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The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. Significance: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression.

The incidence of rectal cancer is increasing in patients younger than 50 years. Locally advanced rectal cancer is still treated with neoadjuvant radiation, chemotherapy and surgery, but recent evidence suggests that patients with a complete response can avoid surgery permanently. To define correlates of response to neoadjuvant therapy, we analyzed genomic and transcriptomic profiles of 738 untreated rectal cancers. APC mutations were less frequent in the lower than in the middle and upper rectum, which could explain the more aggressive behavior of distal tumors. No somatic alterations had significant associations with response to neoadjuvant therapy in a treatment-agnostic manner, but KRAS mutations were associated with faster relapse in patients treated with neoadjuvant chemoradiation followed by consolidative chemotherapy. Overexpression of IGF2 and L1CAM was associated with decreased response to neoadjuvant therapy. RNA-sequencing estimates of immune infiltration identified a subset of microsatellite-stable immune hot tumors with increased response and prolonged disease-free survival.

Supplementary Figures 1-10 and associated legends

The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis.Significance:Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression.

Supplementary Figures 11-14 and associated legends.

Supplemental Table 1: Demographics and clinical data for donor Gife of Life Samples Supplemental Table 2: Putative Ligand-Receptor pairs comparing all cell types from single cell sequencing of tumor samples compared to healthy samples. Supplemental Table 3: Differential gene expression using linear mixed model analysis of cell types from spatial transcriptomic ROIs. Supplemental Table 4: Kras mutational profiling on select donor samples Supplemental Table 5: Antibodies used for multiplex immunofluoresence. Supplemental Table 6: Antibodies and RNAScope probe used for co-IF/ISH.

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Supplementary Table from KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

KRAS mutation in colorectal cancer is associated with aggressive tumor behavior through increased invasiveness and higher rates of lung metastases, but the biological mechanisms behind these features are not fully understood. In this study, we show that KRAS-mutant colorectal cancer upregulates integrin α6β4 through ERK/MEK signaling. Knocking-out integrin β4 (ITGB4) specifically depleted the expression of integrin α6β4 and this resulted in a reduction in the invasion and migration ability of the cancer cells. We also observed a reduction in the number and area of lung metastatic foci in mice that were injected with ITGB4 knockout KRAS-mutant colorectal cancer cells compared with the mice injected with ITGB4 wild-type KRAS-mutant colorectal cancer cells, while no difference was observed in liver metastases. Inhibiting integrin α6β4 in KRAS-mutant colorectal cancer could be a potential therapeutic target to diminish the KRAS-invasive phenotype and associated pulmonary metastasis rate.Implications:Knocking-out ITGB4, which is overexpressed in KRAS-mutant colorectal cancer and promotes tumor aggressiveness, diminishes local invasiveness and rates of pulmonary metastasis.

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HER2 amplification is found in more than 15% of gastric cancers and is associated with poor clinical outcome. Lapatinib, a dual HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has shown promising in vitro results in treating HER2+ cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here, we investigated whether activated MET can confer resistance to lapatinib inhibition of gastric cancer cells. A panel of gastric cancer cell lines was treated with lapatinib, and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT, and extracellular signal-regulated kinase was inhibited by lapatinib and presumably led to cell-cycle arrest as observed with flow cytometry. Hepatocyte growth factor (HGF) activation of MET receptors rescued cells from lapatinib-induced growth inhibition by restimulating the downstream pathways and restoring normal cell-cycle progression. This rescue effect could be abrogated by inhibiting MET with PHA-665752 (a highly specific MET inhibitor) or downregulating MET expression with short interfering RNA. No synergy in growth inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like growth factor 1 and fibroblast growth factor 3 could not uniformly rescue the lapatinib-treated gastric cancer cells. In conclusion, HGF/MET–mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in gastric cancer cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation. Mol Cancer Ther; 11(3); 660–9. ©2012 AACR.

