TS Expression Research Articles

Abstract 2192: DFP-10825 IP delivery provides a new effective treatment option to peritoneal-disseminated cancers

Abstract Objective: Peritoneal disseminated ovarian and pancreatic cancers are the most difficult to be treated with conventional cytotoxic or molecular targeted drugs. The treatment option is very limited although an intraperitoneal (IP) paclitaxel has been available and shown to improve a prognosis in patients. Therefore, it is urgent to develop a new IP chemotherapeutic drug regulating the fast DNA synthesis in peritoneal disseminated tumors originated commonly from the ovary, pancreas and stomach. We have developed a unique RNAi molecule consisting of shRNA (55-mer) against TS and a cationic liposome (DFP-10825) and tested its anti-tumor activity and PK profile in peritoneal disseminated ascetic tumor models. Methods: We developed luciferase gene-transfected ovarian cancer (SKOV3-luc) and pancreatic cancer (PANC-1-luc) models in mice. After IP injection of 5x106 cells, DFP-10825 containing 20 µg TS shRNA (20 mg/mouse) was administered in an IP route (q3d x 4) to the tumor-bearing mice. In combination therapy, paclitaxel (10 mg/kg) was also IP administered to SKOV3-luc model to which the treatment was performed in the same schedule. The anti-tumor effect was assessed by measuring luciferase activity and tumor volume. Furthermore, the TS expression level in both ascetic tumor cells and solid tumors was measured by conventional RT-PCR. For PK study with DFP-10825 (especially TS shRNA), total RNAs were isolated at various time points from ascetic tumor cells and disseminated SKOV3-luc solid tumor models treated with DFP-10825 and TS shRNA levels were determined by Stem-loop RT-PCR. Results: IP DFP-10825 delivery significantly suppressed the growth of ascetic SKOV3-luc and PANC-1-luc tumor cells and extended the survival time of these tumor-bearing mice compared with that of control group. Combination with the IP paclitaxel augmented the efficacy of DFP-10825. After the IP administration, TS shRNA levels derived from DFP-10825 in ascetic fluid were maintained at nM range (0.7 - 4.3-nM) across 24 hours but not detected (below 5 pM) in the plasma, suggesting that TS shRNA be relatively stable in the peritoneal cavity to be able to exert its anti-tumor activity but not in blood. Also, TS expression (TS mRNA) in ascetic tumor cells was significantly suppressed, supporting the notion that the anti-tumor activity and host survival benefit by DFP-10825 in tumor-bearing mice are through MOA to knock down the TS level in tumors specifically. Conclusion: IP administration of newly developed DFP-10825, the TS shRNA conjugated with cationic liposome is localized stably in the peritoneal cavity and provides a new effective treatment option to the intractable peritoneal disseminated ovarian and pancreatic cancers without any systemic adverse events. Citation Format: Cheng Jin, Kenzo Iizuka, Kokoro Eshima, Masakazu Fukushima, Tatsuhiro Ishida, Cheng-Long Huang, Hiromi Wada, Kiyoshi Eshima. DFP-10825 IP delivery provides a new effective treatment option to peritoneal-disseminated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2017-2192

Molecular profiling-guided therapy in gastroesophageal carcinoma: A single-institution experience.

e15582 Background: Gastroesophageal carcinoma (GEC) including carcinoma of stomach (GC), gastroesophageal junction (GEJ), and esophagus (EC) is the 2nd leading cause of cancer death worldwide. Chemotherapy and HER2-targeted therapy (trastuzumab) have shown limited efficacy. We aim to assess the impact of molecular profile (MP)-guided therapy (MPgt) by correlating expression of select biomarkers in GEC patients (pts) with survival. Methods: 27 GEC (11 GC, 9 GEJ, 7 EC) submitted to Caris Life Sciences for molecular profiling between 2011 and 2016 were analyzed and correlated with pt survival. The impact of MPgt was assessed by calculating the ratio of progression-free survival (PFS) on MPgt to PFS on the preceding empiric therapy. MPgt was deemed beneficial if PFS ratio ≥1.3. Results: In-situ hybridization indicated amplification of HER2 (15.4%) and c-MET (7.4%). Immunohistochemistry revealed increased expression of TOPO1 (57.7%), TOP2A (38.5%), HER2 (15.4%) and c-MET (6.9%), as well as decreased expression of TS (69.2%), ERCC1 (42.3%), and PGP (15.4%). These data suggest sensitivity to topoisomerase inhibitors, anthracyclines, trastuzumab, MET-targeted therapy, fluoropyrimidine, platinums, and taxanes, respectively. Expression of these markers was heterogeneous among pts, and a trend toward improved PFS was noted in pts with low/absent ERCC1 expression on platinum-based therapy ( P= 0.06). Of the 13 pts who had sufficient data to assess the benefit of MPgt, 5 (38%) achieved a PFS ratio ≥1.3. One pt with metastatic HER2-amplified EC who had initially demonstrated clinical benefit from trastuzumab-containing chemotherapy, developed a new HER2 and c-MET co-amplified lung metastasis. Conclusions: While MPgt was beneficial in 38% of pts, tumor-associated plasticity, clonal evolution, and adaptive resistance may have limited efficacy. The emergence of HER2 and c-MET co-amplified clones is a potential resistance mechanism in HER2-amplified GEC, and highlights combined inhibition of receptor tyrosine kinases as a therapeutic strategy. Further studies with expanded data sets will be needed to test the hypothesis that actionable targets can be used independently to predict treatment response in GEC pts.

