bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular emboli as a prognostic factor in patients with stage III colorectal cancer undergoing radical surgery.
Authors: Pei Haiping , Pei Qian , Zhu Hong , Tan Fengbo
Published: March 2017

570

Background: The significance of vascular emboli (VE) in stage III colorectal cancer (CRC), the mechanism of their formation and their therapy strategy remain obscure demanding enhanced research. Methods: Data from 323 consecutive patients (192 non-VE, 131 VE) receiving radical surgery and adjuvant chemotherapy in our institution between Jan. 2009 and Nov. 2014 were retrospectively collected. The follow-up deadline was Feb. 2016. Potential prognostic risk factors were tested using univariate and multivariate survival analyses. mRNAs differentially expressed between VE and non-VE stage III CRC were analyzed using Agilent Gene Expression oligo-microarrays (Version 6.5). Patient-derived xenograft (PDX) nude mouse models were constructed to evaluate the efficacy of adjuvant chemotherapy plus targeted drugs (cetuximab or bevacizumab) on VE and non-VE stage III CRC. Results: VE was significantly associated with gross tumor morphology (p = 0.001), histologic type (p < 0.001), lymph node status (p < 0.001), sub-class of stage III (p =0.001), and serum CA199 level (p = 0.022). VE and lymph node status were independent risk factors for overall survival (OS) (p < 0.001, p = 0.008) and disease-free survival (DFS) (p < 0.001, p = 0.007). The median OS and DFS in VE stage III CRC patients were 38 months and 24 months, respectively. Compared with pericarcinous tissue, 809 genes (396 up-, 413 down-regulated) were differently expressed in no-VE tissue, and 1513 genes (898 up-, 615 down-regulated) in VE tissue. The top ten up-regulated protein-coding genes in VE stage III CRC were ITGBL1 (p<0.001), PROCR (p<0.001), CENPW (p<0.001), ZNF485 (p<0.001), VEGFA (p<0.001), DSG3 (p<0.001), CYP24A1 (p=0.001), COL10A1 (p=0.001), HIST3H2A (p=0.001)and PSAT1 (p=0.002). Conclusions: VE appears to be an independent risk factor for the prognosis of stage III CRC patients treated with radical surgery and adjuvant chemotherapy. Differently regulated genes seem to be involved in the formation and progress of VE. The therapy scheme should be more individualized taking into account the VE status.
Safety and tolerability profile of raltitrexed in Chinese patients with locally advanced or recurrent and metastatic colorectal cancer: A phase IV trial.
Authors: Qin Shukui , Li Jin , Liu Yunpeng , Chen Yin , Xu Nong , Xu Jianming , Bai Yuxian , Huang Jianjin , Liang Jun , Zheng Leizhen
Published: March 2017

571

Background: The clinical benefit and safety profile associated with raltitrexed in patients with advanced colorectal cancer (CRC) were established. However, safety information of raltitrexed in Chinese patients is rare. We aimed to assess the safety of raltitrexed monotherapy in Chinese patients with advanced CRC. Methods: This single-arm, open-label, multi-center, phase IV trial was conducted in 13 hospitals across China between March 2011 and July 2014. Patients with locally advanced or recurrent and metastatic colorectal cancer (mCRC) received raltitrexed (3mg/m2) as a 15-min infusion on day 1, every 21 days. Treatment was continued until disease progression or unacceptable toxicity. The primary objective was safety and the secondary objectives were disease control rate (DCR), progression free survival (PFS) and overall survival (OS). Results: A total of 215 patients were enrolled, and the safety was assessed in 205 patients. 92 patients had 394 Adverse events (AEs) during the study. A majority (94.4%) of AEs was grade 1/2 and only 5.3% was grade 3/4. Most AEs were either resolved spontaneously or treated successfully with standard medical interventions. Severe AEs were reported by 5.8% (12/205) of patients. The most commonly reported AEs were increase of transaminases, leukopenia, nausea and vomiting. Serum transaminase elevation, an important safety concern associated with raltitrexed therapy, were transient, self-limiting and manageable. No deaths were reported during the study. The DCR in second-line and third-line therapy by raltitrexed reached 49.5% and 37.4%, respectively. The median PFS in second-line and third-line therapy was 1.43 months and 1.47 months, respectively. The median OS was 13.6 months and 8.9 months, respectively. Conclusions: Raltitrexed monotherapy is a safe, well-tolerated and convenient therapy option for Chinese patients. The treatment-associated toxicity is manageable and does not lead to discontinuation of treatment in oncology practice.
Prevalence of pathogenic mismatch repair mutations in an Irish cohort after clinical and molecular pre-selection.
Authors: Farrell Michael P. , Hearty Kian P , Gallagher David James
Published: March 2017

574

Background: Lynch syndrome (LS) is a highly penetrant, autosomal dominant multi-system disorder characterised by an inherited predisposition to a range of cancers. LS is caused by germline mutations in one of the mismatch repair (MMR) genes ( MSH2, MLH1, MSH2 and PMS2). Tumours from patients with LS have characteristic features resulting from the underlying molecular involvement of defective MMR, that is, the presence of microsatellite instability (MSI) and the absence of MMR protein expression by immunohistochemistry (IHC) corresponding to the mutated gene. MMR mutations are identified in approximately 60% of microsatellite instability-high (MSI-H) cancer patients fulfilling clinical criteria for LS. We sought to define the prevalence of pathogenic MMR mutations among an Irish cohort following molecular pre-selection with IHC/MSI and BRAF/MLH1 hypermethylation testing Methods: The results of diagnostic MMR genetic screening conducted on individuals following genetic counselling, in the Clinical Cancer Genetics Departments of the Mater Campus between January 2011 to September 2016, were correlated with ages at diagnoses and genetic test result. Results: This clinic-based cohort of affected individuals (n = 35) consisted of 22 females and 13 males. Median age at first cancer diagnosis was 44 years, range 23 to 86, standard deviation 12.3. Deleterious mutations were identified in 16 patients (45.7%), 6 in MLH1, 3 in MSH2, 2 in MSH6 and 5 in PMS2. Thirteen patients had a normal genetics test result (37.1%). Variants of uncertain significance (VUS) were identified in 6 patients (17.1%). With additional molecular testing and co-segregation studies in one family an MLH1 VUS was reclassified as likely pathogenic, facilitating pre-symptomatic testing for family members. This reclassification gives a revised prevalence of pathogenic mutation in this cohort of 48.5% and a VUS detection rate of 14.2%. Conclusions: We report a higher than expected VUS rate following molecular preselection of patients likely to have LS. No mutations were identified in 37% of patients supporting the presence of alternative negative regulators of the MMR pathway.
Aspirin in the prevention of colorectal cancer recurrence.
Authors: Kumari Nimisha , Mandaliya Hiren A. , Evans Tiffany , McElduff Patrick , Oldmeadow Christopher , Day Fiona Lee
Published: March 2017

