum-based chemoptherapy. In order to make it possible to conduct an ‘unbiased’ query across the entire genome, a genome-wide association study (GWAS) in 283 lymphoblastoid cell lines (LCLs) was carried out to help identify candidate SNPs associated with variation in cisplatin cytotoxicity. Furthermore, we set out to test the hypothesis that genetic variation resulting in differences in cisplatin cytotoxicity might influence the overall survival of patients with lung cancer treated with platinum-based antineoplastic agents. Material and Methods: We applied ethnically diverse Coriell “Human Variation Panel” LCLs for GWAS. Then, we picked 168 top significant SNPs selected from GWAS in 283 LCLs and genotyped these SNPs in 1183 lung cancer patients (222 SCLC and 961 NSCLC) who received platinum-based chemotherapy. Genotyping was performed using the Illumina Golden Gate platform. The association of SNPs with lung cancer overall survival was analyzed by Cox regression model after adjusting for disease stage. Functional validation of candidate genes was performed by siRNA screening in NSCLC cell lines CRL5872, H1299 and SCLC cell line CRL5823. Results: A total of 157 SNPs were successfully genotyped for 1183 lung cancer patient samples. The Cox regression analysis indicated that 9 and 10 different SNPs were associated with overall survival for NSCLC and SCLC patients, respectively (p value <0.05). Fifteen genes including 7 genes located in the region of 200kb up or downstream of 4 SNPs (rs11169748, rs2440915, rs5952066 and rs7620841) and 8 genes for which the expression was correlated with three SNPs (rs11169748, rs2440915 and rs5952066) were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which the expression levels were correlated with the SNPs rs11169748 and rs2440915, significantly decreased cisplatin sensitivity in lung cancer cell lines CRL5872, H1299 and CRL5823. Conclusions: This series of clinical and complementary laboratory-based functional studies identified several candidate genes and SNPs that might help predict treatment outcomes of platinum-based therapy in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1285. doi:10.1158/1538-7445.AM2011-1285

Abstract Background: Stage IVC (metastatic) anaplastic thyroid cancer (ATC) has a median overall survival (OS) of only 1.9 months. Single agent paclitaxel has modest activity in ATC. In pre-clinical studies, CS-7017 inhibited ATC proliferation through a novel mechanism–activation of PPAR-γ, followed by sequential upregulation of RhoB, then p21. Moreover, CS-7017 augmented the proapoptotic effects of paclitaxel. Based on these results, a multicenter study was conducted to determine if CS-7017+paclitaxel might prove safe and effective in advanced ATC. Design: A Phase 1 study was conducted to determine the Phase 2 dose. In Phase 1, patients received CS-7017 orally BID for one week (run-in phase), followed by a 3 h iv infusion of paclitaxel (175 mg/m2) every 3 weeks in combination with BID CS-7017. CS-7017 doses were escalated (0.15, 0.3, or 0.5 mg). Tissue biopsies were obtained at baseline and at one and three weeks for immunohistochemistry of PPAR-γ responsive proteins; serum CS-7017 PK studies were also performed. Results: Nineteen patients were enrolled, but 4 were not dosed (3 due to progression, 1 ineligible). Seven participated in CS-7017 dose levels 1a/1b (0.15 mg BID), six in dose level 2 (0.3 mg BID), and two in dose level 3 (run-in phase only–0.5 mg BID). Demographics: Of the 15 treated patients, 10 (67%) were women. Median age was 59 years (range: 43-82) Efficacy: Of 15 patients receiving drug, one had a confirmed partial response (PR) lasting from Day 69 to Day 175, and eight had stable disease (SD) as their best response. Median Time to Progression in 7 patients at Dose Level 1 was 49 days, but 70 days in Dose Level 2 (43% prolongation); corresponding median survival was 99 (0.15 mg BID) vs. 140 days (0.3 mg BID, 42% increase). Median peak CS-7017 blood level was 8.6 ng/mL (range: 5.1 to 13.7) for Level 1 and 22.0 ng/mL (17.0 to 31.5) for Dose Level 2. Adverse Events: Ten patients had AEs ≥ Grade 3, with two (anemia and localized edema) related to CS-7017. Thirteen events of fluid retention/edema were reported in 8 patients, with only 2 events of CTCAE grade ≥ 3. Eight patients had ≥ one SAE, with one (anemia) due to CS-7017. One SAE (anaphylactic reaction) was related to paclitaxel. No dose limiting toxicity was observed. Immunohistochemistry: Biopsies were available on 7 patients. PPAR-γ, RXR-α, RhoB were present in all; Angiopoietin-like 4 was induced by CS-7017. Conclusions: Combination therapy with CS-7017 and paclitaxel was tolerated and has biologic activity in patients with metastatic anaplastic thyroid carcinoma, with initial exploratory data suggesting a dose-dependent improvement in time to progression and survival in response to escalating CS-7017 dosage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1278. doi:10.1158/1538-7445.AM2011-1278