Abstract PD8-04: Racial differences in the molecular landscape of breast cancer

Abstract Background:African Americans (AA) have a higher mortality associated with breast cancer (BC) when compared to Caucasians (CC).This has been attributed to diverse factors that include access to care, reproductive factors and different somatic genomic profiles. We aimed to compare the racial mutational landscape of 565 BC samples. Methods:DNA from formalin-fixed paraffin-embedded samples was sequenced using the Illumina NextSeq (Agilent SureSelect XT, 592 gene selected based on COSMIC database) and MiSeq (TruSeq, 47 gene) for mutation and gene amplification analyses. Protein expression was evaluated by Immunohistochemistry (IHC). The exome aggregation consortium database was assessed for known ethnicity associations. Results:Tumor samples from 118 AA and 447 CC female patients were included in this analysis. AA were younger (median age 56 vs58y, p0.005) and had higher proportion of triple negative BC (TNBC) (32% vs17%, p0.001). 50.3% of the samples were obtained from primary tumors and the remainder from metastatic sites. This was similar in AA and CC (48.8% vs51.8% primary tumors, pNS). The two genes with highest mutation prevalence were TP53 and PIK3CA. AA had fewer PIK3CA mutations (14.7% vs28.2%, p0.03). Within HR+/HER2+ and HR+/HER2- subtypes there was a similar trend in the number of PIK3CA mutations but it was no longer significant. The remainder mutation analysis did not differ between races. In terms of protein expression there were significant differences in the androgen receptor(AR), RRM1, EGFR and TS expression (table). AR positivity defined as ≥10% was less frequent in AA (40.0% vs60.4%, p0.0001 and when adjusted for age, p0.005) and associated with PIK3CA mutations in both AA and CC (p0.01 and p0.007). AR expression in TNBC was positive in 17.8% of CC and 5.4% of AA (pNS). Copy number variation (CNV) data assessed by NextGen revealed significantly higher gene copy number in AA compared to CC in CCND1 (16% vs4%, p0.04), FGF19 (16% vs1.3%, p0.01) and FGF4 (16% vs3%, p0.02). When only TNBC was considered, RRM1, TOPO1 and TUBB3 expression was significantly higher in AA than CC (table) and there were no differences in the mutational analyses. Evaluation of other BC subtypes (HER2, HR positive) is currently underway. Conclusions:In this large cohort of AA who underwent genomic profiling there were relatively few differences in the mutation analysis compared to CC. The only significant difference seen was the lower number of PIK3CA mutations in AA, which had been previously reported in a cohort of 105 AA from the TCGA data (Keenan et al. JCO 2015). Protein expression by IHC revealed lower expression of AR in AA, even after adjustment for age, which could have therapeutic implications. Some of the racial differences found in the molecular landscape of BC including PIK3CA mutations, AR, EGFR expression and CNV may contribute to a more aggressive tumor biology in AA. Protein expression by IHCBCTNBCAA %CC %pAA %CC %pHER2/neu912ns00nsAR40600.005180.07ER44620.0000nsPR35460.0900nsEGFR30160.036050nsERCC133460.063626nsMGMT6163ns6571nsPD-150340.066548nsPD-L106ns011nsPGP76ns1015nsPTEN5658ns4247nsRRM139270.0459270.01TLE350590.104357nsTOP2A7164ns86470.08TOPO16358ns69460.03TS44300.016349nsTUBB355410.0677510.03 Citation Format: Lynce F, Xiu J, Nunes MR, Swain SM, Gatalica Z, Isaacs C, Pohlmann P. Racial differences in the molecular landscape of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD8-04.

АНАЛІЗ КОМБІНОВАНОГО ВПЛИВУ ІНТРАТУМОРАЛЬНОЇ ЕКСПРЕСІЇ ТИМІДИЛАТСИНТАЗИ ТА HENT1 НА ЕФЕКТИВНІСТЬ СХЕМИ ГЕМЦИТАБІН+ТЕГАФУР В АД’ЮВАНТОМУ ЛІКУВАННІ ХВОРИХ НА РАК ПІДШЛУНКОВОЇ ЗАЛОЗИ

Рак підшлункової залози (РПЗ) є одним із найагресивніших серед гастроінтестинальних раків. Шанс на довгострокову виживаність, що залишається неприйнятливо низькою, дає тільки радикальна резекція. Оптимізація ад’ювантного лікування хворих на РПЗ є актуальним питанням сучасної онкології. Метою роботи був аналіз комбінованого впливу інтратуморальної експресії hENT1 і тимідилатсинтази (TS) на ефективність схеми гемцитабін+тегафур ГемТег в ад'ювантному лікуванні хворих на РПЗ. До дослідження залучено 62 хворих на РПЗ з R0-cтатусом резекції на І та ІІ стадіях (UICC ver.7). Усі хворі отримували комбінацію ГемТег в якості ад’ювантної терапії. Діагноз у кожному випадку був веріфікований гістологічно, після чого імуногістохімічно визначали експресію hENT1 та TS. Статистичну обробку виконували за допомогою критерію Фішера, методів χі-квадрат, Каплана–Майєра та аналізу ризиків за Коксом. Медіана спостереження становила 34 місяці. За допомогою однофакторного аналізу оцінювали переваги у виживаності, порівняно з когортою хворих із співпаданням низької експресії hENT1 та TS. При цьому виживаність у когортах із високою експресією hENT1, з високою експресією TS та з високою експресією обох маркерів була значно вищою (р=0,053; р=0,03 та р<0,001 відповідно). При багатофакторному аналізі ризиків за Коксом у стратифікованих когортах HR становив 4,65 (СI: 2,12–10,21), 4,82 (СI: 2,13–10,91) та 23,14 (СI: 2,73–195,88) відповідно. Висока інтратуморальна експресія hENT1 та TS незалежно одне від одного може покращувати загальну виживаність хворих на РПЗ при ад’ювантному лікуванні за схемою ГемТег. Хворі, в яких одночасно відзначалася висока експресія hENT1 та TS, отримували найбільшу перевагу в виживаності.