578

Background: Aspirin use reduces the incidence of colorectal adenomas, and of colorectal cancer (CRC) in patients with hereditary non-polyposis colorectal cancer. There is limited data on the effect of aspirin in the secondary prevention of CRC, currently the subject of multiple prospective randomized trials. We aimed to test the hypothesis that aspirin reduces disease recurrence in patients diagnosed with early CRC at a high volume, single institution. Methods: A retrospective analysis was conducted of all patients (pts) treated with at least one cycle of adjuvant chemotherapy for stage II or III CRC over a 5yr period (2009–13). Patients with synchronous CRC were excluded. Aspirin use at the onset of adjuvant chemotherapy was sourced from the universal electronic recording of concomitant medications. Kaplan-Meier curves and the log-rank test were used to compare crude relapse free survival (RFS) between pts who were using aspirin to those who were not. Propensity score analyses was used to balance the groups according to potentially confounding variables: tumor site (colon vs rectum), tumor stage (II vs III), and adjuvant chemotherapy (oxaliplatin-based vs no oxaliplatin). The propensity scores were estimated through a multivariable logistic regression model, and a Cox proportional hazards model used to assess the effect of aspirin use on RFS in the sample weighted according to the inverse probability of receiving aspirin. Results: A cohort of 231 pts met eligibility criteria; median age 64yr; 50% colon, 50% rectal; 19% stage II, 81% stage III; 35% received oxaliplatin; 13% (n=31) were using aspirin and 26% of pts (n=61) developed CRC recurrence in median follow-up of 2.9yr. Unadjusted RFS was not significantly different for those using aspirin (log rank p value 0.88). There was good overlap in propensity scores for the two groups. The hazard ratio for RFS with aspirin use from the weighted Cox regression model was 1.2 (95% CI 0.62-2.29), indicating no statistically significant effect of aspirin on CRC relapse. Conclusions: In this single institution series, we did not find evidence that aspirin use at onset of chemotherapy had an effect on CRC relapse free survival. These results do not exclude an aspirin effect that is modest or restricted to select CRC subgroups.
Evaluating collagen neoepitopes as pharmacodynamic biomarkers of GS-5745, an MMP9 inhibitor, in advanced gastric cancer.
Authors: Brachmann Carrie Baker , Zhang Yafeng , Zavodovskaya Marianna , Hu Jing , Maltzman Julia D. , Smith Victoria , Xiao Yuanyuan , Patterson Scott D
Published: March 2017

58

Background: Cancer is characterized by continuous remodeling of the extracellular matrix (ECM) through a process of degradation and replacement of ECM components, such as collagens. Matrix-metalloproteinase 9 (MMP9) is involved in this remodeling and its inhibition is hypothesized to reduce ECM turnover. MMP9 expression is limited in healthy tissues, but high in tumor epithelia, infiltrating inflammatory cells and fibroblasts. Collagen I and III fragments (neoepitopes C1M and C3M) in blood may provide a measure of tumor MMP activity (BMC Cancer 13:554. 2013). GS-5745 is a monoclonal antibody that specifically inhibits MMP9 and has shown efficacy in combination with chemotherapy in advanced gastric cancer in an ongoing ph I study of multiple cancer types (NCT01803282). Exploratory analyses of collagen neoepitopes from patients in the gastric cancer cohort are reported here. Methods: C1M and C3M were measured by ELISA in serum samples (Nordic Bioscience). Healthy volunteer (HV) samples were age-matched. Pharmacodynamic evaluation and association with clinical outcomes were assessed by non-parametric tests; nominal p-values are reported. Results: Pharmacodynamic Evaluation (Table):Baseline (BL) levels of C1M and C3M were higher in gastric cancer patients than HVs. C1M and C3M displayed a pharmacodynamic response after 1 treatment cycle. Inhibition of C1M continued to decline with treatment. Association with Best Overall Response:There was no association of BL C1M or C3M level with best overall response. 13 of 20 responders had decreased C1M at C2D1; however, of the 3 patients with progressive disease, none had an on-treatment decrease in C1M. Conclusions: These data suggest an association between circulating C1M as a pharmacodynamic biomarker of GS-5745 in combination with chemotherapy in gastric cancer patients. Circulating neoepitope biomarkers will continue to be explored in all ongoing and future oncology GS-5745 studies. Clinical trial information: NCT01803282. [Table: see text]
Blood-based genomic profiling of circulating cell-free tumor DNA (ctDNA) in 1397 patients (pts) with colorectal cancer (CRC).
Authors: Strickler John H. , Banks Kimberly C. , Nagy Rebecca J , Lanman Richard B. , Talasaz AmirAli , Corcoran Ryan Bruce , Kopetz Scott
Published: March 2017