Supplementary Figure from KRAS Mutants Upregulate Integrin β4 to Promote Invasion and Metastasis in Colorectal Cancer

Cellular transformation induces phenotypically diverse populations of tumour-infiltrating T cells

KRAS mutation in colorectal cancer is associated with aggressive tumor behavior through increased invasiveness and higher rates of lung metastases, but the biological mechanisms behind these features are not fully understood. In this study, we show that KRAS-mutant colorectal cancer upregulates integrin α6β4 through ERK/MEK signaling. Knocking-out integrin β4 (ITGB4) specifically depleted the expression of integrin α6β4 and this resulted in a reduction in the invasion and migration ability of the cancer cells. We also observed a reduction in the number and area of lung metastatic foci in mice that were injected with ITGB4 knockout KRAS-mutant colorectal cancer cells compared with the mice injected with ITGB4 wild-type KRAS-mutant colorectal cancer cells, while no difference was observed in liver metastases. Inhibiting integrin α6β4 in KRAS-mutant colorectal cancer could be a potential therapeutic target to diminish the KRAS-invasive phenotype and associated pulmonary metastasis rate. Implications: Knocking-out ITGB4, which is overexpressed in KRAS-mutant colorectal cancer and promotes tumor aggressiveness, diminishes local invasiveness and rates of pulmonary metastasis.

Supplementary Figures S1-S2; Supplementary Table S1.

Purpose: Heat shock protein-90 (HSP-90), a molecular chaperone required by numerous oncogenic kinases [e.g., HER-2, epidermal growth factor receptor (EGFR), Raf-1, v-Src, and AKT] for conformational stability, has attracted wide interest as a novel target for cancer therapy. HSP-90 inhibition induces degradation of HSP-90 client proteins, leading to a combinatorial inhibition of multiple oncogenic signaling pathways with consecutive growth arrest and apoptosis. MET, a tyrosine kinase that is constitutively active in tumor cells with MET oncogene amplification, has recently been identified as another HSP-90 client.Experimental Design: The aim of our study was to assess the efficacy of SNX-2112, a synthetic HSP-90 inhibitor, in 3 different MET-amplified tumor cell lines (GTL-16, MKN-45, and EBC-1) as well as PR-GTL-16 cells, a GTL-16 subline selected for resistance to the highly selective MET kinase inhibitor PHA-665752.Results: In all cell lines, SNX-2112 led to degradation of MET, HER-2, EGFR, and AKT, as well as abrogation of Ras/Raf/MEK/MAPK and PI3K/AKT signaling, followed by complete cell cycle arrest. SNX-5542, an orally bioavailable prodrug of SNX-2112, displayed significant antitumor efficacy in vivo in nude mice bearing MET-amplified tumor xenografts. Importantly, HSP-90 inhibition maintained its antitumor efficacy in PR-GTL-16 cells both in vitro and in vivo, suggesting that HSP-90 inhibition could be a particularly valuable strategy in MET-amplified tumors that have acquired resistance to MET kinase inhibition.Conclusions: Our study provides evidence for the efficacy of HSP-90 inhibition in MET-amplified cancer cells, particularly when MET kinase inhibitor resistance has emerged. Clin Cancer Res; 17(1); 122–33. ©2011 AACR.

PURPOSE To characterize the relationship between tumor-infiltrating lymphocytes (TIL), tumor mutational burden (TMB), and genetic alterations in microsatellite stable (MSS), microsatellite instability (MSI), or mutant POLE/POLD1 colon cancer. MATERIALS AND METHODS Four hundred ninety-nine resected stage I-III colon tumors treated between 2014 and 2019 were assessed for TIL; somatic mutations, copy number alterations, and structural changes in > 400 oncogenes; and MSI status. RESULTS Of the 499 tumors analyzed, 313 were MSS, 175 were MSI, and 11 had POLE/POLD1 pathogenic mutations. Both the percentage of tumors with a high level of TIL (≥ 4 lymphocytes per high-power field) and the median TMB differed significantly between the three phenotypes: MSS, 4.5% and 6 mutations/Mb; MSI, 68% and 54 mutations/Mb; POLE/POLD1, 82% and 158 mutations/Mb ( P < .05). Within each phenotype, TMB did not vary significantly with TIL level. Among MSI tumors, the median number of frameshift indels was significantly higher in tumors with high levels of TIL (20 v 17; P = .018). In the MSS group, significantly higher proportions of tumors with high levels of TIL had mutations in the transforming growth factor-β (36% v 12%; P = .01), RAS (86% v 54%; P = .02), and Hippo (7% v 1%; P = .046) pathways; in contrast, TP53 alterations were associated with low levels of TIL (74% v 43%; P = .01). CONCLUSION The association between TIL, TMB, and genetic alterations varies significantly between MSI, MSS, and mutant POLE/POLD1 colon tumors. These differences may help explain tumoral immunity and lead to predictors of response to immunotherapy.

