TS Expression Research Articles

568P - Tumor Location and Histological Type Related to Intratumoral Expression of 5-Fu Metabolizing Enzymes in Stage III Colon Cancer: a Multicenter, Prospective Cohort Study [B-Cast Study

ABSTRACT Aim: Several oral 5-FU derivatives with different properties are used in adjuvant therapy for colon cancer in Japan. The B-CAST study is a prospective cohort study aimed efficacy prediction of 5-FU derivatives, through evaluating the intratumoral biomarker expressions and treatment outcome. We evaluated relationships between the relevant biomarker expressions and patient characteristics, as baseline analysis of the study. Methods: The frozen specimens of patients with stage III colon cancer who received postoperative adjuvant chemotherapy were examined. The expressions of TP, DPD, EGFR, and VEGF protein and TP, DPD, TS and OPRT mRNA were evaluated by using ELISA and qRT-PCR, respectively. Patients were divided into subgroups according to the biomarker expression levels by using a tree-based regression model with baseline patient characteristics as explanatory factors. Results: Among 2128 patients from the 217 institutions enrolled between Apr. 2009 and Mar. 2012, 2018 were used for analysis. The protein and mRNA expressions were successfully estimated in 100% and 89% of the examined samples, respectively. The logarithmic values of expressions of all examined biomarkers followed normal distributions. In the tree-based model, tumor was the most relevant variable to explain the expression of TP protein and OPRT mRNA (p Conclusions: This large cohort study revealed considerable degrees of correlations between location as well as histological type and the intratumoral expression of TP, DPD, TS, OPRT, and VEGF. The results would provide useful information for future studies in personalization of chemotherapy using 5-FU derivatives. Disclosure: M. Ishiguro: B-CAST study is conducted as a part of joint research of Tokyo Medical and Dental University and the Foundation for Biomedical Research and Innovation (FBRI), with funding from FBRI. All authors declare no relevant competing interest.All other authors have declared no conflicts of interest.

335P - Biomarkers of Targeted Therapies in Malignant Phyllodes Tumors of the Breast

ABSTRACT Aim: Malignant phyllodes tumors constitute 0.1% of all breast tumors, which lack effective treatment options. Comprehensive molecular profiling of rare cancers holds the promise of identifying underlying pathogenetic mechanisms with potentially druggable targets. Methods: Seventeen malignant phyllodes tumors (9 primary, 3 recurrent and 5 metastatic samples) were analyzed (Caris Life Sciences, Phoenix, AZ) using gene sequencing (NGS and Sanger), gene copy number analysis (FISH and CISH), RNA expression (RT-qPCR and whole genome microarrays) and protein expression (immunohistochemistry). Results: Malignant phyllodes tumors showed co-expression of genes involved in angiogenesis (vascular endothelial growth factor A and its receptor KDR) in 6 of 9 cases, and additional 3 cases showed overexpression of VEGFA alone. Amplification of EGFR gene was observed in 4 of 9 tested cases, while EGFR protein overexpression (H-score ≥ 20) was observed in 10 of 11 tested cases. TP53 mutations were identified in the majority of cases tested (8/11). One metastatic tumor harbored increased cMET gene copy number. No other oncogene alterations were identified (e.g. HER2, BRAF, KRAS, PIK3CA, cKIT). DNA repair pathways were altered in 9 out of 17 cases (7/17 low ERCC1; 2/17 low BRCA1) indicating potential response to platinum agents. Low expression of TS and RRM1 (9/17 cases) indicates potential response to fluoropyrimidines and gemcitabine. Steroid receptors were uniformly negative in all cases. Conclusions: Comprehensive tumor profiling identified diverse molecular alterations providing insight into potentially beneficial therapies in all malignant phyllodes tumors. These include novel therapies targeting angiogenesis and the EGFR pathway, as well as biomarkers of the conventional chemotherapy in this rare cancer type. Disclosure: Z. Gatalica: Stock options Caris Life Sciences; R. Bender: Stock options Caris Life Sciences. All other authors have declared no conflicts of interest.

Association between ERCC1 and TS mRNA levels and disease free survival in colorectal cancer patients receiving oxaliplatin and fluorouracil (5-FU) adjuvant chemotherapy.

