Abstract Background Tumor Budding (TB) and Immunoscore are independent prognostic markers in colon cancer (CC). Given their respective representation of tumor aggressiveness and immune response, we examined their combination in association with patient disease-free survival (DFS) in pTNM stage I-III CC. Methods In a series of pTNM stage I-III CCs (n = 654), the Immunoscore was computed and TB detected automatically using a deep learning network. Two-tiered systems for both biomarkers were used with cut-offs of 25% and ten buds for Immunoscore and TB according to clinical guidelines, respectively. Associations of Immunoscore with TB with 5-year DFS were examined using Kaplan–Meier survival analysis in addition to multivariable modeling and relative contribution analysis using Cox regression. Results Immunoscore and TB independently are prognostic with hazard ratio (HR) = 2.0, 95% confidence interval (CI) 1.4–2.8 and HR 2.5, with 95% CI 1.4–4.5, respectively; P value < 0.0001. By combining Immunoscore with TB, patients with Immunoscore Low, TB High tumors had a significantly poorer DFS (HR 5.6, 95% CI 2.6–12.0; P value < 0.0001) than those with Immunoscore High, TB Low tumors. The combined Immunoscore with TB score was independently prognostic (P value = 0.009) in comparison to N-stage, T-stage, and MSI. Immunoscore with TB had the highest relative contribution (35%) to DFS in pTNM stage I-II CCs. Conclusions The association of Immunoscore and TB with patient survival suggests that both biomarkers are complementary and should be interpreted in combination to identify high-risk Stage I-II patients who should be considered for adjuvant therapy or further diagnostic testing.

Background: Talimogene laherparepvec (T-VEC), a first-in-class oncolytic viral immunotherapy, enhances tumor-specific immune activation. T-VEC combined with atezolizumab, which blocks inhibitor T-cell checkpoints, could provide greater benefit than either agent alone. Safety/efficacy of the combination was explored in patients with triple negative breast cancer (TNBC) or colorectal cancer (CRC) with liver metastases.Methods: In this phase Ib, multicenter, open-label, parallel cohort study of adults with TNBC or CRC with liver metastases, T-VEC (106 then 108 PFU/ml; <4 ml) was administered into hepatic lesions via image-guided injection every 21 (+/- 3) days. Atezolizumab 1200 mg was given on day 1 and every 21 (+/- 3) days thereafter. Treatment continued until patients experienced dose-limiting toxicity (DLT), had complete response, progressive disease, needed alternative anticancer treatment, or withdrew due to an adverse event (AE). The primary endpoint was DLT incidence, and secondary endpoints included efficacy and AEs.Results: Between 19 March 2018 and 6 November 2020, 11 patients with TNBC were enrolled (safety analysis set: n = 10); between 19 March 2018 and 16 October 2019, 25 patients with CRC were enrolled (safety analysis set: n = 24). For the 5 patients in the TNBC DLT analysis set, no patient had DLT; for the 18 patients in the CRC DLT analysis set, 3 (17%) had DLT, all serious AEs. AEs were reported by 9 (90%) TNBC and 23 (96%) CRC patients, the majority with grade >3 [TNBC, 7 (70%); CRC, 13 (54%)], and 1 was fatal [CRC, 1 (4%)]. Evidence of efficacy was limited. Overall response rate was 10% (95% confidence interval 0.3-44.5) for TNBC; one (10%) patient had a partial response. For CRC, no patients had a response; 14 (58%) were unassessable.Conclusions: The safety profile reflected known risks with T-VEC including risks of intrahepatic injection; no unexpected safety findings from addition of atezolizumab to T-VEC were observed. Limited evidence of antitumor activity was observed.