Mismatch repair deficient colon cancer is heterogeneous. Differentiating inherited constitutional variants from somatic genetic alterations and gene silencing is important for surveillance and genetic counseling.Determine the extent to which the underlying mechanism of loss of mismatch repair influences molecular and clinicopathologic features of microsatellite instability-high colon cancer.Retrospective analysis.Comprehensive cancer center.Patients with microsatellite instability-high colon cancer of stage I, II, or III.Curative surgical resection.Hypermethylation of the MLH1 promoter, biallelic inactivation, constitutional pathogenic variant, and loss of specific mismatch repair proteins.Of the 157 identified tumors with complete genetic analysis, 66% had hypermethylation of the MLH1 promoter, 18% had constitutional pathogenic variant (Lynch syndrome), 11% had biallelic somatic MMR gene pathogenic variants, and 6% had unexplained high microsatellite instability. The distribution of mismatch repair loss was as follows: MLH1 and PMS2 co-loss, 79% of the tumors; MSH2 and MSH6 co-loss, 10%; MSH6 alone, 3%; PMS2 alone, 2%; other combinations, 2%; no loss, 2%. Tumor mutational burden was lowest in MLH1- and PMS2-deficient tumors. MSH6-deficient tumors had the lowest levels of tumor-infiltrating lymphocytes, lowest MSIsensor scores, and fewest frameshift deletions. Patients with MLH1 promoter hypermethylation were significantly more likely to be older and female and to have right-colon lesions than patients with biallelic inactivation. Mutation was the most prevalent second hit in tumors with biallelic inactivation and tumors of patients with Lynch syndrome.Potential selection or referral bias, missing data for some patients, and relatively small sizes of some subgroups.Clinical characteristics of mismatch repair deficient colon cancer vary with the etiology of microsatellite instability, and its molecular characteristics vary with the affected mismatch repair protein. See Video Abstract at http://links.lww.com/DCR/B984.

We describe a mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation in an immunocompetent setting. This approach uses simple mechanical disruption to allow engraftment, avoiding the use of dextran sulfate sodium. The resulting RC tumors invaded from the mucosal surface and metastasized to distant organs. Histologically, the tumors closely resemble human RC and mirror remodeling of the tumor microenvironment in response to radiation. This murine RC model thus recapitulates key aspects of human RC pathogenesis and presents an accessible approach for more physiologically accurate, preclinical efficacy studies.

We describe a new mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation; the resulting RC tumors invaded inward from the mucosal surface and metastasized to distant organs. Histologically the tumors closely resemble human RC and mirror remodeling of the tumor microenvirnoment (TME) in response to radiation. This murine RC model thus recapitulates the pathogenesis of human RC, thereby fulfilling the need for a physiologically accurate model for preclinical efficacy studies.

Background Total neoadjuvant therapy (TNT) improves tumor response in locally advanced rectal cancer (LARC) patients compared to neoadjuvant chemoradiotherapy alone. The effect of TNT on patient survival has not been fully investigated. Materials and Methods This was a retrospective case series of patients with LARC at a comprehensive cancer center. Three hundred and eleven patients received chemoradiotherapy (chemoRT) as the sole neoadjuvant treatment and planned adjuvant chemotherapy, and 313 received TNT (induction fluorouracil and oxaliplatin-based chemotherapy followed by chemoradiotherapy in the neoadjuvant setting). These patients then underwent total mesorectal excision or were entered in a watch-and-wait protocol. The proportion of patients with complete response (CR) after neoadjuvant therapy (defined as pathological CR or clinical CR sustained for 2 years) was compared by the χ2 test. Disease-free survival (DFS), local recurrence-free survival, distant metastasis-free survival, and overall survival were assessed by Kaplan-Meier analysis and log-rank test. Cox regression models were used to further evaluate DFS. Results The rate of CR was 20% for chemoRT and 27% for TNT (P=.05). DFS, local recurrence-free survival, metastasis-free survival, and overall survival were no different. Disease-free survival was not associated with the type of neoadjuvant treatment (hazard ratio [HR] 1.3; 95% confidence interval [CI] 0.93-1.80; P = .12). Conclusions Although TNT does not prolong survival than neoadjuvant chemoradiotherapy plus intended postoperative chemotherapy, the higher response rate associated with TNT may create opportunities to preserve the rectum in more patients with LARC.