P3.03-048 Profiling Response to Chemotherapy in Malignant Pleural Mesothelioma among Hispanics (MeSO-CLICaP): Topic: Mesothelioma Clinical

Malignant pleural mesothelioma (MPM) is a rare malignant disease, and the understanding of molecular pathogenesis has lagged behind other malignancies. A series of 53 formalin-fixed, paraffin-embedded tissue samples with clinical annotations were retrospectively tested for BAP1 and PI3K mutations and for mRNA expression of TS and EGFR. Immunohistochemistry staining for CD26 (dipeptidyl-peptidase IV, DPP-IV) and Fibulin3 (Fib3) proteins were also performed. Outcomes like progression free survival (PFS), overall survival (OS) and response rate (ORR) were recorded and evaluated according to biomarkers. Cox model was applied to determine variables associated with survival. Median age was 58 years (range 36-76), 27 (51%) were men, 89% were current or former smokers, and six patients had previous contact with asbestos. 77% had a baseline ECOG 0-1 and almost all patients (n=52/98%) received cisplatin or carboplatin plus pemetrexed (Pem) as first line; 58% of them were treated with Pem as maintenance for a mean of 4.7 +/-2.8 cycles. 53.5% and 41.5% of patients were positive for CD26 and fibulin-3, while 49% and 43.4% had low levels of EGFR and TS mRNA, respectively. The majority of epithelioid and biphasic types expressed CD26 (p=0.008), Fibulin3 (0.013) and had lower levels of TS mRNA (p=0.008). Mutations in PI3K (c.1173A> G, c.32G> C and c.32G> T) were found in 5 patients and only one patient had a mutation in BAP1 (c.241T> G). First line PFS were significantly longer in CD26+ (p=0.0001), in those with low EGFR mRNA expression (p=0.001), in patients with positive Fib3 (p=0.006) and lower TS mRNA expression (p=0.0001). OS were significantly higher in patients with CD26+ (p=0.0001), EGFR- (p=0.001), Fib3 + (p=0.0002) and low TS mRNA expression level (p=0.0001). Multivariate analysis found that CD26+ (p=0.012), Fib3 (p=0.020) and TS mRNA levels (p=0.05) were independent prognostic factors. CD26, Fib3 and TS were prognostic factors significantly associated with improved survival in patients with advanced MPM.

Abstract 2735: Clinico-pathological and molecular features associated with TP53 mutation in 3457 molecularly-profiled colorectal cancers (CRCs)

Abstract Deregulation of the p53 tumor suppressor gene (TP53) is a key event contributing to transformation and aggressive metastatic features of CRC. Patients with TP53 mutation are often resistant to therapy and carry a poor prognosis. We investigated TP53 mutation in a cohort of 3457 CRCs to identify molecular features specific to TP53-mutated CRC tumors. The 3457 CRC clinical samples were evaluated for tumor profiling (Caris Life Sciences, Phoenix, AZ). Tests included Sanger or next generation sequencing (NGS), protein expression by immunohistochemistry (IHC) and gene amplification by in situ hybridization (ISH). TP53 mutation was observed in 2106 or 61% of CRCs analyzed. 2018 or 96% of these mutant TP53 tumors carried one TP53 mutation, 83 (4%) carried 2 mutations, 4 carried 3 and 1 tumor carried 4 mutations. Among the ∼2200 mutations found in TP53, 37% were found at one of the six hotspots within the DNA binding domain (R175, G245, R248, R249, R273 and R282). Overall, 1554 (71%) were missense mutations, 367 (17%) nonsense, 209 (9.5%) frameshift, 45 (2%) small in-frame in-dels, and 25 (1.1%) mutations that affect splicing. In this cohort, TP53 mutation was more prevalent in male patients (64% vs. 57%, P<0.0001) and was more likely to occur in tumors that originated from the left colon (69%) as compared to the right colon (45%, p<0.0001). TP53 mutation rate was not correlated with patient age, histology or whether the tumor sample was taken from the primary or metastatic sites. When the molecular features of TP53-mutated tumors were compared to those of wild-type TP53, mutated tumors carried significantly higher Her2 IHC expression (2.5% vs. 1.0%, p = 0.0039) and gene amplification (3.7% vs. 1.4%, p = 0.0002), as well as higher MGMT (61% vs. 53%, p<0.0001) and TOPO2A expression (92% vs. 81%, p<0.0001). On the other hand, lower EGFR expression (57.4% vs. 70%, p<0.0001), PTEN expression (47.9% vs. 61%, p<0.0001), microsatellite instability (2.5% vs. 11.5%, p<0.0001), ERCC1 (18% vs. 24%, p<0.0001) and TS expression (31% vs. 38%, p<0.0001) were associated with TP53-mutated tumors. TP53-mutated CRCs carried higher rates of APC mutation (63% vs. 53%, p<0.0001), but lower rates of KRAS (46% vs. 54%, p<0.0001), PIK3CA (11.6% vs. 22%, p<0.0001), PTEN (2% vs. 5.2%, p<0.0001), GNAS (1% vs. 8.3%, p<0.0001) and AKT1 (0.6% vs. 1.7%, p = 0.0016) mutation. In this cohort of 3457 molecularly profiled CRCs, TP53 mutation was more prevalent in males and tumors that originated from the left colon. Distinct molecular features associated with TP53 mutation in CRC included lower frequency of PI3K/Akt/mTor pathway activation and were more likely to be microsatellite stable.. Our findings suggest differential presence of therapeutic targets in CRC tumors based on TP53 mutation status. Citation Format: Joanne Xiu, Thierry Soussi, Ryan Bender, Sandeep Reddy, Wafik S. El-Deiry. Clinico-pathological and molecular features associated with TP53 mutation in 3457 molecularly-profiled colorectal cancers (CRCs). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2735.

Dasatinib modulates sensitivity to pemetrexed in malignant pleural mesothelioma cell lines.