584

Background: ctDNA is shed into the bloodstream by tumor cells throughout the body, offering a non-invasive means of genomic testing, and a way to detect heterogeneous, subclonal genomic alterations present in distinct tumor lesions within an individual pt. However, a broad comparison of mutation prevalence in CRC ctDNA versus CRC tumor tissue has not yet been performed. Methods: ctDNA from 1397 CRC pts was analyzed using a CLIA-certified digital sequencing assay (Guardant360, Guardant Health) capable of detecting single nucleotide variants (SNV) in up to 70 genes, as well as selected insertions/deletions, amplifications, and fusions. Subclonal mutations were defined as mutations with mutant allele fractions (MAF) ≤ 50% of the greatest somatic MAF in the sample. Frequencies of mutations detected were compared to two large tissue-based sequencing databases (TCGA and NHS/HPS). Results: 1500/1772 (85%) tests had at least one genomic alteration (1397 unique pts). The most common SNV mutations included TP53 (62%), APC (47%), KRAS (39%), PIK3CA (17%), EGFR (11%), SMAD4 (11%), and BRAF (11%); these frequencies were comparable to rates in TCGA and NHS/HPS. In contrast, EGFR extracellular domain (ECD) mutations (42 pts) and JAK2 V617F mutations (16 pts) detected in ctDNA were not seen in tissue sequencing, reflecting acquired resistance to EGFR antibodies and clonal hematopoiesis of indeterminate clinical potential, respectively. 88% of pts with ECD mutations had at least one additional non-ECD resistance alteration detected in ctDNA (range 1-9, median 2.6), including KRAS, NRAS, BRAF, MAP2K1, MET and ERBB2. EGFR mutations were most likely to be detected as subclonal (86%), while mutations most likely to be clonal included KRAS (71%), TP53 (65%), BRAF (65%), and APC(63%). In 84 pts with serial monitoring, 87% had either gain (61%) or loss (63%) of clones over time. Conclusions: Blood-based genomic profiling can effectively detect common genomic alterations in CRC at comparable frequencies as observed in tissue and provide novel insights into tumor clonality and clonal dynamics. Clinical trials to target EGFR ECD mutations may be limited by the multiplicity of resistance mechanisms in each pt.
RAS heterogeneity as a prognostic marker in metastatic colorectal cancer.
Authors: Loree Jonathan M. , Lam Michael , Morris Jeffrey , Overman Michael J. , Raghav Kanwal Pratap Singh , Eng Cathy , Dasari A. , Kee Bryan K. , Fogelman David R. , Wolff Robert A. , Jiang Zhiqin , Davis Jennifer S , Shaw Kenna Rael , Broaddus Russell , Routbort Mark , Luthra Rajyalakshmi , Maru Dipen M. , Menter David , Meric-Bernstam Funda , Kopetz Scott
Published: March 2017

586

Background: The impact of intratumor heterogeneity on prognosis in metastatic colorectal cancer (mCRC) is unclear, however relative variant allele frequency (rVAF) of key mutations within a tumor may impact outcomes. Therefore, we sought to determine whether rVAF of RAS ( KRAS & NRAS) mutant (mt) clones impacts overall survival (OS) in mCRC patients (pts). Methods: Using a next generation sequencing panel of 201 cancer related genes, we tested 200 mCRC tumors / matched normals. Mutations, indels, and copy number variant (CNV) information were obtained. An rVAF of RAS clones was determined by dividing RAS mt VAF by the VAF of the mutated gene with the highest allele frequency. This truncal gene served as a marker of the total malignant population in a specimen. Pts were stratified at an rVAF of 50%. OS was compared with Kaplan-Meier curves, the log-rank test, and Cox regression. We assessed the impact of CNV on our findings by correcting the rVAF for CNVs in RASand truncal mutations. Results: Of 200 pts, 15% had RAS mt rVAF < 50%, 40.5% had rVAF ≥ 50%, and 44.5% were RAS wild type (WT). Age, gender, MSI status, histology, and stage at diagnosis were similar between groups. More RAS WT pts had BRAF mutations (19.1% vs 1.2% and 3.3%, P< 0.0001), left sided (78.7% vs 56.8% and 60%, P= 0.02), or poorly differentiated tumors (27.3% vs 8.6% and 13.3%, P= 0.003) compared to pts with rVAF ≥ 50% or rVAF < 50%, respectively. Mean coverage was 807x for RAS and 602x for truncal mutations. OS was better in pts with an rVAF < 50% compared to pts with rVAF ≥ 50% regardless of whether rVAF was corrected for CNV (HR 0.6; 95% CI 0.39-0.93, P =0.029) or not (HR 0.48; 95% CI 0.31-0.82, P= 0.010). mOS for pts with WT, rVAF < 50% and rVAF ≥ 50% tumors were 65.8, 55.7, and 38.6 months ( P= 0.0025). In multivariate models controlling for stage at diagnosis and BRAF mutation, pts with rVAF < 50% (HR 1.75; 95% CI 1.03-2.97, P = 0.04) and rVAF ≥ 50% (HR 2.46; 95% CI 1.66-3.65, P< 0.0001) had worse OS compared to WT pts. When rVAF was used as a continuous variable, every 1% increase in rVAF RAS mt resulted in a 1% increased hazard of death ( P <0.0001). Conclusions: Our findings suggest that clonal proportion of a tumor with a RAS mutation may impact OS and suggest the prognostic impact of RAS mutations is not an “all or none” phenomenon.
Cancer stemness inhibition and chemosensitization: Phase 1b/II study of cancer stemness inhibitor napabucasin (BBI-608) with FOLFIRI +/- bevacizumab (Bev) administered to colorectal cancer (CRC) patients (pts).
Authors: Bendell Johanna C. , O'Neil Bert H. , Starodub Alexander , Jonker Derek J. , Halfdanarson Thorvardur Ragnar , Edenfield William Jeffery , El-Rayes Bassel F. , Hubbard Joleen Marie , Pitot Henry C. , Hobday Timothy J. , Li Youzhi , Gao Yuan , Grothey Axel , Borodyansky Laura , Li Chiang
Published: March 2017