Abstract Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines (Huh7, HepG2, SNU182, PLC/PRF/5, Hep3B, SNU423 and SNU449) compared to primary hepatocytes. To determine if miR-199a-3p has a tumor suppressive role, miR-199a-3p mimetic was transfected into the 7 HCC cell lines. miR-199a-3p mimetic reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A miR-199a-3p mimetic formulated with chemical modifications to enhance stability while preserving processing reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available miR-199a-3p mimetic. Furthermore, only the duplex miR-199a-3p mimetic, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling (Orian-Rousseau, et al., Genes Dev. 2002) and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Indeed, miRNA target algorithms predict miR-199a-3p to be a putative target of CD44. Immunoblotting confirmed that only the HCC lines that were sensitive to the effects of miR-199a-3p mimetic (SNU449 and SNU423) were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC tissue specimens. The anti-proliferative and anti-invasive properties of miR-199a-3p mimetic on CD44+ HCC may be a useful targeted therapy for CD44+ HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2011-1183

Abstract The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here, we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin that promote α5β1 integrin–dependent fibronectin fibril assembly, matrix stiffness, and tumor growth. Tumor growth and fibronectin fibril assembly were reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in fibronectin fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl that augmented α5β1 fibronectin fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of fibronectin fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment. Cancer Res; 72(16); 4047–59. ©2012 AACR.

Abstract Introduction: Genome-wide association studies of colorectal cancer have identified 18 risk variants that explain ∼25% of the hereditable risk of colorectal cancer. These variants are located in intronic, inter-genic or gene-desert regions. To uncover whether some of these variants may have a regulatory function on the expression of near-by genes, we conducted expression quantitative trait loci (eQTL) analyses of these 18 regions. Methods: DNA and RNA were extracted from 40 fresh-frozen paired colon tumor (microsatellite stable and CpG island methylator phenotype-negative) and adjacent normal tissue samples collected by the Colon Cancer Family Registry. Whole genome gene expression assessment was performed in the tumors and adjacent normal tissue with the Affymetrix GeneChip Human Exon 1.0 ST array. Genome-wide SNP data was generated with DNA extracted from the same normal tissue samples using the Affymetrix Genome-Wide Human SNP 6.0 array. Partek software was used to compare the expression levels by genotype for each gene spanning a 2 Mb region up- and down- stream of each of the 18 established risk variants for colorectal cancer. Results: Three of the 18 risk variants (rs10795668, rs4444235, rs9929218) demonstrated significant cis-eQTLs (FDR q-values<0.05). Specifically, differential gene expression was observed in genes neighboring rs10795668 at 10p14 (ATP5C1, q=0.024), rs4444235 at 14q22.2 (DLGAP5, q=0.041), and rs9929218 at 16q22.1 (NOL3, q=0.017; DDX28, q=0.046). In particular, DLGAP5 (discs, large (Drosophila) homolog-associated protein 5) at 14q22.2 and NOL3 (nucleolar protein 3) at 16q22 are interesting candidate genes for colorectal cancer. DLGAP5 encodes for a microtubule-associated protein and functions as a mitotic regulator that has been reported to be overexpressed in colon tumors. NOL3 (nucleolar protein 3) encodes for an anti-apoptotic protein that down-regulates the expression of caspase 2 and 8, and p53. NOL3 also appears to be overexpressed in colorectal tumors. Conclusion: In summary, these findings suggest putative functional activities for three established colorectal risk variants and highlight potentially interesting candidate genes for this malignancy. If confirmed, these results may help explain the total genetic risk of the disease, as well as further our understanding of the underlying biology of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4732. doi:10.1158/1538-7445.AM2011-4732