BackgroundAim was to explore the association of ERCC1 and TS mRNA levels with the disease free survival (DFS) in Chinese colorectal cancer (CRC) patients receiving oxaliplatin and 5-FU based adjuvant chemotherapy.MethodsTotal 112 Chinese stage II-III CRC patients were respectively treated by four different chemotherapy regimens after curative operation. The TS and ERCC1 mRNA levels in primary tumor were measured by real-time RT-PCR. Kaplan–Meier curves and log-rank tests were used for DFS analysis. The Cox proportional hazards model was used for prognostic analysis.ResultsIn univariate analysis, the hazard ratio (HR) for the mRNA expression levels of TS and ERCC1 (logTS: HR = 0.820, 95% CI = 0.600 - 1.117, P = 0.210; logERCC1: HR = 1.054, 95% CI = 0.852 - 1.304, P = 0.638) indicated no significant association of DFS with the TS and ERCC1 mRNA levels. In multivariate analyses, tumor stage (IIIc: reference, P = 0.083; IIb: HR = 0.240, 95% CI = 0.080 - 0.724, P = 0.011; IIc: HR < 0.0001, P = 0.977; IIIa: HR = 0.179, 95% CI = 0.012 - 2.593, P = 0.207) was confirmed to be the independent prognostic factor for DFS. Moreover, the Kaplan-Meier DFS curves showed that TS and ERCC1 mRNA levels were not significantly associated with the DFS (TS: P = 0.264; ERCC1: P = 0.484).ConclusionThe mRNA expression of ERCC1 and TS were not applicable to predict the DFS of Chinese stage II-III CRC patients receiving 5-FU and oxaliplatin based adjuvant chemotherapy.

Correlation Between TS, MTHFR, and ERCC1 Gene Polymorphisms and the Efficacy of Platinum in Combination With Pemetrexed First-Line Chemotherapy in Mesothelioma Patients

IntroductionThe combination of pemetrexed and platinum compound represents the standard regimen for first-line chemotherapy in malignant pleural mesothelioma patients. Pemetrexed is a multitarget antifolate agent that inhibits folate-dependent enzymes (eg, thymidylate synthase [TS]) and thus synthesis of nucleotides and DNA. Expression of TS and folate availability, regulated by gene polymorphisms, have implications for effectiveness of chemotherapy and the outcome of mesothelioma patients. The aim of this retrospective multicenter study was to assess the correlation between TS, 5,10-methylenetetrahydrofolate reductase (MTHFR) and excision repair cross-complementing group 1 (ERCC1) gene polymorphisms and the efficacy of pemetrexed-based first-line chemotherapy of mesothelioma patients. Patients and MethodsFifty-nine mesothelioma patients (31 men with a median age of 62 years) treated in first-line chemotherapy with platinum in combination with pemetrexed or pemetrexed monotherapy were enrolled. Genomic DNA was isolated from peripheral blood. Using polymerase chain reaction and high resolution melt methods, the variable number of tandem repeat, the G>C single nucleotide polymorphism (SNP) in these repeats, and 6-base pair (bp) insertion/deletion polymorphism of the TS gene, the SNP of 677C>T in MTHFR, and 19007C>T in the ERCC1 gene were analyzed and correlated with disease control rate, progression-free survival (PFS), and overall survival (OS) of mesothelioma patients. ResultsGreater risk of early disease progression (PD), and shortening of PFS and OS were associated with several clinical factors (eg, anemia for early PD and OS), weight loss (for PFS and OS), and previous surgical treatment (for early PD, PFS, and OS). Insertion of 6-bp in both alleles of the TS gene (1494del6) was the only genetic factor that increased the incidence of early progression (P = .028) and shortening of median PFS (P = .06) in patients treated with pemetrexed-based chemotherapy. In multivariate analysis, the 1494del6 in the 3′ untranslated region (UTR) of the TS gene also had a predictive role for PFS (P = .0185; hazard ratio, 2.3258 for +6/+6 homozygotes) in analyzed mesothelioma patients. ConclusionMost analyzed polymorphisms in TS, MTHFR, and ERCC1 genes failed to predict outcome in mesothelioma patients treated with pemetrexed-based chemotherapy. However, different variants of 1494del6 in the 3′ UTR of the TS gene were associated with differences in disease control rate and PFS of our patients.