593

Background: Cancer stem cells or stemness-high cancer cells are considered to be fundamentally important for resistance to therapy, recurrence and metastasis. Napabucasin, a first-in-class cancer stemness inhibitor in clinical development, suppresses cancer stemness by targeting Stat3-driven gene transcription. Preclinical studies suggest that napabucasin sensitizes heterogeneous cancer cells to chemotherapeutics and targeted agents. Methods: This study was performed to assess signs of anti-tumor activity of napabucasin in combination with FOLFIRI +/- Bev in CRC pts. Napabucasin was administered at 240 mg BID. FOLFIRI (5-FU 400 mg/m2 bolus with 2400 mg/m2, irinotecan 180 mg/m2, and leucovorin 400 mg/m2 infusion) +/- Bev 5 mg/kg was administered biweekly until disease progression or other discontinuation criterion. pSTAT3 status of archival tumor tissue was assayed by immunohistochemistry using the H-score with pSTAT3highdefined as ≥ 5% pSTAT3+ cancer cells and tumor stroma staining at ≥ 2+ intensity. Results: 63 CRC pts with an average of >2 prior therapy lines were enrolled. No pharmacokinetic interactions or dose-limiting toxicity was observed. Most common adverse events (AEs) included grade 1/2 diarrhea, nausea, vomiting and fatigue. 25 pts had grade 3 AEs, including diarrhea (14), fatigue (4), dehydration (2), electrolyte imbalance (4), nausea (1) and weight loss (1) resolved with dose reduction and supportive care. Among 63 pts enrolled who received RECIST evaluation, disease control (CR+PR+SD) was observed in 49 pts (86%) with an overall response (CR+PR) of 28% with 1 pt achieving CR. Clinical trial information: NCT02024607. Conclusions: This phase Ib/II study suggests napabucasin may sensitize chemorefractory CRC to FOLFIRI +/- Bev. Encouraging signs of synergistic activity between napabucasin and FOLFIRI was observed in CRC pts regardless of pSTAT3 status.[Table: see text]
Tumor budding is a strong predictor of disease-free survival in stage II colorectal cancer: Validation study based on the International Tumor Budding Consensus Conference (ITBCC) recommendations.
Authors: Dawson Heather , Assarzadegan Naziheh , Riddell Robert , Kirsch Richard , Blank Annika , Zlobec Inti , Lugli Alessandro
Published: March 2017

594

Background: Tumor budding, defined as single tumor cells or small clusters of four or less tumor cells at the invasive front, has been shown to be a strong and independent prognostic factor in colorectal cancer (CRC). However, widespread reporting of tumor budding has been held back largely due to a lack of consensus on scoring methods. 23 experts from the United States, Canada, Japan and Europe participated at the International Tumor Budding Consensus Conference (ITBCC), held in April 2016 in Bern, Switzerland, and agreed on a set of recommendations for assessing and reporting tumor budding in CRC. The aim of this study was to validate the method proposed by ITBCC in the clinically relevant scenario of Stage II CRC. Methods: In 151 Stage II CRC patients, tumor budding was assessed on scanned H&E-stained slides in a single hot spot measuring 0.785mm2. Cutoffs as defined by ITBCC were used: Low (Bd1): 0-4 buds, intermediate (Bd2): 5-9 buds, high (Bd3): ≥ 10 buds. Statistical analysis for associations with clinicopathological features, disease free survival (DFS) and overall survival (OS) was performed. Results: The average number of buds/hotspot was 6.8 (median 5.0). 43.1% of cases were Bd1, 27.2% Bd2 and 29.8% Bd3. Tumor budding as a continuous variable was associated with extramural venous invasion (EMVI) (p = 0.05). Tumor budding was associated with poorer OS in univariate analysis (p = 0.0386, HR 1.048, 95%CI 1.002-1.095). For 3- and 5- year DFS, Bd3 was associated with significantly worse survival in comparison with Bd1/2 (p = 0.0031 and p = 0.0025, respectively). In multivariate analysis adjusting for pT, tumor grade and EMVI, tumor budding retained its adverse prognostic effect for DFS (p = 0.006, HR 3.293, 95%CI 1.66-6.53). Conclusions: This study provides promising data on tumor budding assessed by the ITBCC method in the Stage II scenario. Especially Bd3 shows a detrimental adverse impact on DFS in comparison to Bd1/Bd2. Based on the ITBCC, tumor budding has the potential to strongly affect the management of Stage II CRC patients and should be therefore included in reporting guidelines and staging systems in CRC.
Effect of JAK2 SNP rs2274472 on outcome for mCRC patients treated with first-line FOLFIRI and bevacizumab: Data from FIRE-3 trial.
Authors: Berger Martin D. , Stintzing Sebastian , Heinemann Volker , Yang Dongyun , Cao Shu , Ning Yan , Okazaki Satoshi , Miyamoto Yuji , Suenaga Mitsukuni , Schirripa Marta , Gopez Roel , Hanna Diana L. , Zhang Wu , Lenz Heinz-Josef
Published: March 2017