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS‐mt) and KRAS‐wild‐type (KRAS‐wt) patients. We found that KRAS‐mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment., Oncogenic KRAS is associated with poor outcomes in locally advanced rectal cancer (LARC) but the underlying biologic mechanisms are not fully understood. We investigated differences in gene expression profiles between KRAS mutant and KRAS wild‐type LARC tumors. We found that KRAS mutant tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix.

The gut microbiota is a component of the tumor microenvironment that has the potential to influence sensitivity or resistance to neoadjuvant therapy through modulation of the host immune response in patients diagnosed with locally advanced rectal cancer (LARC, stage II-III). We performed DNA and RNA sequencing of pre-treatment endoscopic biopsies from 89 LARC patients treated with neoadjuvant therapy (NAT). Our cohort included 84 microsatellite stable and 5 microsatellite unstable (MSI) patients. We identified the presence of microbial species by mapping non-human RNA read sequences to a large catalogue of viral and bacterial microorganisms. Our methodology has been validated and determined to have comparable accuracy to reference microbial tests for both viruses and bacteria. We analyzed correlations between presence of specific microbial species and (1) clinicopathological variables such as age, stage, anatomic location and MSI status, (2) genomic variables such as tumor mutational burden, chromosomal instability and somatic alterations and (3) response to NAT. We identified 152 species with at least 100 mapped reads in 10% or more of our cohort. The median number of species per patient was 37 [IQR 16-66], with only 6/89, 7% patients having no detected species at all. The most frequently detected species included well-known intestinal bacteria such as Escherichia coli, Fusobacterium nucleatum, Faecalibacterium prausnitzii and several members of the Bacteroides genus. The Parvimonas micra coccus was more often detected in tumors from the lower rectum (13/22, 59%, 0-4 cm from the anal verge) than the middle (8/36, 22%, 4-8 cm) and upper rectum (5/30, 17%, 8-12 cm) (p Citation Format: Chad M. Vanderbilt, Walid K. Chatila, Danny N. Khalil, Chin-Tung Chen, Jin K. Kim, Fan Wu, Nikolaus Schultz, Martin R. Weiser, Julio Garcia-Aguilar, Francisco Sanchez-Vega. Characterization of the tumor microbiome in patients with locally advanced rectal cancer treated with neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3058.

Colon tumors with deficient DNA mismatch repair (dMMR) are infiltrated more densely by T cells than tumors with proficient mismatch repair (pMMR). However, high tumor-infiltrating lymphocytes (TIL) are found in a subset of pMMR tumors and low TIL are seen in a subset of dMMR colon cancer. In this study, we compared T-cell repertoires in 20 pMMR and 27 dMMR colon tumors with high and low TIL counts. CDR3 regions of the TCR beta chain were sequenced by ImmunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). The fraction T cells was calculated by normalizing TCR beta template counts to the total DNA. Sample richness was calculated as the number of unique productive rearrangements in a sample after computationally down-sampling to a common number of T cells to control for variation in sample depth. Simpson clonality was computed as a measure of how evenly productive rearrangements are distributed amongst the total number of T cells. Statistical analyses were performed using Prism 8.0 (GraphPad, La Jolla, CA). The mean TIL counts per high power field (HPF) for TIL-high and TIL-low dMMR tumors were 17.4 (range, 5-37) and 1.8 (range, 0-4). In pMMR tumors, they were 12.8 (range, 6-37) for TIL-high and 0.3 (range, 0-1) for TIL-low groups. The number of T cells measured by TCR sequencing correlated positively with the TIL count (Spearman rho = 0.391; P = 0.007). When comparing by MMR status, we found that dMMR tumors exhibited a trend towards higher Simpson clonality index (P = 0.09), with significantly lower repertoire richness compared to pMMR tumors (P = 0.030). Within dMMR tumors, TIL-high dMMR tumors had higher density (P = 0.017), higher clonality (P = 0.003), and a less rich repertoire (P = 0.003) compared to TIL-low dMMR tumors. Within pMMR tumors, while the T cell density was higher in the TIL-high tumors, we did not observe differences in T cell clonality or repertoire richness between TIL-high vs TIL-low pMMR tumors. Our study found that T cells in dMMR tumors are more clonal and their repertoire is less rich compared with T cells in pMMR tumors. In the dMMR group, T cells in TIL-high tumors were more clonal and their repertoire was less rich compared with T cells in TIL-low tumors, but in the pMMR group, T-cell diversity in TIL-high tumors was comparable to T-cell diversity in TIL-low tumors. These findings suggest that T cells clonally expand in dMMR tumors, possibly in response to MMR deficiency-induced tumor neoantigens. Citation Format: Jin K. Kim, Chin-Tung Chen, Asama Khan, Neil Segal, Zsofia Stadler, Jinru Shia, Vinod P. Balachandran. Comparison of intratumoral T-cell repertoires in DNA mismatch repair proficient and deficient colon adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1382.