Background: Treatment options for patients with metastatic pancreatic cancer depend on various factors, including performance status, tumor burden and patient preferences. Metastatic pancreatic cancer is incurable and many systemic treatment options have been investigated over the past decades. This analysis of patterns of practice was performed to identify decision criteria and their impact on the choice of first-line management of metastatic pancreatic cancer. Materials and methods: Members of the Swiss Group for Clinical Cancer Research (SAKK) Gastrointestinal Cancer Group were contacted and agreed to participate in this analysis. Decision trees for the first line treatment of metastatic pancreatic cancer from 9 centers in Switzerland were collected and analyzed based on the objective consensus methodology to identify consensus and discrepancies in clinical decision-making. Results: The final treatment algorithms included 3 decision criteria (comorbidities, performance status and age) and 5 treatment options: FOLFIRINOX, FOLFOX, gemcitabine + nab-paclitaxel, gemcitabine mono and best supportive care. Conclusion: We identified multiple decision criteria relevant to all participating centers. We found consensus for the treatment of young (age below 65) patients with good performance status with FOLFIRINOX. For patients with increasing age and reducing performance status there was a decreasing trend to use gemcitabine + nab-paclitaxel. Gemcitabine monotherapy was typically offered to patients in the presence of comorbidities. For patients with ECOG 3-4, most of the experts recommended BSC. (C) 2020 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Background Although rare, the incidence of BM is increasing due to the improvement in metastatic CRC treatment and longer survival. Genomic analyses of small series revealed that BM can harbor potentially unique driver mutations. We aimed to comprehensively characterize the molecular profile of BM and explore the differences between BM vs other distant metastases (OM) and primary tumors (PT) in CRC. Methods Tumor samples from BM (n = 81), PT (n = 6898) and OM (n = 5918) were analyzed using NGS (TruSEQ on 45 genes or NextSEQ on 592 genes), in situ hybridization and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. Results The most frequently mutated genes in BM were TP53 (80%), APC (73%), KRAS (68%), ARID1A (18%), PIK3CA (13%) and FBXW7 (9%). The most prevalent copy number increase was seen in CDX2 (20%), CCND2 (7%), FLT1 (7%), FLT3 (7%) and FOXO1 (7%). When compared to OM and PT, mutations in KRAS (BM: 68%, OM: 49%, PT: 48%), CDKN2A (5%, 1%, 1%), ERCC2 (2%, 0, 0) and HRAS (1%, 0, 0) were significantly higher in BM (P 17mut/MB) and MSI-high were seen in BM vs PT (2 vs 9% and 1 vs 8%, P=.049 and .031, respectively) but not compared to OM. No RSPO3 nor NTRK1 fusions were seen in BM (n = 5). Female gender was associated with younger age in BM (53.5 vs 62 yr, P=.0014) and OM (58.8 vs 60.2 yr, P Conclusions This is the largest and most extensive profiling study to investigate the molecular makeup of BM and the differences with PT and OM in CRC. Our data show distinct mutations and CNA characterizing BM and lower expression of immune related markers, supporting the rationale to develop tailored approaches to the treatment of this metastatic site. Legal entity responsible for the study The authors. Funding National Cancer Institute grant number P30CA014089, Gloria Borges WunderGlo Foundation-The Wunder Project, Dhont Family Foundation, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Call to Cure Research Fund and Fong Research Project. Disclosure J. Xiu: Full / Part-time employment: Caris Life Sciences. Y. Baca: Full / Part-time employment: Caris Life Sciences. R.M. Goldberg: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. A. Grothey: Travel / Accommodation / Expenses: Caris Life Sciences. A.F. Shields: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. A. Seeber: Advisory / Consultancy: Caris Life Sciences. M.E. Salem: Travel / Accommodation / Expenses: Caris Life Sciences. P.A. Philip: Travel / Accommodation / Expenses: Caris Life Sciences. J.L. Marshall: Advisory / Consultancy: Caris Life Sciences. W..M. Korn: Full / Part-time employment: Caris Life Sciences. H.J. Lenz: Travel / Accommodation / Expenses: Caris Life Sciences. All other authors have declared no conflicts of interest.