595

Background: The Jak2 (Janus kinase 2) / Stat5a (signal transduction and activators of transcription 5a) pathway plays a key role in regulating cell survival, proliferation and immune function. JAK2 is a non-receptor tyrosine kinase which is stimulated by a variety of cytokines. Moreover the JAK2 / STAT5 axis is activated by hypoxia and involved in regulating angiogenesis. We therefore hypothesize that variations in the JAK2 gene may predict outcome in patients with metastatic colorectal cancer (mCRC) treated with first-line FOLFIRI and bevacizumab (bev). Methods: Theimpact of 2 functional SNPs within the JAK2 and STAT5a genes on outcome was evaluated in 295 pts with mCRC treated with first-line FOLFIRI / bev in the randomized phase III FIRE-3 trial. 227 pts receiving FOLFIRI and cetuximab (FIRE-3) served as a negative control. Genomic DNA was extracted from formalin fixed paraffin embedded tissue and the SNPs were analyzed by PCR-based direct sequencing. Results: Baseline characteristics in the FOLFIRI / bev arm were as follows: female:male ratio = 1/3:2/3; median age = 65y (31-76) andmedian PFS/OS = 10.1/24.2 months. The JAK2 rs2274472 SNP showed significant association with progression-free survival (PFS). C allele carriers had a shorter median PFS compared to those with a T/T genotype (9.9 vs 11.7 months) in both univariate (HR 1.30, 95% CI 1.00-1.69, p = 0.045) and multivariate analysis (HR 1.39, 95% CI 1.06-1.83, p = 0.018). However, this association could not be observed in patients treated with FOLFIRI and cetuximab. Here, patients harboring any C allele and T/T genotype carriers showed an equal median PFS (10.0 vs 9.9 months, HR 1.00, 95% CI 0.75-1.33, p = 1.00). Conclusions: Our results provide the first evidence that the JAK2 polymorphism rs2274472 might serve as a predictive marker in pts with mCRC treated with FOLFIRI and bev in the first line setting. Targeting the JAK2 / STAT5A axis might be a promising approach to further enlarge our treatment options against mCRC and to overcome resistance to anti-angiogenic therapy.
Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer.
Authors: Kawakami Hisato , Huang Shengbing , Pal Krishnendu , Mukhopadhyay Debabrata , Sinicrope Frank A.
Published: March 2017

603

Background: Oncogenic BRAFV600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAFV600E mutant CRCs, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. Methods: BRAF isogenic RKO CRC cells and RKO, HT29, WiDr cell lines were treated with cobimetinib ± the small molecule MCL-1 inhibitor, A-1210477. Apoptosis was measured by Annexin V staining and caspase cleavage. Gene knockdown or overexpression was achieved by lentivirus; ERK siRNA was utilized. Competitive RT-PCR was performed for MCL-1 mRNA expression. HT-29 cells with control or MCL-1 shRNA were xenografted into SCID mice, treated with cobimetinib or vehicle, and tumor volume was measured. Results: We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by ERK siRNA that suppressed MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively.MEK /ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced pro-apoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown vs control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. A-1210477 also enhanced cobimetinib-induced apoptosis in vitrothat was due to disruption of the interaction of MCL-1 with pro-apoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. Conclusions: BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism.
Targeting inflammatory monocytes in human metastatic colorectal cancer.
Authors: Grossman Julie G. , Nywening Timothy M. , Belt Brian , Ahlers Michael , Hawkins William G. , Strasberg Steven M. , Goedegebuure Peter S. , Linehan David , Fields Ryan C.
Published: March 2017

605

Background: Colorectal cancer (CRC) is the most common gastrointestinal malignancy. 60% of CRC patients are diagnosed with metastatic CRC (mCRC) and the 5-year survival is < 20%. CCR2+ inflammatory monocytes (IM) are recruited from the bone marrow to the tumor microenvironment by the CCL2/CCR2 chemokine axis. At the tumor, they become tumor associated macrophages (TAM) and play a crucial role in promoting tumor progression, metastasis, and chemoresistance. While the importance of IM has been shown in other malignancies, little is known about their role in mCRC. Methods: Flow cytometry was performed on human and murine PBMCs, adjacent normal tissue, and tumors. Qualitative RT-PCR, ELISA, and confocal microscopy were performed for CCL2 expression. T-cell suppression assays were performed using CD14+ IM isolated from patient PBMCs and liver metastasis (LM). A murine model of CRC LM was created by hemispleen injection of luciferase-labelled MC38 CRC cells. Mice were treated with a CCR2 inhibitor and/or FOLFOX. Results: Prior to liver resection, mCRC patients have a higher percentage of IM in the peripheral blood compared to healthy donors (p < 0.0001), and on multivariate analysis elevated monocytes were prognostic of poor survival. We also found higher levels of CCL2 in the serum of mCRC patients (p < 0.01). Additionally, there was increased expression of CCL2 in LM compared to uninvolved tissue (p < 0.01), with the production of CCL2 localized to mCRC cells. FACS analysis showed CCR2+ TAM were elevated in LM compared to adjacent normal liver (p < 0.05) with a paucity of effector T-cells. CD14+ TAMs isolated from mCRC inhibited T-cell proliferation, illustrating the immune suppressive phenotype of these cells. In a murine model of CRC LM, treating mice with a CCR2 inhibitor alone or in combination with FOLFOX chemotherapy resulted in decreased tumor burden. FACS analysis of treated tumors showed increased effector T-cells in mice treated with CCR2 inhibitor. Conclusions: IM are involved in the progression of mCRC. Targeting these immunosuppressive cells with a CCR2 inhibitor decreases tumor burden in a murine model. This represents a potential novel treatment for mCRC.
MicroRNA regulation of radiation sensitivity in colorectal cancers.
Authors: Rana Shushan Rajesh , Kelley Katherine , Ruhl Rebecca , Thomas Charles R. , Tsikitis Vassiliki Liana , Anand Sudarshan
Published: March 2017