BackgroundThymidylate synthase (TS), one of the key enzymes for thymidine synthesis, is a target of pemetrexed (PEM), a key agent for the systemic therapy of malignant pleural mesothelioma (MPM) and its overexpression has been correlated to PEM-resistance. In MPM, experimental data report activation of the c-SRC tyrosine kinase suggesting it as a potential target to be further investigated.ResultsMPM cell lines showed different sensitivity, being MSTO the most and REN the least sensitive to PEM. REN cells showed high levels of both TS and SRC: dasatinib inhibited SRC activation and suppressed TS protein expression, starting from 100 nM dose, blocking the PEM-induced up regulation of TS protein levels. Dasatinib treatment impaired cells migration, and both sequential and co-administration with PEM significantly increased apoptosis. Dasatinib pretreatment improved sensitivity to PEM, downregulated TS promoter activity and, in association with PEM, modulated the downstream PI3K-Akt-mTOR signaling.Cell lines and MethodsIn three MPM cell lines (MPP89, REN and MSTO), the effects of c-SRC inhibition, in correlation with TS expression and PEM sensitivity, were evaluated. PEM and dasatinib, a SRC inhibitor, were administered as single agents, in combination or sequentially. Cell viability, apoptosis and migration, as well as TS expression and SRC activation have been assessed.ConclusionsThese data indicate that dasatinib sensitizes mesothelioma cells to PEM through TS down-regulation.

Abstract P3-07-27: Distinct biomarker features in triple-negative breast cancer metastases to the brain, liver and bone

Abstract Background: Triple-negative breast cancer (TNBC) is characterized by its aggressive nature and accounts for a disproportionate number of metastatic disease cases and breast cancer-related deaths. Despite recent improvements, TNBC patients who develop metastatic diseases have limited treatment options. We investigated biomarkers from brain, liver and bone metastases collected from TNBC patients to identify therapeutic options and to examine molecular differences between the metastatic sites. Method: Triple-negative breast cancer tumors referred to Caris Life Sciences (Phoenix, AZ) between 2009 and 2015 were tested with a combination of immunohistochemistry, fluorescent/chromogenic in-situ hybridization and sequencing (Next-generation and Sanger). Result: 1570 TNBC tumors were analyzed, including 1297 tumors taken from breast, 54 from brain, 172 from liver and 47 from bone. Select biomarker frequencies of protein overexpression (IHC), gene amplification (ISH) and mutations (SEQ) are summarized in Table 1. Brain metastases showed the highest protein expression of TOPO2A and PDL1; liver metastases showed the highest expression of AR and SPARC, as well as the highest mutation rate of PIK3CA. Bone metastases showed the lowest expression of TS, RRM1 and ERCC1. BRCA1 and BRCA2 mutation rates ranged from 0-11% in various specimen sites. Table 1Biomarker and MethodBreast Metastases (%)Brain Metastases (%)Liver Metastases (%)Bone Metastases (%)p value[pound]TOP2A IHC76100[sect]7339<0.0001PDL1 IHC1540[sect]8170.03AR IHC151036[sect]260.0005SPARC IHC173040[sect]150.0027PIK3CA SEQ165.329[sect]250.036TS IHC[dagger]49542415[sect]<0.0001RRM1 IHC[dagger]39433216[sect]0.006ERCC1 IHC[dagger]35554816[sect]0.002BRCA1 SEQ708n/ansBRCA2 SEQ11114n/ans[sect]:the group with the highest frequency of actionable results; [pound]:p values are calculated from comparing the group with the highest frequency with the lowest frequency using two tailed Fisher-Exact test, further detailed statistical analysis will be presented;[dagger]:low levels predict response to associated drugs; Ns: non-significant, i.e., p >0.05; n/a: data not available due to low N Conclusion: Distinct biomarker features identified in different metastatic sites in TNBC present the rationale to investigate differential treatment strategies. Based on biomarker results, etoposide, immune-modulatory agents may seem promising for brain metastases; anti-androgen therapies and nab-paclitaxel may be promising in treating liver metastases; while fluoropyrimidines, gemcitabine and platinum may be considered for TNBC patients with bone metastases. Citation Format: Xiu J, Gatalica Z, Reddy S, Waisman J, Link J. Distinct biomarker features in triple-negative breast cancer metastases to the brain, liver and bone. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-27.

Molecular characterization of colorectal tumors in young patients compared with older patients and impact on outcome.

505 Background: The incidence of colorectal cancer (CRC) among young individuals is rising. The effect of an individual’s age on their tumor molecular profile is unknown. Methods: Molecular profiles of 4,821 tumors obtained from younger and older CRC patients (pts) were reviewed and correlated with pt outcome. Protein expression (IHC), gene amplification (ISH), sequencing (NGS and Sanger), and fragment analysis were performed. T and Fisher’s exact tests determined differences between age groups; Kaplan-Meier methodology estimated survival. Results: Tumors from 1,277 younger (median age 40; range 15-45 yr) and 3,544 older (72; 65-98) pts were studied. Most frequently mutated genes included TP53, APC, KRAS, PIK3CA, SMAD4, and BRCA1/2. Mutation rates for BRAF (14.4% vs. 4.8%, p < 0.001), APC (62% vs. 54%, p = 0.0034), FBXW7 (9.6% vs. 5.5%, p = 0.01), and KRAS (45% vs. 41%, p = 0.02) were higher in older pts; NRAS mutation rates (4.5% vs. 3.9%) were similar in both groups. Younger pts had higher overexpression rates of HER-2/neu (3.2% vs. 1.8%, p = 0.017) and MGMT (64% vs. 58%, p = 0.001). Microsatellite instability (MSI), determined by IHC (MLH1, MSH2, MLH6 and PMS2) and fragment analysis, was similar between cohorts (10.3% vs. 8.1%), but somatic MSI high (determined by concurrent BRAF mutation) was higher in older pts (6% vs. 0%, p < 0.0001). There was no difference in TS (37% vs. 34%), ERCC1 (25% vs. 26%) or TOPO1 (49% vs. 46%) expression between age groups. Older pts showed a trend toward higher PD-L1 expression (2.9% vs. 0.7%, p = 0.0512) but there was no difference in the frequency of PD-1 expression on tumor-infiltrating lymphocytes (39% vs. 43%). Outcome was evaluable for 82 pts (younger, 47; older, 35). Most pts received FOLFOX + Bev as first line Rx. Median OS was worse in younger pts (p = 0.03). Low ERCC1 expression was associated with prolonged survival in older (p < 0.01) but not in younger (p = 0.3) pts. There was no association between TOPO1 expression and OS (p = 0.8). Pts with low TS expression showed a trend toward longer OS (p = 0.08). Conclusions: Young pts with CRC may carry genetic alterations that are distinct from older pts. A better understanding of disease biology may help to identify therapeutic targets for younger pts.