SMAD4 and TS expression might predict the risk of recurrence after resection of colorectal liver metastases

Colorectal liver metastases (CLM) have significant molecular heterogeneity, which contributes to the risk of recurrence following surgery. Most of the traditional scores intended to predict recurrence is based on clinicopathological variables and it is unclear whether incorporating molecular biomarkers might improve our assessment of the risk of recurrence. Our aim was to determine if molecular biomarkers might be associated with the risk of recurrence after surgery of CLM. A total of 121 patients diagnosed with colorectal cancer (CRC) with resected liver metastases were included. The role of several clinicopathological variables to predict patient's outcome after resection of liver metastases was analyzed. Eighteen genes related to CRC pathogenesis were also included in the analyses. Univariate and multivariate stepwise Cox regression analyses were performed to identify factors associated with recurrence and the risk of death. Eight prognostic factors for progression-free survival and nine factors for overall survival were identified in the univariate analyses. After adjusting for other risk factors, only the expression of two molecular factors was associated with the risk of recurrence: TS (HR 0.631, 95 % CI 0.422-0.944) and SMAD4 (HR 1.680, 95 % CI 1.047-2.695). None of the variables was significantly associated with the risk of death in the multivariate analyses. The prognostic significance of most traditional clinicopathological variables might be insufficient to define patients at risk for recurrence after liver metastases resection. Molecular biomarkers might improve the identification of patients with higher risk of recurrence.

Molecular profiling in gastric cancer: Examining potential targets for chemotherapy

Current NCCN guidelines recommend epirubicin (E), cisplatin (C), and 5-fluorouracil (F) as a first-line therapeutic approach for operable gastric adenocarcinoma (GC). Molecular profiling (MP) was used to evaluate the expression of chemotherapy targeted biomarkers associated with ECF therapy and other first-line cytotoxic regimens for GC. GC specimens were analyzed by immunohistochemistry (IHC) for TOP2A, TS, ERCC1, PGP, and TOPO1 expression (Caris Life Sciences, Phoenix, AZ) from 2009 to 2012. A total of 230 GC specimens were analyzed. The median age of patients was 61 (IQR: 50-72) years with the majority being male (n = 139, 60%). IHC actionable targets included: 60% (n = 138) high TOP2A, 55% (n = 127) negative ERCC1, and 63% (n = 145) negative TS, indicating potential benefit from E, C, and F, respectively. Simultaneous expression analysis demonstrated only 24% (n = 55) of patients had gene expression levels that suggested uniform sensitivity to ECF. Biomarker results of 6.5% (n = 15) of patients revealed a potential complete lack of sensitivity to first-line ECF. MP of GC has the potential to define patients who would derive the greatest benefit from current therapies. Prospective controlled studies are required to validate the role of biomarkers in the management of GC patients.

Variability of Proliferation and Diffusion in Different Lung Cancer Models as Measured by 3'-Deoxy-3'-¹⁸F-Fluorothymidine PET and Diffusion-Weighted MR Imaging.

Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts. Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by (18)F-FDG and (18)F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2'-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography-mass spectrometry. Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. (18)F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in (18)F-FDG uptake were only marginal. No clear relationship between (18)F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence (18)F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death. A direct relationship of (18)F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings.

P21 and CD166 as predictive markers of poor response and outcome after fluorouracil-based chemoradiotherapy for the patients with rectal cancer

BackgroundPre-operative chemoradiotherapy (CRT) is the standard treatment in clinical stage T3/4 or node positive rectal cancer. However, there are no established biomarkers that can predict the pathological response and clinical outcome to CRT.MethodsImmunohistochemical staining was performed in tissue arrays constructed from core tissue specimens taken before treatment and from operative specimens from 112 patients who received 5-FU based pre-operative CRT and surgery. Expression of Ki67, TS, BAX, EpCAM, p53, p21, EGFR, CD44, CD133, CD166, HIF1α and ALDH1 were assessed and correlated with tumor regression grades and disease free survival.ResultsOf the 112 patients (M/F 74/38, median age: 62), 20 (17.9%) patients achieved pathologic complete remission (pCR). In analyzing the associations between marker expressions and tumor regression grades, high p21 expression at the pretreatment biopsy was significantly associated with non-pCR (p = 0.022) and poor disease free survival (median DFS - low vs high p21: 75.8 vs 58.1 months, p = 0.002). In the multivariate analysis, high p21 expression level at the pre-treatment biopsy was significantly associated with poor DFS (p = 0.001, HR 6.14; 95% CI 2.03, 18.55). High CD166 expression level at the pretreatment biopsy was also associated with poor DFS (p = 0.003; HR 5.61; 95% CI 1.81, 17.35).ConclusionThese show high p21 and CD166 expression at the pretreatment biopsy were associated with tumor regression and poor prognosis in patients treated with 5-FU based CRT. Larger, prospective and functional studies are warranted to determine the role of p21 and CD166 as predictive biomarker of response to CRT.