608

Background: Patients with locally advanced colorectal cancer (T3/T4/Node positive) receive neoadjuvant chemoradiation therapy (CRT) and subsequent surgery. While 10-25% of patients have complete response to CRT, the remaining patients undergo extensive tumor excision that leads to quality of life issues. Response to CRT is an independent predictor of overall survival highlighting the importance of improving CRT response rates. Several tumor intrinsic factors govern responses to CRT including specific gene expression programs. Emerging evidence suggests that microRNAs (miRs) modulate gene expression programs in response to radiation. Moreover, miR-processing machinery is frequently mutated in colorectal cancers (TCGA, 2016 provisional). miRs have been implicated in several pathological processes associated with colorectal cancer progression including cancer stemness and epithelial-to-mesenchymal transition (EMT). In this context, we hypothesized that differential expression of miRs regulates colorectal cancer radiation sensitivity and therefore can be used as a biomarker to predict therapeutic responses to radiation. Methods: To investigate the differences in miR profiles between rectal cancer patients that had either a pathological partial response (PR) or no response (NR), we isolated RNA from FFPE biopsies using the miRvana microRNA isolation kit (Life Technologies). We used the Nanostring miR profiling platform and obtained absolute counts for > 700 human miRs of which ~500 miRs were expressed above detection limits (cut-off of 20 counts after normalization to the top-100 miRs). We performed in vitro gain and loss of studies with miR transfections in human CRC cell lines with RNAimax reagent and used a luminescence-based assay for proliferation (Cell titer glo, Promega). We performed surviving fraction assays by seeding cells on 6 well plate and counting colonies stained with Methylene Blue. Results: We identified seventeen miRs that were differentially expressed. Among the most upregulated in this group, miR-451a, inhibited proliferation and colony formation in 2D and 3D assays in the presence of radiation. Conclusions: Our data suggests miRs may alter cell survival pathways and affect tumor radiosensitivity.
A phase IB study of the combination of selumetinib (AZD6244; ARRY-142886) and cyclosporin A (CsA) in patients with advanced solid tumors with an expansion cohort in metastatic colorectal cancer (mCRC).
Authors: Krishnamurthy Anuradha , Dasari A. , Lockhart Albert C. , Stein Mark N. , Sanoff Hanna Kelly , Lee James J. , Hansen Aaron Richard , Bekaii-Saab Tanios S. , Malhotra Usha , Rippke Sarah , Davis Sarah Lindsey , Messersmith Wells A. , Yao James C. , Meric-Bernstam Funda , Eckhardt S. Gail , Lieu Christopher Hanyoung
Published: March 2017

609

Background: Targeting MEK is of interest in the development of novel agents for treatment of many malignancies. However, better strategies are needed to overcome acquired resistance to MEK inhibitors. Preclinical studies have shown Wnt pathway overexpression in KRAS mutant cell lines resistant to the MEK inhibitor, Selumetinib. The combination of selumetinib and cyclosporin A (CsA), a non-canonical WnT pathway modulator, demonstrated antitumor activity in patient-derived xenograft (PDX) models. We are conducting an NCI CTEP-approved Phase I/IB trial (NCI # 9571/COMIRB # 13-2628) of selumetinib and CsA combination. Biomarkers of response to therapy are being co-developed. We hypothesize that this combination will be safe and potentially effective in patients with mCRC and that upregulation of FZD2 may predict for sensitivity. Methods: Phase I trial with initial dose escalation investigating the combination of selumetinib and CsA in patients with advanced solid tumors (n = 18) followed by an expansion cohort in patients with irinotecan and oxaliplatin-refractory mCRC (n = 20). The expansion cohort utilizes a selumetinib “run-in” to evaluate efficacy in RAS-WT and RAS-MT mCRC to identify those patients most likely to respond to the combination. Results: 18 patients were enrolled in the dose escalation phase and 10 patients have been enrolled in the dose expansion phase as of September 2016. Grade 1 or 2 nausea and rash were reported as the most common AEs. Most commonly reported Grade 3 or 4 toxicities were hypertension, elevated liver enzymes and rash. Three DLTs were reported with Grade 3 hypertension noted at dose level 1 and 2 and grade 3 rash reported at dose level 2. The maximum tolerated dose was defined as Selumetinib 75 mg BID and CsA 2 mg/kg BID on a 28-day cycle. Two partial responses and sixteen stable disease responses have been observed. Six patients have exhibited progressive disease. Conclusions: Selumetinib in combination with cyclosporin A appears to be well-tolerated with evidence of activity in solid tumors. Expansion cohort will complete enrollment this month. Clinical trial information: NCT02188264.
Intratumoral heterogeneity in colorectal cancer: Can histology be used as a guidance for molecular testing?
Authors: Sagaert Xavier , Tejpar Sabine , Desmedt Linde , Spaepen Marijke , Matthijs Gert , Andel Daan
Published: March 2017

611

Background: Intratumour heterogeneity is a key challenge in colorectal cancer management. We have recently shown high levels of morphologic heterogeneity in microsatellite unstable (MSI) colorectal tumours (CRC). In this study we verified whether morphologic intratumour heterogeneity can be used as a guidance to test for molecular heterogeneity. Methods: 15 MSI CRCs tumour, characterized by a heterogenous morphology, were selected together with the associated metastasis. Macrodissection of the distinct morphologic tumour components was followed by MSI testing according to the Bethesda guidelines and Sanger sequencing for BRAF V600E mutations. In addition, 2 MSI CRC underwent macrodissection of the distinct morphologic components, followed by nanostring ncounter analysis. Results: MSI status varies between MSI-H and MSI-L in the primary tumours and 35% of the metastasis of MSI-H primary tumours presented as MSI-L or MSS. BRAF mutations status was constant throughout the primary tumours, but two BRAF mutated tumours presented with a BRAF wild type metastasis. Nanostring analysis revealed that ùorphologically identical clones clustered separately from tumour clones with a different morphologic display. Conclusions: Morphologically heterogeneous tumour compartments have less related gene expression profiles, linking morphologic homogeneity to molecular homogeneity. Our findings support the hypothesis that morphologic heterogeneity reflects molecular intratumour heterogeneity and can be used as a guidance for molecular testing of colorectal cancers.
Antitumor activity of panRAF inhibition in BRAF V600E metastatic colorectal cancer.
Authors: Sorokin Alex , Bitner Lea , Wu Ji , Menter David , Kopetz Scott , Morris Van Karlyle
Published: March 2017