Expression of ERCC1, TUBB3, BRCA1, and TS as predictive markers of neoadjuvant chemotherapy for squamous cell carcinoma of the esophagus.

47 Background: No predictive biomarker of the response to neoadjuvant chemotherapy with docetaxel and cisplatin plus 5-fluorouracil (NAC-DCF) is available for patients with squamous cell carcinoma of the esophagus (SCCE) in a clinical setting. The aim of this study was to identify the biomarkers associated with chemotherapeutic efficacy and long-term survival for patients with advanced SCCE who had received NAC-DCF followed by surgery. Methods: This study included 45 patients with advanced SCCE who received NAC-DCF between January 2008 and December 2012. The NAC-DCF was conducted as a phase II study (UMIN000007408). The expressions of excision repair cross-complementing-1 (ERCC1), class III beta-tubulin, breast cancer susceptibility gene I (BRCA1), and thymidylate synthase were investigated simultaneously in the pre-treatment endoscopic tumor biopsy samples. Results: The multivariate logistic regression analysis indicated that pathological responses were significantly associated with tumors with low ERCC1 expression (P = 0.016) and with tumors with high BRCA1 expression (P = 0.014). The incidence of pathological responses to DCF in patients with low ERCC1 and high BRCA1 expressions was 100%. The multivariate Cox proportional hazard model analysis for relapse-free survival (RFS) revealed tumors with high BRCA1 expression (P = 0.031, hazards ratio, 4.39) as independent prognostic factors. Conclusions: Low ERCC1 expression and high BRCA1 expression in patients with SCCE were predictive biomarkers for chemotherapeutic efficacy. High BRCA1 expression was considered as prognostic factors for long-term RFS. These results may be a helpful tool for tailoring chemotherapy of patients with SCCE. Prospective validation of this biomarker panel is warranted. Clinical trial information: UMIN000007408.

Tumor profiling of 431 pancreatic tumors from elderly patients.

257 Background: Pancreatic cancer (PC) is frequently a disease of the elderly and controversy exists as to whether these patients benefit from aggressive treatments. We aimed to investigate biomarker features from tumors taken from elderly PC patients to identify therapeutic implications. We also compared the results to those from younger patients to assess differences. Methods: PC tumors were tested at Caris Life Sciences between 2009 and 2015 by immunohistochemistry, fluorescent/chromogenic in-situ hybridization and sequencing. De-identified biomarker data were analyzed. Results: A total of 431 tumors from PC patients aged > = 75 were analyzed: 50% were samples from pancreas, 50% were from metastatic sites. 26 of 47 genes sequenced carried mutations with frequencies ranging from 0.6% to 84%. The highest mutation rates were seen in KRAS (84%), TP53 (55%), BRCA2 (21%), SMAD4 (12%), ATM (4.4%), BRCA1 (4.2%) and PIK3CA (3.8%). Overexpression of TOPO1 and low expression of ERCC1, RRM1 and TS were seen in 45%, 73%, 86% and 79%, respectively, indicating potential benefit from irinotecan, platinums, gemcitabine and fluoropyrimidine, respectively. Tumor expression of PD-L1 was seen in 14% and tumor-infiltrating lymphocyte expression of PD-1 was seen in 45%. Overexpression of EGFR and cMET were seen in 91% and in 62%, respectively. When compared to 470 tumors from PC patients aged < = 50, KRAS mutations were significantly more common in the elderly than in younger patients (84% vs. 74%, p = 0.0018), while MLH1 mutations were seen exclusively in younger patients (0% vs. 4%, p = 0.0241). Moreover, expression of TOP2A (53%vs. 45%), ERCC1 (36% vs. 27%), RRM1 (20% vs. 14%), SPARC (20% vs. 14%) and PR (5% vs. 1%) were significantly more prevalent in the younger group (all p < 0.05). Conclusions: Tumor profiling of 431 tumors from elderly PC patients suggests therapeutic opportunities including cytotoxic, biological as well as targeted agents for this patient group. A comparison with younger patients indicated that irinotecan, fluoropyrimidine and taxanes may provide equal benefit to elderly and younger patients, while platinums and gemcitabine may be more likely to benefit elderly patients.

Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model.

Malignant pleural mesothelioma (MPM) is an incurable cancer with an increasing incidence. Currently, pemetrexed (PMX)-based chemotherapy is the mainstay of chemotherapy for MPM, however, the outcome of PMX-based chemotherapy in patients with MPM is dismal. RNA interference (RNAi) technology has been considered as an effective tool to substantially enhance the therapeutic efficacy of chemotherapeutic agents in many preclinical and clinical settings. In this study, therefore, we investigated whether non-viral anti-thymidylate synthase RNAi embedded liposome (TS shRNA lipoplex) would effectively guide the downregulation of TS in human malignant mesothelioma MSTO-211H cells. Consequently, it enhanced the antitumor effect of PMX both in vitro and in vivo. TS shRNA effectively enhanced the in vitro cell growth inhibition upon treatment with PMX via downregulating TS expression in the MSTO-211H cell line. In in vivo orthotopic tumor model, the combined treatment of PMX and TS shRNA lipoplex efficiently combated the progression of orthotopic thoracic tumors and as a result prolonged mouse survival, compared to each single treatment. Our findings emphasize the pivotal relevance of RNAi as an effective tool for increasing the therapeutic efficacy of PMX, a cornerstone in the treatment regimens of MPM, and thereby, raising the possibility for the development of a novel therapeutic strategy, combination therapy of TS-shRNA and PMX, that can surpass many of the currently applied, but less effective, therapeutic regimens against lethal MPM.