Molecular profiling in gastric cancer: Examining potential targets for chemotherapy.

131 Background: Current NCCN guidelines recommend perioperative epirubicin (E), cisplatin (C), and 5-fluorouracil (F) along with other triple agent derivations as first line therapeutic approaches for operable gastric adenocarcinoma (GC). In this study, we utilized molecular profiling to evaluate expression of chemotherapy targeted biomarkers associated with ECF therapy for GC. Methods: Surgically obtained GC specimens were analyzed by immunohistochemistry for TOP2A, ERCC1, and TS expression (Caris Life Sciences, Phoenix) from 2009-2012. Actionable gene targets were analyzed for mutually exclusive or simultaneous expression. Results: A total of 230 GC specimens were analyzed. The median age of patients was 61 (IQR: 50-72) years with the majority being male (n= 139, 60%). IHC actionable targets included: 60% (n=138) high TOP2A, 55% (n=127) negative ERCC1, and 63% (n=145) negative TS, indicating potential benefit from E, C and F respectively. When analyzing for simultaneous expression profiles of the three genes, only 24 % (n=55) of patients had gene expression levels that suggested sensitivity to all three agents (ECF). Moreover, biomarker results of 6.5% (n=15) of patients demonstrated a potential complete lack of sensitivity to first line ECF therapy. Overall, 61% (n=140) of patients had molecular profiles that indicated sensitivity to two or more agents. Conclusions: Biomarker analysis of GC suggests that there is potential for TOP2A, ERCC1and TS to define patients who have the greatest likelihood of deriving benefit from ECF therapy. 93.5% of patients had the biomarker profile that predicted sensitivity to at least one of these agents. Prospective controlled studies are required to validate the role of TOP2A, TS and ERCC1 in routine management of GC patients.

Detection of potential actionable targets in appendiceal cancer detected by immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and mutational analysis.

469 Background: Appendiceal cancers are rare and there are no established guidelines for adjuvant treatment and metastatic disease. Methods: Samples were identified from &gt; 60,000 global tumors analyzed at a referral molecular profiling CLIA certified laboratory (Caris Life Sciences) for biomarkers of drug sensitivity, under an approved protocol. 285 samples with primary tumor sites of appendix were identified (M/F ratio of 38%/62%; mean age of 55). Approximately 80% of samples were adenocarcinomas, 10% carcinoids, and 10% of indeterminate histology. IHC assays (n=273) were performed with up to 30 biomarkers; FISH (n=39) for c-Myc, cMET, EGFR, HER2 and/or TOPO2A gene copy amplifications; and sequencing done ( n= 223) for KRAS, EGFR, PIK3CA, and BRAF. Results: Targets on IHC: 97% high BCRP; 16% high KIT; 80% high COX-2; 50%high EGFR; 30% low MGMT, 81% high MRP1, 35% high PDGFR, 82% low PTEN, 27% high RRM1, 39% high SPARC; 67% high TLE3, 27% high TOPO2A ; 63% high TOPOI, and 99% negative TS. Targets on FISH: 5% amplified EGFR and 0% amplified Her2/Neu. Sequencing results: 53% mutated KRAS and 2% mutated BRAF. Conclusions: The high levels of drug resistance proteins (BCRP and MRP1) highlights the difficulty in treating these tumors. The incidence of KRAS mutations (53%) and low expression of TS (99%) is noteworthy, and points towards using 5FU/capecitabine as a backbone of therapy. Therapeutic options are varied and options include TOPO1 inhibitors (irinotecan/topotecan), EGFR inhibitors (erlotinib, cetuximab), PDGFR antagonists (regorafenib, axitinib), MGMT (temazolamide), BRAF inhibitors (vemurafenib, dabrafenib) and SPARC (nab-paclitaxel). These findings indicate the need to evaluate patient samples for patterns in marker expression and alteration, in order to better understand the molecular biology and formulate a personalized therapy approach in these difficult to treat cancers, and to pursue whole genomic sequencing. Supported by a grant from Caris Life Sciences.