616

Background: BRAF V600E mutations, present in <10% of patients with metastatic colorectal cancer (mCRC), are associated with low responses to chemotherapy and poor survival outcomes. Targeted therapies against BRAF and EGFR have shown promising clinical activity. The panRAF inhibitor (PRI) LSN3074753 inhibits dimerization of all RAF isoforms to impede downstream MEK activation, with no reflexive MAPK reactivation common with other BRAF inhibitors. Anti-tumor activity of PRI has not been compared to BRAF + EGFR inhibition in patient-derived xenograft (PDX) models of BRAF V600E mCRC. Methods: Two PDX models of BRAF V600E mCRC (B1003 and C0999) were generated. C0999 featured a concomitant KRAS G12D mutation following resistance to the BRAF V600E kinase inhibitor vemurafenib. Mice were treated daily with oral PRI or with the combination of vemurafenib + intraperitoneal cetuximab. Tumor volumes were measured twice weekly. B1003 and C0999 cell cultures were established to test the interaction between PRI and palbociclib or BYL319 (PI3K inhibitor). Results: PRI was tolerated at a dose of 60 mg/kg and demonstrated a reduced tumor volume in the B1003 model after 28 days when compared to untreated controls (P=.03). No difference in tumor volume was seen between PRI and vemurafenib + cetuximab (P=.08). Assessment of anti-tumor activity by PRI in the vemurafenib-resistant BRAF V600E/KRAS G12D C0999 model will be reported. Cell culture from both the B1003 and C0999 models demonstrated synergism for PRI with palbociclib (ED50 .41 and .62 for the 2 models, respectively) and with BYL319 (ED50 .48 and .86, respectively). Conclusions: panRAF inhibition demonstrates similar anti-tumor activity to BRAF + EGFR inhibition in PDX models of BRAF V600E and represents a promising treatment strategy for further combination studies targeting additional critical signaling pathways in mCRC.
Detecting NTRK, ROS1, and ALK gene fusions in gastrointestinal tumor patients.
Authors: Potts Steven James , Dean Emma Jane , Polikoff Jonathan , Pacey Simon , Chiorean E. Gabriela , Chee Cheng Ean , Sigal Darren , Aslam Maria , Pauwels Patrick , Christiansen Jason H. , Multani Pratik S. , Lamoureux Jennifer , Murphy Danielle
Published: March 2017

619

Background: The STARTRK-2 trial is a potentially registration-enabling Phase 2 global basket trial of the tyrosine kinase inhibitor entrectinib in patients with solid tumors harboring NTRK1, NTRK2, NTRK3, ROS1, or ALK gene fusions. Phase 1 studies of entrectinib reported a 79% ORR across multiple histologies in patients with gene fusions of these targets who were naïve to inhibitors of these targets, received an effective dose, and had extracranial disease. Patients harboring these fusions are typically rare in the cancer population ( < 3%); however, they have been seen across a wide variety of GI tumors. In this presentation we report on the occurrence of these fusions in GI tumors, based on Ignyta’s internal and partner testing. We discuss the development of an assay for GI samples and immunohistochemistry (IHC) screening efficiency rates in a clinical cohort of 157 GI samples. Methods: The occurrence of NTRK, ROS1, and ALK gene fusions in GI tumors was studied from Ignyta internal and partner collaborations. We developed a 2-step diagnostic test to identify fusions in FFPE clinical specimens. IHC screening using a pan-receptor tyrosine kinase cocktail of antibodies precedes an RNA-based anchored multiplex PCR next generation sequencing (NGS) assay. Results: IHC positivity rates ranged from 0 to 24 percent depending on histology and averaging 8% positivity rate across 15 tumor locations. The screen is particularly effective in cholangiocarcinoma, colon, esophagus, gastric, peritoneum, rectum and small bowel. It was highest in pancreas (24%). Out of 157 GI samples, no instances were detected where the IHC was screened negative and gene fusions were observed by NGS. The IHC cocktail enriches detection of the patient population for gene fusions over 11-fold and has a 100% negative predictive value. Conclusions: This 2-step assay is an efficient method for detection of gene rearrangements in both high-volume clinical testing and studies of archival formalin-fixed paraffin-embedded specimens. Depending on how it is implemented in clinical trials, the staged approach can substantially decrease the costs and tissue expended for NGS. Screening efficiency may be improved even further as additional diagnostic antibodies become available. Clinical trial information: NCT02568267.
Comparison of irinotecan and oxaliplatin as adjuvant chemotherapy for patients with resectable synchronous colon cancer plus liver-confined metastases: A retrospective nationwide database study.
Authors: Liang Yi-Hsin , Shao Yu Yun , Chen Ho-Min , Cheng Ann-Lii , Lai Mei-Shu , Yeh Kun-Huei
Published: March 2017

624

Background: Although irinotecan and oxaliplatin are both standard treatment for advanced colon cancer, it was unclear whether either was effective for patients with resectable synchronous colon cancer plus liver-confined metastasis (SCCLM) after curative surgery. Methods: We established a population-based cohort of patients diagnosed with de novo SCCLM between 2004 and 2009 by searching the database of Taiwan Cancer Registry and National Health Insurance Research Database of Taiwan. Patients who received curative surgery as the first therapy followed by chemotherapy doublets were classified into two groups: irinotecan group, or oxaliplatin group. Patient who received radiotherapy or did not receive chemotherapy doublets were excluded. Results: We included 6533 patients with de novo stage IV colon cancer, and 309 of them received chemotherapy doublets after surgery; 77 patients received irinotecan, and 232 patients received oxaliplatin as adjuvant chemotherapy. Patients in the irinotecan group and patients in the oxaliplatin group exhibited similar overall survival (OS) (median: not reached vs. 40.8 months, p = 0.151) and time to the next line of treatment (median: 16.5 months vs. 14.3 months, p = 0.349) in both univariate and multivariate analyses. Patients with resectable SCCLM had significant shorter median OS than patients with stage III colon cancer who received curative surgery and subsequent adjuvant chemotherapy but longer than patients with de novo stage IV colon cancer who received surgery only to the primary site and standard systemic chemotherapy ( p< 0.001). Conclusions: Either irinotecan or oxaliplatin exhibited similar efficacy for patients who received curative surgery for resectable SCCLM. [Table: see text]
Systematic assessment of intra- and peri-metastatic tumor budding in patients with stage IV colorectal cancer.
Authors: Blank Annika , Burren Sandra , Zlobec Inti , Dawson Heather , Lugli Alessandro
Published: March 2017