Molecular profiling of 6,892 colorectal cancer samples suggests different possible treatment options specific to metastatic sites

Metastatic colorectal cancer (mCRC) carries a poor prognosis with an overall 5-year survival of 13.1%. Therapies guided by tumor profiling have suggested benefit in advanced cancer. We used a multiplatform molecular profiling (MP) approach to identify key molecular changes that may provide therapeutic options not typically considered in mCRC. We evaluated 6892 mCRC referred to Caris Life Sciences by MP including sequencing (Sanger/NGS), immunohistochemistry (IHC) and in-situ hybridization (ISH). mCRC metastases to liver, brain, ovary or lung (n = 1507) showed differential expression of markers including high protein expression of TOPO1 (52%) and/or low RRM1 (57%), TS (71%) and MGMT (39%), suggesting possible benefit from irinotecan, gemcitabine, 5FU/capecitabine and temozolomide, respectively. Lung metastases harbored a higher Her2 protein expression than the primary colon tumors (4% vs. 1.8%, p = 0.028). Brain and lung metastases had higher KRAS mutations than other sites (65% vs 59% vs 47%, respectively, p = 0.07, <0.01), suggesting poor response to anti-EGFR therapies. BRAF-mutated CRC (n = 455) showed coincident high protein expression of RRM1 (56%), TS (53%) and low PDGFR (22%) as compared with BRAF wild-type tumors. KRAS-mutated mCRC had higher protein expression of c-MET (47% vs. 36%) and lower MGMT (56% vs. 63%), suggesting consideration of c-MET inhibitors and temozolomide. KRAS-mutated CRC had high TUBB3 (42% vs. 33%) and low Her2 by IHC (0.5%) and HER2 by FISH (3%, p <0.05). CRC primaries had a lower incidence of PIK3CA and BRAF mutations in rectal cancer versus colon cancer (10% and 3.3%, respectively). MP of 6892 CRCs identified significant differences between primary and metastatic sites and among BRAF/KRAS sub-types. Our findings are hypothesis generating and need to be examined in prospective studies. Specific therapies may be considered for different actionable targets in mCRC as revealed by MP.

ERCC1 and TS Expression as Prognostic and Predictive Biomarkers in Metastatic Colon Cancer.

In patients with metastatic colon cancer, response to first line chemotherapy is a strong predictor of overall survival (OS). Currently, oncologists lack diagnostic tests to determine which chemotherapy regimen offers the greatest chance for response in an individual patient. Here we present the results of gene expression analysis for two genes, ERCC1 and TS, measured with the commercially available ResponseDX: Colon assay (Response Genetics, Los Angeles, CA) in 41 patients with de novo metastatic colon cancer diagnosed between July 2008 and August 2013 at the University of California, San Diego. In addition ERCC1 and TS expression levels as determined by RNAseq and survival data for patients in TCGA were downloaded from the TCGA data portal. We found that patients with low expression of ERCC1 (n = 33) had significantly longer median OS (36.0 vs. 10.1 mo, HR 0.29, 95% CI .095 to .84, log-rank p = 9.0x10-6) and median time to treatment to failure (TTF) following first line chemotherapy (14.1 vs. 2.4 mo, HR 0.17, 95% CI 0.048 to 0.58, log-rank p = 5.3x10-4) relative to those with high expression (n = 4). After accounting for the covariates age, sex, tumor grade and ECOG performance status in a Cox proportional hazard model the association of low ERCC1 with longer OS (HR 0.18, 95% CI 0.14 to 0.26, p = 0.0448) and TTF (HR 0.16, 95% CI 0.14 to 0.21, p = 0.0053) remained significant. Patients with low TS expression (n = 29) had significantly longer median OS (36.0 vs. 14.8 mo, HR 0.25, 95% CI 0.074 to 0.82, log-rank p = 0.022) relative to those with high expression (n = 12). The combined low expression of ERCC1/TS was predictive of response in patients treated with FOLFOX (40% vs. 91%, RR 2.3, Fisher’s exact test p = 0.03, n = 27), but not with FOLFIRI (71% vs. 71%, RR 1.0, Fisher’s exact test p = 1, n = 14). Overall, these findings suggest that measurement of ERCC1 and TS expression has potential clinical utility in managing patients with metastatic colorectal cancer.

Expression of TS, RRM1, ERCC1, TUBB3 and STMN1 Genes in Tissues of Non-small Cell Lung Cancer and its Significance in Guiding Postoperative Adjuvant Chemotherapy.

To explore the expression of TS, RRM, ERCC1, TUBB3 and STMN1 genes in the tissues of patients with non-small cell lung cancer (NSCLC) and its significance in guiding the postoperative adjuvant chemotherapy. Real time polymerase chain reaction (PCR) was applied to detect the expression of TS, RRM, ERCC1, TUBB3 and STMN1 genes in the tissues of NSCLC patients so as to analyze the relationship between the expression of each gene and the clinical characteristics and to guide the postoperative individualized chemotherapy according to the detection results of NSCLC patients. Expression of TS gene was evidently higher in patients with adenocarcinoma than those with non-adenocarcinoma (P=0.013) and so was the expression of ERCC1 (P=0.003). The expression of TUBB3 gene was obviously higher in NSCLC patients in phases I/II and IV than those in phase III (P1=0.021; P2=0.004), and it was also markedly higher in patients without lymph node metastasis than those with (P=0.008). The expression of STMN1 gene was apparently higher in patients in phase I/II than those in phase IV (P=0.002). There was no significant difference between the rest gene expression and the clinical characteristics of NSCLC patients (P>0.05). Additionally, the disease- free survival (DFS) was significantly longer in patients receiving gene detections than those without (P=0.021). The selection of chemotherapeutic protocols based singly on patients' clinical characteristics has certain blindness. However, the detection of tumor-susceptible genes can guide the postoperative adjuvant chemotherapy and prolong the DFS of NSCLC patients.