To characterize the relationship between tumor-infiltrating lymphocytes (TIL), tumor mutational burden (TMB), and genetic alterations in microsatellite stable (MSS), microsatellite instability (MSI), or mutant POLE/POLD1 colon cancer. MATERIALS AND METHODS: Four hundred ninety-nine resected stage I-III colon tumors treated between 2014 and 2019 were assessed for TIL; somatic mutations, copy number alterations, and structural changes in > 400 oncogenes; and MSI status. RESULTS: Of the 499 tumors analyzed, 313 were MSS, 175 were MSI, and 11 had POLE/POLD1 pathogenic mutations. Both the percentage of tumors with a high level of TIL (≥ 4 lymphocytes per high-power field) and the median TMB differed significantly between the three phenotypes: MSS, 4.5% and 6 mutations/Mb; MSI, 68% and 54 mutations/Mb; POLE/POLD1, 82% and 158 mutations/Mb (P < .05). Within each phenotype, TMB did not vary significantly with TIL level. Among MSI tumors, the median number of frameshift indels was significantly higher in tumors with high levels of TIL (20 v 17; P = .018). In the MSS group, significantly higher proportions of tumors with high levels of TIL had mutations in the transforming growth factor-β (36% v 12%; P = .01), RAS (86% v 54%; P = .02), and Hippo (7% v 1%; P = .046) pathways; in contrast, TP53 alterations were associated with low levels of TIL (74% v 43%; P = .01). CONCLUSION: The association between TIL, TMB, and genetic alterations varies significantly between MSI, MSS, and mutant POLE/POLD1 colon tumors. These differences may help explain tumoral immunity and lead to predictors of response to immunotherapy.

Because permanent-magnet synchronous linear motors (SLM) still exhibit nonlinear friction, ending effects and time-varying dynamic uncertainties, better control performances cannot be achieved by using common linear controllers. We propose a backstepping approach with three adaptive laws and a beating function to control the motion of permanent-magnet SLM drive systems that enhance the robustness of the system. In order to reduce greater vibration in situations with uncertainty actions in the aforementioned control systems, we propose an adaptive modified recurrent Rogers&ndash, Szego polynomials neural network backstepping (AMRRSPNNB) control system with three adaptive laws and reimbursed controller with decorated gray wolf optimization (DGWO), in order to handle external bunched force uncertainty, including nonlinear friction, ending effects and time-varying dynamic uncertainties, as well as to reimburse the minimal rebuild error of the reckoned law. In accordance with the Lyapunov stability, online parameter training method of the modified recurrent Rogers&ndash, Szego polynomials neural network (MRRSPNN) can be derived by utilizing an adaptive law. Furthermore, to help reduce error and better obtain learning fulfillment, the DGWO algorithm was used to change the two learning rates in the weights of the MRRSPNN. Finally, the usefulness of the proposed control system is validated by tested results.