Abstract Background: Exemestane, a steroidal aromatase inhibitor, reduced the incidence of invasive breast cancers by 65% among 4560 post-menopausal randomized to exemestane or placebo for 5 years on MAP.3. Differences in quality of life (QOL) were judged to be minimal, but only summary information was reported. Purpose: To provide more detailed information about effects of exemestane on menopause-specific and health-related qualities of life. Method: Participation in quality of life assessment was an eligibility criterion. Menopause-specific and health-related qualities of life were assessed using the MENQOL (4 scales; physical, vasomotor, psychosocial, sexual) and SF-36 (8 scales; physical health, role function — physical, bodily pain, general health, vitality, social function, role function — emotional, mental health, and 2 summary scales) instruments, respectively at baseline, 6 months and then yearly after randomization. Compliance with QOL questionnaire completion at each follow-up visit ranged from 93–98%, and did not differ by group. Change scores for each MENQOL and SF-36 scale, calculated for each assessment time relative to baseline, were compared using the Wilcoxon Rank-Sum test. Summary scores were used to summarize the QOL scores observed at each time point for each SF-36 dimension and overall mental (MCS) and physical component summaries (PCS) and MENQOL domains. Clinically important worsening of MENQOL change scores was defined as an increase of ≥0.5/8 points. SF-36 change scores were considered worsened if scores decreased by ≥ 5 points from baseline. Results: Both groups were balanced on scores for MENQOL and SF-36 at baseline. Median follow-up was 35 months and the proportion of women who stopped study medication early for toxicity reasons was 15% in the exemestane arm and 11% in the placebo arm. There was a statistically significant difference in change scores for vasomotor symptoms among women on exemestane during the first 4 years (p-values <0.01), compared to placebo. However, no between-group differences in vasomotor change met the criterion for clinical importance. Women on exemestane had statistically poorer sexual functioning (mean change = −0.02, SD=1.37) compared to placebo (mean change = −0.12, SD=1.32) during the first 6 months on study (p-value = 0.03) but the differences were not statistically significant thereafter or clinically important at any time. Among the 8 SF-36 scales, only bodily pain was statistically different between exemestane and placebo for the first 24 months on study medication (p-value <0.01), but no between-group difference in change scores exceeded 5 points. Overall SF-36 PCS and MCS assessing changes in overall physical and mental health-related QOL did not differ significantly by group at any assessment. Conclusion: Our assessment that early differences in vasomotor symptoms and pain were probably not clinically important is supported by the observation of no between-group differences when overall physical and mental health-related QOL changes were compared. Exemestane does not appear to have a major negative impact on the quality of life among these women. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-1.

Abstract Background: We have previously reported the results of surgical excision without irradiation for selected patients with DCIS in ECOG E5194, where the 5-year rates of local recurrence varied with age, grade, and lesion size (Hughes et al. J Clin Oncol 27:5319, 2009). New methods are needed to provide more accurate and reproducible assessment of recurrence risk. Methods: ECOG E5194 included 670 eligible patients with DCIS treated with surgical excision (≥ 3 mm negative margins) without irradiation, 228 of whom received tamoxifen. Patients had low or intermediate grade DCIS ≤ 2.5 cm, or high grade DCIS ≤ 1 cm. The Oncotype DX® assay was performed by quantitative RT-PCR using formalin fixed paraffin embedded tumor specimens from 327 patients (49% of the parent study). Recurrence Score® (RS) was calculated using the published algorithm. A new, prespecified DCIS Score™ was designed to predict recurrence using an optimized gene expression algorithm. The primary objective was to determine whether there was a significant association between the risk of an ipsilateral breast event (IBE) and the continuous DCIS Score in Cox models. 46 patients had an IBE (defined as ipsilateral local recurrence of DCIS [n=20] or invasive cancer [n=26]). Median follow-up was 8.8 years. Results: The 10-year IBE rates were 15.4% for low/intermediate grade DCIS and 15.1% for high-grade DCIS (as determined by central pathology review), and for invasive IBE, 5.6% and 9.8%, respectively. Comparison between local and expert grading showed substantial disagreement. Continuous DCIS Score was significantly associated with IBE (HR 2.34 per 50 units; 95% CI 1.15, 4.59; p=0.02) when adjusted for tamoxifen use (prespecified primary analysis) and with invasive IBE (HR 3.73; CI 1.34, 9.82; p=0.01). DCIS Score was significantly associated with outcome when evaluated by the prespecified risk groups (see Table). Similar results were observed with and without adjustment for tamoxifen use or for negative margin width. Features associated with IBE in multivariate models included menopausal status (HR 0.49; 95% CI 0.27, 0.90; p=0.02), tumor size (HR 1.52 per 5 mm; 95% CI 1.11, 2.01; p=0.01), and continuous DCIS Score (HR 2.41; 95% CI 1.15, 4.89; p=0.02). The standard RS, which is calculated using thresholding of many genes unlike the DCIS Score, was not associated with IBE or invasive IBE (p > 0.6). Conclusions: We have prospectively validated a multigene assay that quantifies recurrence risk and complements traditional clinical and pathologic factors in selected patients with DCIS treated with surgical excision without irradiation. The DCIS Score provides a new clinical tool for individualized selection of treatment for patients with DCIS. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S4-6.