Background One-carbon metabolism (1CM) comprises the folate and the methionine cycles and is involved in nucleotide synthesis and methylation. 1CM is known to be crucial in CRC development and progression. However, the impact of its genetic variants on prognosis in mCRC patients has not been clarified yet. We hypothesized that single nucleotide polymorphisms (SNPs) in genes related to the 1CM pathway may predict first-line treatment outcomes in mCRC patients. Methods Genomic DNA from blood samples of patients enrolled in two independent randomized phase III trials, TRIBE and FIRE-3, was genotyped through the OncoArray, a custom array manufactured by Illumina, including approximately 530K SNP markers. The impact on outcome of SNPs in six genes of the 1CM pathway (MTHFR, MTR, MTRR, MAT2A, SHMT, TYMS) was analyzed. Results A total of 451 patients were included. TRIBE FOLFIRI/bevacizumab (bev) arm served as discovery cohort (N = 215, mPFS/OS: 9.7/26.2 months), FIRE-3 FOLFIRI/bev arm as validation (N = 107, mPFS/OS: 11.5/31.4 months) and FOLFIRI/cetuximab arm as control (N = 129, mPFS/OS: 12.8/49.8 months). In the discovery cohort, the overall population carrying the SHMT rs1979277 A/A variant showed a shorter median PFS (8.1 vs 10.3 months) compared to patients with any G alleles both in univariate (HR 2.13; 95%CI, 1.19-3.79; P = 0.007) and in multivariable analysis (HR 2.03; 95%CI, 1.10-3.73; P = 0.023). Additionally, A/A carrier showed a shorter median OS (18.0 vs 27.9 months) both in univariate (HR 1.74; 95%CI, 1.00-3.01; P = 0.045) and in multivariable analysis (HR 2.14; 95%CI, 1.17-3.90; P = 0.013). These findings were validated in overall patients in FIRE-3 bev cohort in median OS (24.9 vs 36 months) both in univariate (HR 2.18; 95%CI, 0.96-4.96; P = 0.049) and in multivariable analysis (HR 3.61; 95%CI, 1.47-8.86; P = 0.005). No significant association was observed in the control arm. Conclusions Our results suggest for the first time that SNPs in the SHMT gene may have a prognostic and predictive value in mCRC patients. These findings may provide novel perspectives on the role of 1CM signaling in CRC and possibly contribute to open novel therapeutic options. Legal entity responsible for the study The authors. Funding This manuscript was partly supported by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation-The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, the Call to Cure Research Fund, and the Fong Research Project. Disclosure F. Loupakis: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck Serono. S. Stintzing: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Lilly; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Merck KGaA; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Sanofi; Honoraria (self), Travel / Accommodation / Expenses: Sirtex Medical; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy, Travel / Accommodation / Expenses: Takeda. F. Battaglin: Travel / Accommodation / Expenses: Amgen; Travel / Accommodation / Expenses: Bayer. M.D. Berger: Travel / Accommodation / Expenses: Astellas Pharma; Research grant / Funding (institution): Merck KGaA. C. Cremolini: Honoraria (self): Amgen; Honoraria (self), Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Lilly; Research grant / Funding (self), Research grant / Funding (institution): Merck. A. Falcone: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Servier; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bayer; Advisory / Consultancy: Bristol-Myers Squibb; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Sanofi. V. Heinemann: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy: Baxalta; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Celgene; Honoraria (self): Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: SERVIER; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Sirtex Medical; Honoraria (self): Taiho Pharmaceutical; Advisory / Consultancy, Travel / Accommodation / Expenses: Bristol-Myers Squibb; Advisory / Consultancy: Halozyme; Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Research grant / Funding (institution): Shire. H.J. Lenz: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bayer; Honoraria (self): Boehringer Ingelheim; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Merck Serono; Advisory / Consultancy: Pfizer; Travel / Accommodation / Expenses: CARIS. All other authors have declared no conflicts of interest.

Background: Disruption of epigenetic regulation in CRC, particularly aberrant histone methylation, has been shown to correlate with poorer survival rates in mCRC. We aimed to highlight the molecular differences between CRC harboring pathological mutations in genes involved in histone modification (KMT family). Methods: Tumors were examined by NextGen sequencing, protein expression, and gene amplification. Tumor mutational burden (TMB) was calculated based on somatic missense mutations (TMB-high ≥ 17 mt/MB) and microsatellite instability (MSI) was evaluated on known MSI loci in the target regions. Chi-square and t-tests were used for comparative analyses. Results: In total, 3621 CRC were examined, and 224 (6.2%) showed a pathological mutation in KMT2A, KMT2C, or KMT2D. Demographics were as follow: F/M 47%/53%, median age 60. KMT mutations were strongly associated with MSI-H (66.5% vs 2.5%, P<.0001), TMB-high (72% vs 3%, P<.0001), BRAF mutation (24.7% vs 7.5%, P<.0001), PD-L1 overexpression (14.2% vs 2.7%, P<.0001), right-sided (11.7% vs 3.4% in left-sided tumors, P = 0.001) and POLE mutations (6.7% vs 0.1%, P<.0001). Furthermore, the rate of mutations among several genes such as ATM, ATRX, BRCA1/2, MLH1, MSH2/6, PMS2 (all P < 0.001) were more frequently seen in the KMT mutated tumors. All remained significant after multiple test correction. When we analyzed MSS tumors and POLE non-mutant tumors excluding MSI-H or POLE mutation, TMB-H remains more frequent in tumors with KMT mutations (8.3% vs 0.9%, P = 0.004), as well as several genes were more frequently mutated in KMT mutated tumors compared to WT tumors (CDC73, 1.7% vs 0.0%, P=.003; KDR, 1.7% vs 0.03%, P=.011; SDHB, 1.7% vs 0.1%, P=.023; PRKDC, 1.7% vs. 0.1%, P=.046; CHEK2, 5.6% vs 1.4%, P=.049). Conclusions: Our findings provide the first exploratory data on KMT genes mutations and their association with clinical and molecular features in CRC, in a large population of patients. Further investigations are warranted to elucidate the association between TMB-H and histone modifications, potentially leading to a better understanding of epigenetic alterations in CRC and to identify novel targets for therapeutic options for CRC patients. Legal entity responsible for the study: Heinz-Josef Lenz. Funding: Has not received any funding. Disclosure: K. Poorman, M. Winerip, W.M. Korn: Employee: Caris Life Sciences. J.L. Marshall: Consultant: Caris Life Sciences. All other authors have declared no conflicts of interest.