Due to a good ability of learning for nonlinear uncertainties, a mixed modified recurring Rogers-Szego polynomials neural network (MMRRSPNN) control with mended grey wolf optimization (MGWO) by using two linear adjusted factors is proposed to the six-phase induction motor (SIM) expelling continuously variable transmission (CVT) organized system for acquiring better control performance. The control system can execute MRRSPNN control with a fitted learning rule, and repay control with an evaluated rule. In the light of the Lyapunov stability theorem, the fitted learning rule in the MRRSPNN control can be derived, and the evaluated rule of the repay control can be originated. Besides, the MGWO by using two linear adjusted factors yields two changeable learning rates for two parameters to find two ideal values and to speed-up convergence of weights. Experimental results in comparisons with some control systems are demonstrated to confirm that the proposed control system can achieve better control performance.

This diagnostic study explores the diagnostic accuracy of differentially expressed miRNAs between stage I and stage II or III rectal cancers., Key Points Question Can the expression levels of microRNA (miRNA) in the primary tumor distinguish locally advanced disease among nonmetastatic rectal cancers? Findings This diagnostic study of 2 independent cohorts found that a set of 19 miRNAs can effectively distinguish locally advanced disease (stage II or III) among nonmetastatic rectal cancers. Within this set of miRNAs, 3 miRNA-mRNA functional interactions were identified by integrative transcriptome analysis. Meaning These results suggest that the identified miRNAs in this study have potential to serve as diagnostic biomarkers in rectal cancer., Importance Accurate clinical staging is important in rectal cancer because it determines the appropriate treatment and prognosis. Despite the use of multiple diagnostic imaging tools, it is sometimes difficult to clinically distinguish stage I tumors from stage II or III locally advanced disease. Identification of differentiating microRNAs (miRNAs) between these 2 groups may improve the clinical diagnostic power and provide insight into the biology of tumor progression. Objectives To investigate differences in the expression of miRNAs in stage I vs stage II or III rectal cancers and integrate matched mRNA profiling data to identify possible functional roles of these miRNAs. Design, Setting, and Participants The primary tumor specimens from patients who were enrolled in 2 prospective clinical trials between March 24, 2004, and November 16, 2012 (American College of Surgeons Oncology Group [ACOSOG] Z6041 and Timing of Rectal Cancer Response to Chemoradiation [TIMING]) were sequenced to arrive at a set of 127 cases (41 stage I and 86 stage II or III tumors) with matched miRNA and messenger RNA (mRNA) profiling data. These findings were also evaluated in an independent cohort of 127 patient specimens (29 stage I and 98 stage II or III tumors) from The Cancer Genome Atlas Rectum Adenocarcinoma (TCGA-READ) that also had matched miRNA and mRNA data. Data analysis was performed from September 1, 2019, to September 1, 2020. Main Outcomes and Measures Alterations in miRNA expression between stage I and stage II or III tumors and their potential gene targets. Results A total of 254 pretreatment rectal adenocarcinoma specimens were analyzed in this study as 2 distinct cohorts: 127 samples in the ACOSOG/TIMING (stage I group: 27 [66%] male; mean [SD] age, 64.4 [10.8] years; stage II or III group: 47 [55%] male; mean [SD] age, 57.0 [11.4] years), and another 127 samples from TCGA-READ (stage I group: 17 [59%] male; mean [SD] age, 63.6 [12.0] years; stage II or III group: 48 [49%] male; mean [SD] age, 64.5 [11.4] years). A total of 19 miRNAs were overexpressed in stage II or III vs stage I tumors in both cohorts. This miRNA signature had an excellent discriminative value for distinguishing stage II or III from stage I rectal tumors (area under the curve, 0.88; 95% CI, 0.83-0.94 in ACOSOG/TIMING cohort and area under the curve, 0.84; 95% CI, 0.77-0.91 in the TCGA-READ cohort). Integrative analysis revealed 3 miRNA-mRNA pairs that exhibited significant correlations in both cohorts: miR-31-5p-SATB2, miR-143-3p-KLF5, and miR-204-5p-EZR. Conclusions and Relevance This diagnostic study found that many of the dysregulated miRNAs in stage II or III vs stage I rectal cancers have biological implications for tumor progression. The results of this study suggest that these miRNAs could assist as diagnostic biomarkers to better identify patients with locally advanced rectal cancer.