ERCC1 and TS expression and prognosis in colon cancer after postoperative adjuvant chemotherapy

ERCC1 and TS expression and prognosis in colon cancer after postoperative adjuvant chemotherapy

PO125 INFLUENCE OF HER2 STATUS ON METASTATIC PATTERN IN POSTMENOPAUSAL HORMONE RECEPTOR POSITIVE BREAST CANCER PATIENTS TREATED WITH ADJUVANT TAMOXIFEN ONLY

were the viceral in 25 patients, bone in 9 patients and soft tissue in 17 patients. The overall response rate (RR) was 22.9% (11/48) and clinical benefit rate was 45.8% (22/48). The median time to tumor progression (TTP) was 14.3 months (range 6.2 22.4). ER, PgR and HER2 status was not significantly correlated with RR (P=0.724, 0.288, 1.000, respectively). TS expression was significantly associated with clinical efficacy of S-1. Moreover, it is notable that triple negative breast cancer (TNBC) revealed lower TS expression than luminal type (TNBC vs luminal type = 1.03 (0.32 4.70) vs 2.45 (0.42 10.1), P= 0.020). According to the correlation between metastatic sites and RR, better RR was noted for soft tissue metastases (soft tissue vs visceral = 25% (5/20) vs 84% (5/6), OR 15.0 (95%CI 1.40 161.05). Conclusion: This study demonstrated that TS can be a significant predicitve factor of S-1 in patients with MBC.

Components of homologous recombination and translesion synthesis (TLS) in pemetrexed/cisplatin-treated non-small-cell lung cancer (NSCLC) patients (p).

11028 Background: REV3, the catalytic subunit of the TLS polymerase ξ, can continue replication past DNA adducts. Depletion of REV3 sensitizes A549 lung cancer cells to cisplatin. REV3 expression is part of a gene signature that predicted pemetrexed sensitivity in 17 NSCLC cell lines. Homologous recombination and TLS pathways have non-redundant functions in response to cisplatin. We hypothesized that low REV3 mRNA expression – alone or in combination with low expression levels of genes involved in homologous recombination – could correlate with better outcome to cisplatin/pemetrexed in NSCLC. Methods: REV3, BRCA1, RAP80, TS and AEG1 mRNA was examined by quantitative RT-PCR and categorized by terciles. Expression of each gene was correlated with outcome in 47 cisplatin/pemetrexed-treated NSCLC p. Results: 63.8% male; 47% smokers; 80.9% ECOG PS 1; 80.8% adenocarcinoma. Overall response rate was 51%, with no differences according to expression levels of any of the genes. Progression-free survival (PFS) for p with low, intermediate and high BRCA1 levels was 13.4, 5.5 and 3.9 months (m), respectively (P=0.005). Similar differences in PFS were observed according to TS (P=0.003) and AEG1 (P&lt;0.001) expression. Hazard ratio (HR) for PFS for p with high BRCA1 levels was 4 (P=0.002). Overall survival (OS) for p with low, intermediate and high BRCA1 levels was 29.7, 7.4 and 6.3 m, respectively (P=0.05). Similar differences in OS were observed according to TS (P=0.005) and AEG1 (P=0.001) expression.HR for OS for p with high BRCA1 levels was 3.6 (P=0.004). There were no differences in PFS or OS according to REV3 or RAP80 levels. However, the joint effect of BRCA1 and REV3 was significant for predictive modeling. PFS for p with low, intermediate and high levels of both genes was 14.9, 7.2 and 2.8 m, respectively (P=0.001). OS for p with low, intermediate and high levels of both genes was 29.7, 7.8 and 6.3 m, respectively (P=0.04). Conclusions: Low BRCA1 expression predicts longer PFS and OS in pemetrexed/cisplatin-treated NSCLC p. Low TS and AEG1 levels have similar predictive value. Analysis of these genes could be useful for customizing pemetrexed/platinum chemotherapy.

Gene expression profiles and tumor locations in colorectal cancer (left vs. right vs. rectum).