Background: HER3 is a tyrosine kinase receptor and involved in cell proliferation and carcinogenesis. Preclinical data suggest that HER3 plays a critical role in the escape mechanism from anti-EGFR directed treatment in metastatic colorectal cancer (mCRC) patients. Additionally, HER3 downstream signaling has shown to stimulate angiogenesis. We therefore hypothesize that variations in genes involved in the HER3 signaling pathway may predict outcome in patients with mCRC treated with first-line FOLFIRI and bevacizumab (bev) or FOLFIRI and cetuximab (cet). Methods: The impact of 4 functional SNPs within the HER3, NRG1, NEDD4 and BTC genes on outcome was evaluated in 585 pts with mCRC treated with either first-line FOLFIRI/bev (n = 293) or FOLFIRI/cet (n = 292) in the randomized phase III FIRE-3 trial. Genomic DNA was extracted from FFPE and the SNPs were analyzed by PCR-based direct sequencing. Results: Baseline characteristics in the FOLFIRI/bev and FOLFIRI/cet cohorts were as follows: female:male ratio=1/3:2/3 in both arms; age >65y (48% and 47%) and median PFS/OS=10.1/23.7 and 9.6/26.5 months. The HER3 rs2271189 SNP showed significant association with PFS. A/A genotype carriers treated with FOLFIRI/bev had a shorter median PFS compared to those harboring any G allele (7.0 vs 10.3 months) in both univariate (HR 1.60, 95% CI 1.09-2.36, p = 0.014) and multivariate analysis (HR 1.64, 95% CI 1.09-2.46, p = 0.018). The same effect on PFS could be seen in patients receiving FOLFIRI/cet. Here again, A/A carriers showed a shorter median PFS than those having any G allele (7.9 vs 10.0 months) in both univariate (HR 1.56, 95% CI 1.13-2.15, p = 0.005) and multivariate analysis (HR 1.40, 95% CI 1.00-1.96, p = 0.05). Conclusions: Our results provide the first evidence that the HER3 polymorphism rs2271189 might serve as a predictive/prognostic marker in pts with mCRC treated with FOLFIRI/bev and FOLFIRI/cet in the first line setting. Targeting HER3 might be a promising approach to further improve treatment options against mCRC and to overcome resistance to both anti-EGFR and anti-angiogenic therapy. Legal entity responsible for the study: Heinz-Josef Lenz. Funding: This work was supported by the National Institute of Health P30CA014089, the Gloria Borges Wunderglo Project, and the Daniel Butler Research Fund. Martin D. Berger received a grant from the Werner and Hedy Berger-Janser foundation for cancer research. Yuji Miyamoto received a grant from the Japan Society for the Promotion of Science (S2606). Ryuma Tokunaga received a grant from the Uehara Memorial Foundation. Disclosure: S. Stintzing: Advisory board member: Amgen, Bayer, Lilly, Merck KgaA, Nordic Pharma, Roche, Sanofi V. Heinemann: Advisory board member: Amgen, Baxalta, Boehringer Ingelheim, Lilly, Merck KgaA, Merrimack, Roche AG, Sanofi, Servier, Sirtex, Taiho. H-J. Lenz: Advisory board member: Merck Serono, Roche. All other authors have declared no conflicts of interest.