Purpose To validate the finding of an association between single nucleotide polymorphisms (SNPs) and toxicity during chemoradiotherapy (CRT) in rectal cancer patients, in an independent population. Methods and Materials The cohort consisted of 165 patients who received CRT for rectal cancer from 2006 to 2012. Prospectively recorded toxicity information, graded according to the Common Terminology Criteria for Adverse Events version 3.0, was retrieved from the medical record. Additionally, a subset of 52 patients recorded their gastrointestinal symptoms weekly during CRT, using the 7-item Bowel Problems Scale. Deoxyribonucleic acid was extracted from normal tissue in the proctectomy specimens and screened for 3 SNPs: XRCC1 R399Q, XPD K751Q, and TGFβ1 R25P. Univariable and multivariable logistic regression models were constructed. Results The median radiation dose was 50.4 Gy, and all patients received concurrent chemotherapy. Toxicities measured by the Common Terminology Criteria for Adverse Events were closely associated with patient-reported outcomes for the patients who completed the 7-item Bowel Problems Scale. Grade ≥3 toxicity occurred during CRT in 14 patients (8%). All 14 patients had either XRCC1 R399Q or TGFβ1 R25P polymorphisms. The TGFβ1 R25P polymorphism was significantly associated with grade ≥3 toxicity (odds ratio [OR] 3.47, P =.04) and, in patients who completed the Bowel Problems Scale, with grade ≥4 toxicity (OR 5.61, P =.02). The latter finding persisted in a multivariable logistic regression model controlling for ethnicity, age, and sex (adjusted OR 1.83, P =.02). Conclusions We have validated the correlation between the TGFβ1 R25P SNP and acute toxicity during CRT in an independent cohort using both clinician- and patient-reported toxicity. The information from our study could be used as a basis to formulate a prospective trial testing the utility of this SNP as a biomarker of acute toxicity during neoadjuvant treatment in locally advanced rectal cancer.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to individual patients. We established a biorepository of 32 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and theirex vivoresponses to clinically relevant chemotherapy and radiation treatment correlate well with responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive rectal cancer followed by metastasis to lung and liver. Importantly, engrafted tumors closely reflected the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based,in vitroplatform coupled with endoluminal propagation in animals.

Locally advanced rectal cancer (LARC) is treated with chemoradiation prior to surgical excision, leaving residual tumors altered or completely absent. Integrating layers of genomic profiling might identify regulatory pathways relevant to rectal tumorigenesis and inform therapeutic decisions and further research. We utilized formalin-fixed, paraffin-embedded pre-treatment LARC biopsies (n=138) and compared copy number, mRNA, and miRNA expression with matched normal rectal mucosa. An integrative model was used to predict regulatory interactions to explain gene expression changes. These predictions were evaluated in vitro using multiple colorectal cancer cell lines. The Cancer Genome Atlas (TCGA) was also used as an external cohort to validate our genomic profiling and predictions. We found differentially expressed mRNAs and miRNAs that characterize LARC. Our integrative model predicted the upregulation of miR-92a, miR-182, and miR-221 expression to be associated with downregulation of their target genes after adjusting for the effect of copy number alterations. Cell line studies using miR-92a mimics and inhibitors demonstrate that miR-92a expression regulates IQGAP2 expression. We show that endogenous miR-92a expression is inversely associated with endogenous KLF4 expression in multiple cell lines, and that this relationship is also present in rectal cancers of TCGA. Our integrative model predicted regulators of gene expression change in LARC using pre-treatment FFPE tissues. Our methodology implicated multiple regulatory interactions, some of which are corroborated by independent lines of study, while others indicate new opportunities for investigation.