Abstract Bisphosphonates reduce the incidence of skeletal-related events (fractures, pain, hypercalcemia) in patients (pts) with bone metastases from breast cancer. By inhibiting osteoclast function and subsequent bone turnover they may inhibit the growth of bone (and other) metastases. Their role in preventing or delaying the development of bone (or other) metastases in pts with early breast cancer is uncertain. Three previous trials of oral clodronate have given mixed results. The largest trial (placebo-controlled) and a smaller open-label trial suggested that oral clodronate benefitted pts with improved bone metastases-free survival (BMFS) and overall survival (OS), but a third open-label study showed no benefit with an apparent detrimental effect on survival. Studies of IV zoledronate in open-label trials in early breast cancer have also shown mixed results: the AZURE trial in pts with node positive breast cancer showed no benefit in disease-free survival (DFS) or OS with a possible effect in pts aged over 60. The ABCSG-12 trial in GnRH (plus tamoxifen or anastrozole) treated women with early breast cancer showed a small DFS benefit for those receiving IV zoledronate but no BMFS or OS benefit. Methods: B-34 is a prospective, randomized, double-blind, phase III clinical trial in pts with stage 1, 2 or 3 breast cancer assessing oral clodronate 1600mg daily for 3 years compared to placebo given alone or in addition to adjuvant chemo- or hormone therapy. Stratification is by age (<50, +50), number of positive nodes (0, 1–3, 4+) and ER/PR status. The primary end-point is DFS. Secondary endpoints are the incidence of skeletal metastases, OS, relapse-free survival, incidence of non-skeletal metastases, and incidence of skeletal morbid events. Results: 3323 patients were accrued — 1662 in Group A and 1661 in Group B. As of March 31, 2011, 54 pts were declared ineligible: 23 in Group A and 31 in Group B. The average time on study is 101.3 months. Patient characteristics were evenly distributed throughout both groups. Approximately 75% of pts in each group had negative axillary lymph nodes and some 78% in each group were ER and/or PR positive. About 64% of pts were age 50 or over. Oral clodronate was generally tolerable, toxicities observed being mainly due to the concomitant systemic chemotherapy. One pt in Group A had a 1 mm area of osteonecrosis on the palatal taurus. Compliance, as expected with trials of oral medications, has been a problem. 1910 pts completed 3 years of therapy. Event rates were slower than anticipated. This final analysis assesses a total of 598 events in the two arms. Conclusions: This trial is the largest placebo-controlled study of an oral bisphosphonate in patients with early breast cancer and will provide further information on the role of bisphosphonates in breast cancer management. Supported for NSABP by NCI U10CA12027, −37377, 69651, 69974; and Bayer Schering Pharma Oy; for ECOG by U10CA021115; for NCCTG by U10CA25224; for SWOG by U10CA38926. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S2-3.