The MAPK-interacting kinase 1 (MKNK1) is localized downstream of the RAS/RAF/ERK and the MAP3K1/MKK/p38 signaling pathway. Through phosphorylation MKNK1 regulates the function of eukaryotic translation initiation factor 4E, a key player in translational control, whose expression is often upregulated in metastatic colorectal cancer patients (mCRC). Preclinical data suggest that MKNK1 increases angiogenesis by upregulating angiogenic factors. We therefore hypothesize that variations in the MKNK1 gene predict outcome in mCRC patients treated with first-line FOLFIRI and bevacizumab (bev). A total of 567 patients with KRAS wild-type mCRC in the randomized phase III FIRE-3 and TRIBE trials treated with first-line FOLFIRI/bev (discovery and validation cohorts) or FOLFIRI and cetuximab (cet) (control cohort) were included in this study. Five single-nucleotide polymorphisms in the MAPK signaling pathway were analyzed. AA genotype carriers of the MKNK1 rs8602 single-nucleotide polymorphism treated with FOLFIRI/bev in the discovery cohort (FIRE-3) had a shorter progression-free survival (PFS) than those harboring any C (7.9 versus 10.3 months, Hazard ratio (HR) 1.73, P = 0.038). This association could be confirmed in the validation cohort (TRIBE) in multivariable analysis (PFS 9.0 versus 11.0 months, HR 3.04, P = 0.029). Furthermore, AA carriers in the validation cohort had a decreased overall response rate (25% versus 66%, P = 0.049). Conversely, AA genotype carriers in the control group receiving FOLFIRI/cet did not show a shorter PFS. By combining both FOLFIRI/bev cohorts the worse outcome among AA carriers became more significant (PFS 9.0 versus 10.5 months) in univariable (HR 1.74, P = 0.015) and multivariable analysis (HR 1.76, P = 0.022). Accordingly, AA carriers did also exhibit an inferior overall response rate compared with those harboring any C (36% versus 65%, P = 0.005). MKNK1 polymorphism rs8602 might serve as a predictive marker in KRAS wild-type mCRC patients treated with FOLFIRI/bev in the first-line setting. Additionally, MKNK1 might be a promising target for drug development.

Background Tri-phosphorylated trifluridine (FTD) incorporation into DNA is TAS-102's main anti-tumor action. We tested whether genetic polymorphisms in homologous recombination (HR) and cell cycle checkpoint pathway for DNA repair is associated with outcomes in refractory metastatic colorectal cancer (mCRC) patients treated with TAS-102. Patients and methods We analyzed genomic DNA extracted from 233 samples of three cohorts: an evaluation cohort of 52 patients receiving TAS-102, a validation cohort of 129 patients receiving TAS-102 and a control cohort of 52 patients receiving regorafenib. Single nucleotide polymorphisms of genes involved in HR (ATM, BRCA1, BRCA2, XRCC3, FANCD2, H2AX, RAD51) and cell cycle checkpoint (ATR, CHEK1, CHEK2, CDKN1A, TP53, CHE1, PIN1, PCNA) were analyzed by PCR-based direct sequencing. Results In univariate analysis for the evaluation cohort, patients with any G allele in ATM rs609429 had longer overall survival (OS) than those with the C/C variant (8.7 vs. 4.4 months, HR 0.37, 95% CI: 0.14-0.99, P = 0.022). Patients carrying any A allele in XRCC3 rs861539 had significantly longer progression-free survival (PFS) (3.8 vs. 2.3 months, HR 0.44, 95% CI: 0.21-0.92, P = 0.024) and OS (15.6 vs. 6.3 months, HR 0.25, 95% CI: 0.08-0.79, P = 0.012) than those with the G/G variant. In multivariable analysis, ATM rs609429 remained significant for OS (P = 0.020). In the validation cohort, patients having ATM rs609429 with any G allele showed longer OS and PFS; the G/A variant in XRCC3 rs861539 showed longer OS, though without statistical significance. Conclusion Genetic variants in the HR pathway may predict clinical outcome in mCRC patients receiving TAS-102.