626

Background: Tumor budding is a strong independent prognostic factor in primary colorectal cancer (CRC). The prognosis of stage IV CRC is still poor and there is need for biomarkers to facilitate the clinical management. Data on tumor budding in colorectal cancer distant metastases (CRDM) are still missing. Therefore, the aim of this study was to analyze its role in tumor progression. Methods: 73 stage IV CRC with clinico-pathological data were retrieved from a cohort of 331 CRC patients, surgically treated between 2002 and 2013 at the University Hospital of Bern. Tumor budding was visualized immunohistochemically by pan-cytokeratin staining. Tumor budding was defined as intra- and peri-tumoral budding in the primary tumor (ITB = buds in the tumor center; PTB = buds at the tumor margin), intra- and perinodal budding (INB and PNB) and intra- and peri-metastatic tumor budding (IMB and PMB). Overall tumor budding was defined as tumor buds independent of the center and margin (OTB, ONB, OMB). The tumor bud count was assessed by calculating the mean number of tumor buds in 10 high power fields. For survival analysis a cut off of 10 tumor buds was applied to subdivide the tumors with low and high numbers of tumor buds. Results: In lymph nodes and CRDM, the tumor bud count was lower compared to primary tumors (PNB: 13; INB: 18; ONB: 21 vs PMB: 10; IMB: 21; OMB: 22 vs PTB: 23; ITB: 24; OTB: 33). For PTB/PNB and OTB/ONB, there were significant differences between primary tumor and lymph node metastasis (PTB/PNB: p < 0.001; OTB/ONB: 0.008) and primary tumor and distant metastases (PTB/PMB: p < 0.001; OTB/OMB: 0.007). The presence of PMB was predicted by INB, PNB and V1 (p = 0.032, p = 0.001 and p = 0.003). A trend towards a worse prognosis in patients with a high OMB number was observed. Conclusions: Tumor budding is present in lymph node and distant metastases of CRC, but its number is lower compared to the corresponding primary tumor. As tumor budding in local and distant metastases seems to be a predictor of tumor progression, these novel data should be considered as a basis for further analysis in large and multicentric clinical trials.
The role of sidedness, EGFR gene copy number (GCN) and EGFR promoter methylation in RAS/BRAF wild type (WT) colorectal cancer (CRC) patients receiving irinotecan/cetuximab.
Authors: Puzzoni Marco , Giampieri Riccardo , Demurtas Laura , Pusceddu Valeria , Mandolesi Alessandra , Faloppi Luca , Loretelli Cristian , Zaniboni Alberto , Falcone Alfredo , Cascinu Stefano , Scartozzi Mario
Published: March 2017

628

Background: Data from randomized trials suggested that tumour sidedness could represent a prognostic and predictive factor in CRC patients. Distinctive molecular and clinical features are related to tumour sidedness, however their predictive role has not yet been clearly defined. Methods: Tumour samples from RAS/BRAF WT metastatic CRC patients treated with second-third line irinotecan-cetuximab were analised for EGFR GCN and promoter methylation. Study objective was to evaluate the correlation of tumour sidedness, EGFR promoter methylation and EGFR GCN with clinical outcome. Results: Eighty-eightpatients were included in the study, 24/88 (27.3%) had right sided CRC(RSCC), 64/88 (72.7%) had left sided CRC(LSCC); 32/88 (36.4%) had EGFR GCN < 2.12 tumour, 56/88 (63.6%) had EGFR GCN ≥ 2.12 tumour; 44/88 (50%) had EGFR promoter methylated tumour. RSCC were associated with reduced ORR (4.2% for RSCC vs 35.9% for LSCC, p:0.0030), with shorter PFS (3.0 vs 10.29 months, HR:2.74, p < 0.0001) and shorter OS (8.0 vs 13.6 months, HR:2.83, p < 0.0001). EGFR GCN < 2.12 tumours were associated with reduced ORR (6.2% for EGFR GCN < 2.12 vs 39.3% for ≥ 2.12 tumours, p:0.0009), with shorter PFS (3.8 vs 7.0 months HR:2.15, p = 0.0010) and shorter OS (8.5 vs 14.0 months HR:2.59, p < 0.0001). EGFR methylated tumours were associated with reduced ORR (9.1% for methylated vs 45.5% for unmethylated, p = 0.0001), with shorter PFS (3.0 vs 7.67 months HR:3.23, p < 0.0001) and shorter OS (8.0 vs 17.0 months HR:3.57, p < 0.0001). At multivariate analysis EGFR GCN and EGFR promoter methylation mantained their independent role for ORR (respectively p:0.0082 and p:0.0025), PFS (respectively Exp(b):0.40, p:0.0048 and Exp(b):4.52, p < 0.0001) and OS (respectively Exp(b):0.23, p:0.0001 and Exp(b):5.58, p < 0.0001). Conclusions: In our study an accurate molecular selection based on an all RAS and BRAF analisys along with EGFR GCN and EGFR promoter methylation status resulted more relevant than tumour sidedness for the prediction of clinical outcome during cetuximab/irinotecan. We then suggest that molecular selection might overcome the importance of tumour sidedness in this setting.