3527 Background: Recent data suggests that CRC from different locations show distinct genetic profiles. Right-sided tumors have a worse prognosis and may have less benefit from targeted therapies. We investigated the tumor locations and genetic profiles (KRAS and BRAF mutation status and ERCC1, TS, EGFR and VEGFR2 mRNA expression) in 580 CRC tumors. Methods: FFPE tumor specimen from 580 patients with advanced CRC adenocarcinoma were microdissected and DNA and RNA were extracted. Specifically designed primers and probes were used to detect 7 different base substitutions in codon 12 and 13 of KRAS, V600E mutations in BRAF and the mRNA expression levels of ERCC1, TS, EGFR and VEGFR2 by RT-PCR. These values were analyzed according to tumor location (left vs. right vs. rectum). Results: BRAF mutations were significantly more common in the right colon (15%), followed by rectum (3.8%) and left colon (2.5%). KRAS mutations occurred at similar frequencies throughout the colon. Gene expression of ERCC1 was significantly higher in right-sided than left-sided colon tumors in KRAS wild-type colon cancers. The highest expression levels for all genes were seen in rectum. These differences reached significant levels for ERCC1 (rectum vs. right and rectum vs. left, p&lt;0.001), TS (rectum vs. left, p&lt;0.036) and VEGFR2 (rectum vs. right and rectum vs. left, p&lt;0.001). Conclusions: Tumor location in CRC is associated with specific mutation and expression profiles. Differences in chemosensitivity may be explained by mutation status and mRNA levels in right vs. left CRC. Rectum cancers showed a distinct genetic profile when compared to colon which indicates different tumor biology and may be related to differences in the microflora.

Reply to association of EGFR mutation or ALK rearrangement with expression of DNA repair and synthesis genes in never‐smoker women with pulmonary adenocarcinoma

We thank Uramoto and Tanaka for their comments on our study.1 We think there are a few potential reasons for the discrepancy between their results and ours. First, the study population differed. Lung adenocarcinoma in nonsmoking Asian females has been found to be a unique entity in that it has a higher frequency of activating epidermal growth factor (EGFR) mutation and better prognosis.2 Our study further showed that 70% of subjects had activated EGFR mutation, and the remaining 30% had anaplastic lymphoma kinase (ALK) fusion.1, 3 Similarly, the different demographic characteristics in our study might also result in a different TS expression level. In addition, our selected population eliminated the impact of sex on TS expression, since TS expression differed between male and female subjects.4 Second, as Uramoto and Tanaka mention, disparities in sampling species (messenger RNA vs protein level) and experimental methods (real-time polymerase chain reaction vs immunohistochemistry) might have led to different results. Most importantly, the comparative population differed. We found that TS expression was significantly lower in patients with EGFR mutation than in those with both EGFR wild type and negative ALK fusion status.1 Uramoto and Tanaka compared TS expression in patients with EGFR mutation with patients who had EGFR wild-type. As we know, the EGFR wild-type group is heterogeneous, including the ALK fusion subgroup, which is not only associated with lower TS expression but also makes up a substantially large part of the wild-type group.3 As a result, Uramoto and Tanaka would be more likely to see a negative result. However, because Chinese and Japanese patients have a similar mutation spectrum and a similar therapeutic efficacy, ethnic difference would contribute little to the discrepancy. Hopefully, the worldwide multiplex mutation driver examination and pharmacogenomics detection efforts currently underway will shed light on the relationship between EGFR mutation and TS expression in the future. This study was supported by the Amory Company and was supported in part by the National Science Foundation of China (grants 81172108 and 81201707). The authors made no disclosures. Shengxiang Ren, MD and Caicun Zhou, MD, PhD Department of Medical Oncology, Shanghai Pulmonary Hospital Tongji University School of Medicine, Tongji University Medical School Cancer Institute, Shanghai, People's Republic of China