A recent genome-wide association study identified seven single-nucleotide polymorphisms (SNPs) in region 16q24, near the Forkhead box-F1 ( FOXF1 ) gene, which confer susceptibility to esophageal adenocarcinoma. We examined whether these SNPs are associated with clinical outcomes in gastric cancer (GC) patients in Japan and the United States. A total of 362 patients were included in this study: 151 Japanese GC patients treated with first-line S1 plus CDDP (training cohort) and 211 GC patients from Los Angeles County (LAC; validation cohort). Genomic DNA was isolated from whole blood or tumor tissue and analyzed by PCR-based direct DNA sequencing. Cox proportional hazard regression analyses were used to assess relationships between FOXF 1 SNPs and progression-free survival (PFS) and overall survival (OS). FOXF1 rs3950627 was significantly associated with survival in both the training and validation cohorts. Japanese patients with the C/C genotype had a longer PFS (median 8.2 vs 5.3 months, hazard ratio (HR) 1.44, P =0.037) and OS (median 16.4 vs 12.2 months, HR 1.44, P =0.043) compared to patients with any A allele. Similarly, LAC patients with the C/C genotype had improved OS (3.9 vs 2.3 years, HR 1.5, P =0.022). Subgroup analyses showed these associations were specific to male patients and primary tumor subsite. Our findings suggest that FOXF1 rs3950627 might be a promising prognostic marker in GC patients.

We analyzed associations between CXCR4/CXCL12 single-nucleotide polymorphisms and outcomes in metastatic colorectal cancer (mCRC) patients who underwent first-line bevacizumab-based chemotherapy. A total of 874 patients were included in this study: 144 treated with bevacizumab and FOLFOX or XELOX (training cohort), 653 treated with bevacizumab and FOLFIRI or FOLFOXIRI (validation cohort A or B) and 77 treated with cetuximab-and oxaliplatin-based regimens (control cohort). One CXCR4 polymorphism (rs2228014) and two CXCL12 polymorphisms (rs1801157 and rs3740085) were analyzed by PCR-based direct sequencing. Patients with a C/C genotype had a prolonged progression-free survival (PFS) compared with those with any T allele (P = 0.030) in the training cohort. Similarly, patients with the C/C genotype had a superior PFS in the validation cohorts, but not in the control cohort. Our findings suggest that a common genetic variant, CXCR4 rs2228014, could predict PFS and may guide therapeutic decisions in mCRC patients receiving first-line bevacizumab-based chemotherapy.

HIC1 (Hypermethylated in Cancer 1) is a transcription repressor, which cooperates with several partners to suppress the expression of multiple target genes. Among HIC1 targets, SIRT1 (Sirtuin1) plays a critical role in promoting the nucleotide excision repair (NER) pathway, which is the main oxaliplatin-induced damage repair system. HIC1 expression might be influenced by the number of variations in a tandemly-repeated sequence, situated close to the promoter region. We tested the hypothesis that variable number of tandem repeat (TR) in HIC1 will be associated with outcome in metastatic colorectal cancer patients (mCRC pts) receiving 1st-line chemotherapy with oxaliplatin. This study enrolled 3 independent cohorts. Pts treated with FOLFOXIRI + bevacizumab in the phase III TRIBE study served as a training set (TRIBE-B cohort, n = 218). Pts receiving FOLFOXIRI + bevacizumab in the phase II MOMA study served as a validation set (MOMA cohort, n = 176). Pts treated without oxaliplatin (FOLFIRI + bevacizumab) in the TRIBE study served as a control set (TRIBE-A cohort, n = 215). Genomic DNA was isolated from blood samples. Variations in the number of TR were analyzed by PCR and Gel electrophoresis, and tested for the association with PFS and OS. Main patients characteristics' were the following: TRIBE-A; M/F 60/40%, median age 60, TRIBE-B; M/F 60/40%, median age 60, MOMA; M/F 57/43%, median age 61. Median follow-up times were 49.9, 48.0, and 25.3 months, respectively. Pts with number of TR ≤4 or ≥5 were 90/10% (TRIBE-A), 91/9% (TRIBE-B), and 95/5% (MOMA), respectively. In the training cohort, pts with TRs ≥5 showed a significantly shorter PFS compared to those with TRs ≤4 (9.5 vs. 11.6 mo, HR 1.93, P = 0.012), which retained statistical significance in multivariate analysis (HR 2.00, 95%CI: 1.13-3.54, P = 0.018). This preliminary association was confirmed in the validation cohort, and pts with TRs ≥5 showed a worse PFS compared to others (7.9 vs. 9.8 mo, HR 1.85, P = 0.044). This correlation was not observed in the control cohort. Our findings suggest that variable number of TRs in HIC1 could be a predictive marker for oxaliplatin-containing chemotherapy in mCRC pts.