Abstract Background: The BRCA1 tumour suppressor gene is mutated in the germline of women who are predisposed to developing breast cancer. Gene expression profiling has identified at least five different breast cancer subtypes with BRCA- mutated breast tumours clustering with triple negative breast cancers. The majority of BRCA1-mutated breast tumours are characterised as being negative for the estrogen receptor-α (ER-α), however, the underlying molecular biology of these tumours has not yet been fully determined. The aim of this study is to identify and characterise novel proliferation-associated BRCA1 target genes that are activated in BRCA1 mutated estrogen receptor negative (ER-ve) breast tumours. Methods: Gene expression profiling and data analysis was performed on a cohort of 46 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA1 mutated (ER-ve) breast tumours and matched sporadic controls using the Almac Diagnostics Breast DSA research tool. Profiling was also performed on a panel of 15 breast cancer cell lines. Bioinformatics analysis was performed using Oncomine, DAVID and Metacore. High throughput siRNA screens using HiPerFect were performed on the Qiagen Flexiplate siRNA. Validation of gene targets was performed by qRT-PCR and ChIP assay. Clonogenic assays were performed to independently validate the effect of selected target genes on cell survival. Results: A list of differentially expressed transcripts was derived from the comparison of 23 (ER-ve) BRCA1 mutated breast tumours and 23 matched sporadic controls. A genome-wide ChIP-Chip promoter analysis was also performed in MCF7 breast cancer cells. By overlapping these two datasets, a list of tumour derived BRCA1 promoter bound target genes was identified. Functional analysis of this gene list has identified the main pathways and processes that are deregulated in BRCA1 mutated (ER-ve) breast cancer including: (1) immune response (2) induction of the epithelial to mesenchymal transition (EMT) (3) cell cycle regulation and (4) apoptosis and survival. Hierarchical clustering of these 46 breast tumours and 15 breast cancer cell lines was performed and the BRCA1 mutated (ER-ve) breast cancer cell lines (MDA436 and SUM149) were identified as those that best reflect the biology BRCA1 mutated (ER-ve) tumours. High throughput siRNA screening in these cell lines has identified a set of transcripts that when inhibited have a negative impact on cellular proliferation. Independent validation by qRT-PCR, ChIP assay, western blotting and clonogenic assays have confirmed HE4 (WFDC2) as a novel BRCA1 target gene that provides a growth advantage in BRCA1 mutated (ER-ve) breast cancer cells. Conclusions: Gene expression profiling of an extensive cohort of BRCA1 mutated (ER-ve) breast tumours and matched sporadic controls has identified a profile of BRCA1 mutated (ER-ve) breast cancer. This list has been further refined to generate a list of BRCA1 promoter bound transcriptional target genes. High throughout siRNA screening has revealed a panel of genes that are implicated in the proliferation of BRCA1 mutated (ER-ve) breast cancer. HE4 has been identified as a novel BRCA1 transcriptional target gene that promotes the survival of BRCA1 mutated (ER-ve) breast tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD10-05.

Abstract Background: Unstained recuts from formalin-fixed paraffin-embedded tissues are commonly collected for cooperative group studies. There is concern among pathologists that improper storage conditions can lead to antigen degradation. In an effort to quantify this effect, we compared the expression of HER1 and HER2 on two sets of identical cohort tissue microarrays (TMAs) from the N9831 HER2+ adjuvant phase III trial (NCT00005970; www.clinicaltrials.gov); one freshly cut set (cut April 18, 2011) and a second set stored at 4 degrees for over two years (cut between Nov, 2007 and Jan, 2008). Methods: The two sets of TMA slides containing 1580 tumor samples from the N9831 cohort were treated identically using the AQUA method of quantitative immunofluorescence. HER1 was tested with D38B1 (rabbit monoclonal, Cell Signaling Technology, Inc.) and HER2 with CB11 (mouse monoclonal, Biocare, Inc.) on tumors from 695 patients (712 specimens) in the fresh TMAs and 779 patients (800 specimens) in the old TMAs in up to three-fold redundancy per specimen. Results: Frequency distributions of the expression of HER2 revealed bimodality in the fresh TMAs compared to an attenuated distribution of the old cases. The average score of the entire cohort was significantly lower in old TMAs compared to fresh cuts (paired t-test, p<0.0001). Linear regression of the average HER2 scores from new TMAs versus the average scores from old TMAs showed a slope term of 0.52, which is statistically significantly different from the hypothetical value of 1 (p<0.0001). Regressions between any two fresh slides showed slopes close to 1.0. Similar results were seen for HER1, but fewer positive cases made the changes less dramatic. Conclusions: The storage condition of tissue slides is a critical pre-analytical variable that can dramatically lower the score of HER1 and HER2, artificially. Thus, studies done on inadequately stored slides, either whole sections or TMAs, must be interpreted with caution. Tissue collection and analysis of biomarkers for cooperative group studies should not include unstained recuts, but rather, entire blocks or large cores from tissue blocks. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-04.