Thymidylate synthase inhibitors for thoracic tumors

Abstract Thymidylate synthase (TS) is a valid target for treatment of non-small cell lung cancer (NSCLC) and mesothelioma (MPM). The TS inhibitor pemetrexed (PMX) is now commonly used in combination with cisplatin (CDDP) or carboplatin, in the first-line setting for both of these lethal diseases. A combination of another TS inhibitor, Tomudex with CDDP, demonstrated similar activity in MPM patients. However, the efficacy of TS inhibitors is limited by uptake, metabolism and target affinity. While uptake of most antifolates is mediated by the reduced folate carrier, PMX is also a good substrate for the proton-coupled folate transporter (PCFT), which displays optimal activity at the acidic pH of the tumor microenvironment. NSCLC and MPM have a variable expression of PCFT, which might be caused by the differential methylation status of the PCFT promoter, resulting in decreased anticancer activity. PMX and TDX activity also depends on polyglutamylation, catalyzed by folylpolyglutamate synthetase (FPGS), which results in intracellular retention and thus enhanced cytotoxicity. We demonstrated that resistance to the classical antifolate methotrexate in human leukemia cells with a marked loss of FPGS activity was associated with impaired splicing of FPGS pre-mRNA. Moreover, we recently showed that the sensitivity of MPM patients to PMX-carboplatin is related to tumor TS expression. Patients with low TS mRNA levels had a significantly longer overall survival (20 vs. 7 months) compared with patients with high expression. Intriguingly, recent trials demonstrated that histology plays an important role in the sensitivity of NSCLC patients, and PMX-CDDP is now approved only for adenocarcinoma patients. These results might be partly explained by the higher TS expression detected in lung tumors of squamous histology. In summary, the clinical success of TS inhibitors may depend on biomarker-driven patient selection, and further studies should evaluate the genetic/epigenetic factors involved in the modulation of these biomarkers in the preclinical and clinical setting.

Reduced Folate Carrier and Folylpolyglutamate Synthetase, but not Thymidylate Synthase Predict Survival in Pemetrexed-Treated Patients Suffering from Malignant Pleural Mesothelioma

Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.

Abstract 4019: Regulatory role of Gli motifs in thymidylate synthase expression .

Abstract Fluoropyrimidine analogue 5-Flurorouracil (5-FU) is the mainstay of CRC treatment. It was designed as a competitive inhibitor of the enzyme thymidylate synthase [TS; TYMS]. All the new agents (Irinotecan, CPT-11, Avastin etc) are effective only in combination with 5-FU. The enzyme thymidylate synthase is pivotal for DNA replication. It controls cell survival and proliferation. Increases in TS level that follows 5-FU administration renders the treatment ineffective. Considerable efforts have been made to reverse 5-FU resistance by down-regulating TS expression. In this context, we demonstrated that arsenic trioxide (ATO) sensitized 5-FU resistant colorectal cancer cells by inhibiting TS mRNA expression both in vitro and in clinic. However, the precise mechanism of TS reduction by arsenic trioxide is not known. Many investigators, including ourselves, have shown a direct correlation between Glioma associated oncogene homolog (Gli) and TS expression in cultured cells as well in patient samples, who received arsenic trioxide and 5-FU combination therapy. However, the link between ATO, Gli and TS is not known. Gli is an important molecule in hedgehog (hh) signaling. It plays a vital role in development and cancer. As a transcription factor, Gli binds the sequence 5’ GACCACCCA 3’ a conserved 9 bp DNA motif. We hypothesized that TS promoter may harbor one or more of the 9 bp Gli motifs. We queried for Gli motifs in the TS gene regulatory region. Position specific base frequencies for the TF consensus sequence for Gli1-3, Ci, cECF, TGM were prepared using the data from Hallikas et al. (2006). We used FIMO (Find Individual Motif Occurrences) tool in the Meme Suite (http://meme.nbcr.net) to search for Gli motifs in the query sequence. Anti-Gli antibodies and non-specific IgG were used to precipitate chromatin immuno complex. The immuno precipitates were probed with TS specific primer. This search yielded 35 possible Gli motifs in TS promoter. From among the 35 motifs with significant homology (p≤0.001), only top eight matches had passed FDR threshold (q≤0.05). However, these eight matches represented three distinct binding sites on TS promoter. Thus we computationally determined that TS promoter has three unique Gli motifs viz BS1, BS2 and BS3. BS-2 in TS promoter is identical to BS-3 on the Jun and BS1 is identical to the BS 1 of Fgf15 which had been experimentally confirmed to be regulated by Gli1. The binding site was subsequently confirmed by ChIP assays. It appears that the inhibition of Gli1 by ATO down regulates TS expression. This cascade of reactions initiated by ATO reverts refractory tumors, once again sensitive to 5-FU. Taken together the results strongly indicate a direct role of Gli in TS transcription. Citation Format: Subbarayan R. Pochi, Nikesh Doshi, Xiaoqing Han, Zhiqiang Wang, Anthony Capobianco, David Robbins, Bach Ardalan. Regulatory role of Gli motifs in thymidylate synthase expression . [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 4019. doi:10.1158/1538-7445.AM2013-4019