Using approved methods, circulating tumor cells (CTCs) are only isolated from blood in 30%–50% of metastatic colorectal cancer (mCRC) patients. We previously validated a technique to isolate circulating tumor cells (CTCs) in a cohort of mCRC patients by combining immunomagnetic enrichment of EpCAM + /CD45 − cells with qRT-PCR amplification of CK20 and survivin expression. Here, we examined the prognostic utility of CTC epithelial–mesenchymal transition (EMT) and stem cell gene expression. An 8 ml blood sample was collected from 78 consecutive mCRC patients before treatment with investigational and standard chemotherapeutics. The mRNA expression of EMT ( PI3Kα , Akt-2, Twist1 ) and stem cell ( ALDH1 ) markers was measured. Associations between CTC gene expression and progression-free survival (PFS) and overall survival (OS) were determined using Cox regression models. Among patients without CK20 or survivin -expressing CTCs ( n =17), 55% had expression of ALDH1 , PI3Kα and/or Akt-2 . Patients with positive CTC Akt-2 expression had a significantly shorter median PFS (3.0 versus 4.0 months) compared with those without CTC Akt-2 expression in univariable (hazard ratio (HR)=1.61; log-rank P =0.034) and multivariable analyses (HR=1.70; adjusted P =0.041). In univariable analysis, CTC ALDH1 expression was associated with shorter OS (10.0 versus 38.6 months; HR=2.04, P =0.021). Patients with CTCs expressing ALDH1 , PI3Kα and/or Akt-2 had a significantly inferior PFS (3.0 versus 7.7 months; HR=1.88, P =0.015) and OS (10.0 versus 26.8+ months; HR=2.25, P =0.050) in univariable, but not multivariable, analysis. Conclusions: CTC Akt-2 expression may serve as a clinically useful prognostic marker in mCRC patients and warrants further evaluation in prospective trials.

Immunomodulator-targeting therapies are under development in gastric cancer (GC). However, the role of genes modulating antitumor immunity in GC remains poorly understood. We investigated the association of variations in genes involved in immunomodulatory pathways with overall survival (OS) in locoregional GC patients. Extracted genomic DNA was analyzed for 35 functional single-nucleotide polymorphisms in genes, PDCD1, CD274, CTLA4, FOXP3, LAG3, ADORA2A, NT5E and IDO1, in 162 Japanese patients as discovery set and 277 US patients as validation set. The C allele of PDCD1 rs10204525 had univariate and multivariable associations with shorter OS in Japanese cohort (P = 0.015, P = 0.043, respectively). In US cohort the C allele predicted worse OS (P = 0.007). Univariate and multivariable analyses revealed IDO1 rs9657182 associated with OS in the Japanese cohort; moreover, the association was confirmed in the US cohort. Genetic predisposition of the host in the immunomodulators may serve as a prognostic biomarker in patients with locoregional GC.

The Hippo pathway is involved in colorectal cancer (CRC) development and progression. The Hippo regulator Rassf1a is also involved in the Ras signaling cascade. In this work, we tested single nucleotide polymorphisms within Hippo components and their association with outcome in CRC patients treated with cetuximab. Two cohorts treated with cetuximab plus chemotherapy were evaluated (198 RAS wild-type (WT) patients treated with first-line FOLFIRI plus Cetuximab within the FIRE-3 trial and 67 Ras WT patients treated either with first-line mFOLFOX6 or SOX plus Cetuximab). In these two populations, Rassf1a rs2236947 was associated with overall survival (OS), as patients with a CC genotype had significantly longer OS compared with those with CA or AA genotypes. This association was stronger in patients with left-side CRC (hazard ratio (HR): 1.79 (1.01–3.14); P =0.044 and HR: 2.83 (1.14–7.03); P =0.025, for Fire 3 and JACCRO cohorts, respectively). Rassf1a rs2236947 is a promising biomarker for patients treated with cetuximab plus chemotherapy.