Impact of intratumoral expression levels of fluoropyrimidine-metabolizing enzymes on treatment outcomes of adjuvant S-1 therapy in gastric cancer.

We analyzed the expression levels of fluoropyrimidine-metabolizing enzymes (thymidylate synthase [TS], dihydropyrimidine dehydrogenase [DPD], thymidine phosphorylase [TP] and orotate phosphoribosyltransferase [OPRT]) to identify potential biomarkers related to treatment outcomes in gastric cancer (GC) patients receiving adjuvant S-1 chemotherapy. In this study, 184 patients who received curative gastrectomy (D2 lymph node dissection) and adjuvant S-1 were included. Immunohistochemistry and quantitative reverse transcription polymerase chain reaction were performed to measure the protein and mRNA levels of TS, DPD, TP, and OPRT in tumor tissue. In univariate analysis, low intratumoral DPD protein expression was related to poorer 5-year disease-free survival (DFS; 78% vs. 88%; P = 0.068). Low intratumoral DPD mRNA expression (1st [lowest] quartile) was also related to poorer DFS (69% vs. 90%; P < 0.001) compared to high intratumoral DPD expression (2nd to 4th quartiles). In multivariate analyses, low intratumoral DPD protein or mRNA expression was related to worse DFS (P < 0.05), irrespective of other clinical variables. TS, TP, and OPRT expression levels were not related to treatment outcomes. Severe non-hematologic toxicities (grade ≥ 3) had a trend towards more frequent development in patients with low intratumoral DPD mRNA expression (29% vs. 16%; P = 0.068). In conclusion, GC patients with high intratumoral DPD expression did not have inferior outcome following adjuvant S-1 therapy compared with those with low DPD expression. Instead, low intratumoral DPD expression was related to poor DFS.

Biomarker patterns of localized and metastatic prostate cancer.

192 Background: Presence or absence of metastases is a determining factor for prostate cancer prognosis. Common sites of metastasis include bone (B), lymph nodes (LN) or visceral organs (V). We sought to determine theranostic biomarker differences between primary (P) and metastatic (M) specimens, with subset analysis for differences between metastatic sites. Methods: 497 prostate cancer cases referred to Caris Life Sciences between 2009 thru 2014 were evaluated. Specific testing was performed and included a multiplatform approach: sequencing (Sanger, NGS), protein expression (IHC) and gene amplification (CISH/FISH). Results: 58% (287/497) were M specimens, of which 23% (66), 24% (68) and 52% (149) were from B, LN and V, respectively. Significant differences for EGFR amplification/overexpression, low MGMT expression, TOPO1/2A overexpression and higher PTEN mutation rate were found in the M subgroup. These alterations indicate a role for EGFR/PTEN in progression of prostate cancer, and potential role of MAPK/PAM-targeted therapies, alkylating agents and topoisomerase inhibitors in M disease. Mutational profiles of the M subgroup are more genetically unstable exhibiting mutations in 60% of genes tested (notable events include APC &amp; β-Catenin, PTEN, TP53 and BRCA1/2) vs. 30% observed in the P subgroup (p=0.0067). In the subset analysis, EGFR amplification was higher in B (29%) and V (24%) compared to LN (13%); (not significant). Low TS expression was more frequent in B vs. LN (p=0.004), TOPO2A overexpression was higher in V vs. B (p=0.0001) and there was a trend for PD1 positive infiltrating lymphocytes being more abundant in LN vs B (p=0.09). Interestingly, cMET overexpression (7/230) and amplification (1/162) were rarely observed across the cohort, providing support for the surprising failure of cabozantinib in castration-resistant prostate cancer. Conclusions: Our data indicate a role for multiplatform profiling to identify therapeutic options that are appropriate for subsets of patients. Molecular changes associated with M disease including EGFR, PTEN, MGMT and TOPO1/2A, present opportunities for developing therapeutic strategies which may include a combination of targeted therapies, as well as traditional cytotoxic chemotherapies.

Comprehensive multiplatform biomarker analysis of 313 hepatocellular carcinoma to identify potential therapeutic options.

283 Background: Effective treatment strategies for hepatocellular carcinoma (HCC) remain limited. Identification of additional therapies remains paramount as currently available agents have resulted in marginal improvements in overall survival. Methods: 313 HCC sampleswere evaluated using a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ), including gene sequencing (Sanger, NGS [N=79]), protein expression (IHC) and gene amplification (ISH). Results: Biomarker changes of interest are shown. See Table. TP53 was mutated in 28%, CTNNB1 in 23%, and BRCA2 in 20%; other gene mutation rates were &lt; 5%.TP53-mutated tumors show significantly higher TOPO2A (89% vs. 39%, p&lt;0.0001), TS (70% vs. 32%, p=0.0067) and RRM1 expression (40% vs. 12%, p=0.017), implying high rates of proliferation and DNA synthesis. CTNNB1-mutated tumors showed significantly higher SPARC (67% vs. 21%, p=0.0013) and AR expression (53% vs. 22%, p=0.025). Primary HCC (N=209) exhibited significantly higher PD-1 (89% vs. 33%, p=0.01) and TS expression (35% vs. 13%, p&lt;0.0001) than metastatic (N=105). Patient history/outcomes relative to biomarker status are being evaluated. Conclusions: These data suggest potential therapeutic targets, such as tyrosine kinase inhibitors, anti-PD1 agents, or PI3 kinase pathway inhibitors. Although no evidence shows that cytotoxics are effective in patients with HCC, irinotecan, alkylating agents, fluoropyrimidines, anthracyclines, nab-paclitaxel, gemcitabine, or taxanes may be therapeutically relevant. The protein changes associated with CTNNB1-mutated tumors suggest potential benefit of targeting WNT pathway in combination with nab-paclitaxel or anti-androgens. Immuno-modulatory agents may be a therapeutic option in primary HCC, based on the higher levels of PD-1. Multiplatform tumor profiling reveals molecular heterogeneity HCC, similar overall to previous reports, and identifies different potential treatment options for molecular subtypes. [Table: see text]