Evidence for predictive role of BRCA1 and bTUBIII in gastric cancer

Sensitivity of gastric cancer (GC) to conventional cytotoxic therapy may be at least in part attributed to molecular features of the tumor cells. We analyzed all patients with metastatic GC treated in the N.N. Petrov Institute of Oncology (St. Petersburg) within years 1999-2010 and identified 65 cases with evaluable treatment response and available biological material. Two of 65 patients (3 %) carried germ-line BRCA1 5382insC mutation and demonstrated particularly pronounced response to the treatment; both of their tumors showed loss of the remaining BRCA1 allele, thus confirming the causative role of BRCA1 heterozygosity in GC predisposition. RNA expression of TS, DPD, BRCA1, ERCC, TOP2A and bTUBIII was analyzed in the remaining 63 tumors. Low BRCA1 expression was associated with increased response rate [6/9 (67 %) vs. 17/54 (32 %), p = 0.04]. Low bTUBIII level correlated with the improved probability of tumor response [21/49 (43 %) vs. 1/13 (8 %), p = 0.02] and prolonged overall survival (10.5 vs. 7.1 months, p = 0.02); this trend was maintained both for taxane-containing and for taxane-free drug combinations. We conclude that GC should be considered as a part of BRCA1-related hereditary cancer syndrome. Tumors with BRCA1 inactivation and low bTUBIII expression demonstrate improved response to cytotoxic therapy.

Analysis of predictive and prognostic value of clinical and pathological factors in locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiotherapy (CRT): Bologna multidisciplinary rectal cancer group study (BMRG-B01-Study).

421 Background: Preoperative fluoropyrimidine based CRT is standard treatment in LARC patients. The aim of this study was to evaluate prognostic and predictive role of clinical and pathological factors in this setting Methods: Between December 2001 and January 2012 we evaluated 149 pts with cT3-T4 N-/+ rectal adenocarcinoma located ≤12 cm from the anal margin. Preoperative CRT consisted of radiotherapy 50.4 Gy in 28 daily fractions + 5-fluorouracil or capecitabine +/- oxaliplatin. Rectal surgery with total mesorectal excision was performed 6-8 weeks after the end of neoadjuvant treatment. Pathological examination of surgical specimens included TRG according to the Dworak criteria. TS, EGFR, Ki-67, p53, Bcl-2, MLH1 and MSH2 were immunohistochemically determined in pre-treatment biopsies and surgical specimens. For immunohistochemistry evaluation serial sections of formalin-fixed, paraffin-embedded tissues were stained with specific antibodies using a biotin-free ready-to-use amplification system Results: After a median follow-up of 60 months (2-122) we observed 4.7% local recurrences, 12.7% distant recurrences, and 13.4% deaths. In surgical specimens TRG 1, 2, 3 and 4 were observed respectively in 22.5%, 35.3%, 25.6% and 16.6% of patients. ypN and TRG were independent prognostic factors of DFS (p=0.020, p=0.027). CRM and TRG were independent prognostic factors of OS (p=0.016, p=0.010). High pre-treatment biopsy expression of TS was associated with poor TRG (0-1) (p=0.007); high biopsy expression of Ki-67 was associated with good TRG (2-4) (p=0.039); decrease between pre-treatment biopsy and surgical specimen of Ki-67 (Δ Ki-67 ≥ 0) was associated with good TRG (2-4) (p=0.02). Decrease in Ki-67 (Δ Ki-67 ≥ 0) was associated with good DFS (p=0.011) and confirmed by multivariate analysis as an independent prognostic factor (p=0.035). Conclusions: In our analysis ypN, CRM and TRG were independent prognostic factors; baseline expression of TS, Ki-67 and decrease in Ki-67 were predictive of TRG; decrease in Ki-67 was an independent prognostic factor of DFS.