Genetic variations in genes regulating tumor-associated macrophages (TAMs) may serve as a predictor of bevacizumab efficacy in metastatic colorectal cancer. In addition, our study suggests that some TAM-related gene variations may be associated with clinical outcome of bevacizumab treatment in KRAS status-dependent manner.Background: Tumor-associated macrophages (TAMs) with the M2-like phenotype are regulated by mainly NF-kB pathway including TBK1, which can influence tumor progression by secretion of proangiogenic factors such as vascular endothelial growth factor. The CCL2/CCR2 axis, histidine-rich glycoprotein (HRG), and placenta growth factor (PIGF) play a critical role in the polarization of M1/M2 phenotypes and the recruitment of TAMs to tumor microenvironment. We therefore hypothesized that variations in genes involved in regulating TAMs may predict clinical outcomes of bevacizumab treatment in patients with metastatic colorectal cancer (mCRC).Patients and methods: We analyzed genomic DNA extracted from samples of patients receiving bevacizumab plus FOLFIRI as a first-line treatment using PCR-based direct sequencing. Twelve functional single-nucleotide polymorphisms in eight genes (CCL2, CCR2, HRG, PIGF, NFKB1, TBK1, CCL18, and IRF3) were tested for associations with clinical outcomes in a discovery cohort of 228 participants in TRIBE trial (NCT00719797), then validated in 248 KRAS exon2 (KRAS) wild-type participants in FIRE3 trial (NCT00433927). FIRE3-cetuximab cohort served as a negative control.Results: TBK1 rs7486100 was significantly associated with overall survival in 95 KRAS wild-type patients of TRIBE cohort in univariate analysis and had a strong trend in multivariable analysis; furthermore, the association of the T allele was observed for progression-free survival (PFS) in both univariate and multivariable analyses in FIRE3-bevacizumab but not cetuximab cohort. CCL2 rs4586, CCL18 rs14304, and IRF3 rs2304205 had univariate and multivariable correlations with PFS in KRAS mutant patients of the TRIBE cohort, whereas they had no correlations in KRAS wild-type patients of the TRIBE cohort. No association was seen in control cohort.Conclusions: Our study demonstrates for the first time that variations in genes regulating TAMs-related functions are significantly associated with clinical outcomes in mCRC patients treated with bevacizumab-containing chemotherapy. These results also suggest that some TAM-related gene variations may predict outcomes of bevacizumab treatment in KRAS status-dependent manner.

Estrogen has been shown not only to reduce the incidence of colorectal cancer but also gastric cancer (GC). Polymorphisms in estrogen receptor β gene, ESR2, correlate with colorectal cancer survival. To better understand the role of ESR2 in GC, genomic DNA extracted from 169 Japanese patients and 172 patients from Los Angeles County (LAC) was analyzed for association of overall survival (OS) with three ESR2 polymorphisms, which are of biological significance using multivariable Cox proportional hazard regression. ESR2 rs1271572 (C>A) and rs3020443 (T>G) had univariate and multivariable associations with OS in the Japanese cohort, whereas the C allele of ESR2 rs2978381 (T>C) predicted favorable OS in the Japanese cohort but worse OS in the LAC cohort. The interaction term of the ESR2 rs2978381 and cohort group reached statistical significance. Our study provides evidence that genetic variations in ESR2 gene are significantly associated with survival in patients with locally advanced GC.

Colorectal brain metastases (BM) are rare (1-2%) and a late-stage disease manifestation. Molecular mechanisms for BM development are not well understood. We tested whether variants within genes involved in overcoming the blood-brain barrier (BBB) are associated with BM susceptibility and survival in patients with BM. Germline single-nucleotide polymorphisms (SNPs, n=17) in seven genes (CXCR4, MMP9, ST6GALNAC5, ITGAV, ITGB1, ITGB3, KLF4) were analyzed from germline DNA in patients with resected BM (n=70) or no clinical evidence of BM after at least 24 months from diagnosis (control group, n=45). SNPs were evaluated for association with BM susceptibility and overall survival (OS) from BM diagnosis. ST6GALNAC5 rs17368584 and ITGB3 rs3809865 were significantly associated with BM susceptibility. In multivariable analysis adjusted for patient characteristics, KLF4 rs2236599, ITGAV rs10171481, ST6GALNAC5 rs1883778, CXCR4 rs2680880 and ITGB3 rs5918 were significant for OS. This study shows for the first time that variants within genes involved in breaching the BBB are associated with BM susceptibility and survival. These findings warrant further validation to develop better screening guidelines and to identify novel therapy targets for patients with BM.