Abstract 2927: Rapamycin decreases expression of thymidylate synthase and enhances the response to pemetrexed in preclinical studies and a Phase I/II clinical study of subjects with non-small cell lung cancer

Abstract Non-small-cell lung cancer (NSCLC) accounts for 80-85% of lung cancers and almost half of patients with newly diagnosed NSCLC have metastatic disease. Platinum-based chemotherapy regimens given as first-line treatment for advanced NSCLC patients with metastasis are standard but have modest response rates. Pemetrexed is well tolerated and inhibits several folate-dependent enzymes including thymidylate synthase, TS. Increased expression of TS confers resistance to pemetrexed in vitro and is a predictive factor for poor response to pemetrexed. Rapamycin is a mTOR inhibitor and suppresses protein synthesis. Here, we show that rapamycin decreases endogenous expression of TS in NSCLC cells. The combination of rapamycin and pemetrexed synergistically inhibits NSCLC cell proliferation and enhances the inhibition of 4E-binding protein 1 (4E-BP1) activation that is regulated by mTOR. Although pemetrexed as a single agent upregulated expression of TS, pretreatment with rapamycin suppressed pemetrexed-upregulated TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited the growth of NSCLC xenografts, which correlated with decreased mTOR activity and inhibition of pemetrexed-induced upregulated expression of TS. Based on these preclinical data, we have conducted a Phase I/II clinical trial combining rapamycin and pemetrexed in subjects with relapsed NSCLC. This regimen was well tolerated, and resulted in a 22% partial response rate that is above what is expected for response to single agent pemetrexed (9%). In biomarker analyses, pretreatment with rapamycin decreased the expression of TS in peripheral blood mononuclear cells, which correlated with increased progression free survival. Collectively, these studies identify rapamycin and pemetrexed as an effective combination in NSCLC, and suggest that rapamycin enhances the efficacy of pemetrexed by suppressing TS expression. Citation Format: Shigeru Kawabata, Chun-Te Chiang, Regan M. Memmott, Takefumi Komiya, Joell J. Gills, Phillip A. Dennis. Rapamycin decreases expression of thymidylate synthase and enhances the response to pemetrexed in preclinical studies and a Phase I/II clinical study of subjects with non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2927. doi:10.1158/1538-7445.AM2014-2927

Abstract 2042: Deregulated TS promotes tumor progression in diverse hematopoietic and mesenchymal cell lineages

Abstract Our laboratory recently showed that elevated TS plays a direct causal role in tumorigenesis of NIH3T3 cells in vitro and in the development of endocrine pancreatic mouse tumors in vivo. This observation changed the paradigm of TS as a passive biomarker and has significance for cancer treatment as it refocuses attention on the importance of TS as a tumor-promoting signal. To address whether TS plays an early role in tumorigenesis that is restricted to specific tumor types or if TS plays a late role associated with enhanced progression of established tumors of diverse histologic origin, we have now cross-bred human TS (hTS) transgenic mice with Ink4a/Arf null mice, a common somatic mutation frequently found in adult human cancers. Median survival of hTS/Ink4a/Arf+/- (n=15) mice was reduced 24% (313 vs. 411 days, p &amp;lt;0.05). Both hTS/Ink4a/Arf+/- and hTS/Ink4a/Arf-/- mice (n=16) showed an increase in lymphoma, histiocytic sarcoma (HS) and soft tissue sarcoma (SS). For example, the incidence of HS in hTS/Ink4a/Arf-/- male mice was 31% versus 12% in Ink4a/Arf-/- minus (p&amp;lt;0.01), and tumor cells had spread to multiple tissues (as many as 9 different organs) as compared to hTS minus mice where tumors were localized in lymph nodes and spleen. Strikingly, the tumor burden was markedly increased in hTS/Ink4a/Arf-/- mice: average spleen weight 1.11 vs. 0.49 g, p&amp;lt;0.001, lymph nodes, 1.06 g vs. 0.42 g, p&amp;lt;0.001. We also detected an increase in size and extent of tumor mass of SS (average weight 5.8 g). We detected increased aneuploidy in hTS/Ink4a/Arf-/- HS cell lines as compared to Ink4a/Arf-/- HS cell lines: chromosomal loss 32.1% vs. 16.7%, p&amp;lt;0.05, gain 24.5% vs. 0%, p&amp;lt;0.001 and translocations 9.4% vs. 0%, p&amp;lt;0.05 by spectral karyotyping analysis. Treatment of TS positive HS tumor cells with lenti-TS shRNAs downregulated TS expression and resulted in tumor growth inhibition in vitro as well as in mouse orthotopically transplanted xenograft. In addition, combined TS shRNAs with gemcitabine or pemetrexed treatment showed synergistic inhibition of HS tumor cell growth. These data demonstrate that deregulated TS enhanced genome instability and promote tumor progression in diverse hematopoietic and mesenchymal tumor cells. Citation Format: Min Chen, Akbar Nawab, Frederic J. Kaye, Maria Zajac-Kaye. Deregulated TS promotes tumor progression in diverse hematopoietic and mesenchymal cell lineages. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2014-2042