The study was undertaken to determine if microsatellite instability (MSI) status influences the composition of the tumor microbiome in primary colon carcinoma. 426 unique primary, stage I-III colon biopsy or resection specimens (234 right-sided, 192 left-sided) were sequenced by MSK-IMPACT, a large panel next generation sequencing (NGS) assay. MSI status [150 MSI-H; 276 microsatellite stable (MSS)] was determined using the MSISensor algorithm, which is clinically validated to detect MSI status from NGS data. We have developed a technique for identifying the presence of microbial species in tumor tissue being tested with NGS by analysis of non-human DNA read sequences. The method has been validated and determined to have comparable accuracy to reference microbial tests for both viruses and bacteria. We identified 13 bacterial species that are enriched in MSI-H colon carcinoma compared to MSS (odds ratio, 95% confidence interval greater than 1 with Bonferonni adjustment) (Table 1). We also observed that MSI-H is more common in right-sided tumors compared to left-sided tumors (53% vs. 13%, p=1.0E-4). To understand if sidedness of the tumor was a significant confounder, we performed stratified analysis of bacteria enrichment for right and left sided colon carcinoma. 8 of the 13 species remained significantly enriched in MSI-H tumors independent of sidedness; 9 of 13 in right-sided tumors; and 12 of 13 in left-sided tumors. Non-significant confidence intervals are underlined in Table 1. While Fusobacterium nucleatum has previously been reported to be enriched in MSI-H colon cancer, the broader spectrum of species enriched in MSI-H colon carcinoma we observed in our study has not been reported. The different microbiome suggests that either these bacteria play a role in development of MSI-H tumors or a unique immune environment exists in MSI-H tumors. The relationship between this unique microbiome and MSI status is the focus of further investigation. Table 1:Bacteria enriched in MSI-H colon cancerMSI-HMSI-HMSI-HMSSMSSMSSBacteria speciesLocationPositiveNegativeTotalPositiveNegativeTotalOdds ratioBonferroni adjusted 95% confidence intervalFusobacterium nucleatumTotal/right/left54/44/1096/81/15150/125/2538/20/18238/89/149276/109/1673.5/2.4/5.5(1.6-7.7)/(1.3-4.4)/(2.2-14.1)Bacteroides fragilisTotal/right/left47/35/12103/90/13150/125/2537/16/21239/93/146276/109/1672.9/2.3/6.4(1.3-6.5)/(1.2-4.4)/(2.6-15.9)Prevotella intermediaTotal/right/left27/22/5123/103/20150/125/2514/10/4262/99/163276/109/1674.1/2.1/10.2(1.4-12.5)/(1.01-4.7)/(2.5-41.1)Prevotella melaninogenicaTotal/right/left20/18/2130/107/23150/125/256/3/3270/106/164276/109/1676.9/5.9/4.8(1.5-31.9)/(1.7-20.8)/(0.8-30.0)Odoribacter splanchnicusTotal/right/left26/20/6124/105/19150/125/2513/5/8263/104/159276/109/1674.2/4.0/6.3(1.4-13.3)/(1.4-11.0)/(2.0-20.0)Selenomonas sputigenaTotal/right/left33/31/2117/94/23150/125/2522/15/7254/94/160276/109/1673.3/2.1/2.0(1.3-8.4)/(1.04-4.1)/(0.4-10.2)Bacteroides xylanisolvensTotal/right/left32/25/7118/100/18150/125/2521/8/13255/101/154276/109/1673.3/3.2/4.6(1.3-8.6)/(1.4-7.3)/(1.6-13.0)Prevotella denticolaTotal/right/left20/16/4130/109/21150/125/259/3/6267/106/161276/109/1674.6/5.2/5.1(1.2-17.2)/(1.5-18.3)/(1.3-19.6)Leptotrichia buccalisTotal/right/left17/13/4133/112/21150/125/256/5/1270/104/166276/109/1675.8/2.4/31.6(1.2-27.2)/(0.8-7.0)/(3.4-296.4)Prevotella sp. oral taxon 299Total/right/left14/12/2136/113/23150/125/252/1/1274/108/166276/109/16714.1/11.5/14.4(1.2-161.7)/(1.5-89.7)/(1.3-165.6)Porphyromonas gingivalisTotal/right/left13/10/3137/115/22150/125/252/0/2274/109/165276/109/16713.0/19.9/11.3(1.1-150.8)/(1.1-343.0)/(1.8-71.1)Streptococcus anginosusTotal/right/left19/16/3131/109/22150/125/2510/4/6266/105/161276/109/1673.9/3.9/3.7(1.1-14.1)/(1.2-11.9)/(0.9-15.7)Fretibacterium fastidiosumTotal/right/left15/13/2135/112/23150/125/256/3/3270/106/164276/109/1675.0/4.1/4.8(1.03-24.3)/(1.1-14.8)/(0.8-30.0) Citation Format: Chad Michael Vanderbilt, Ajaratu Keshinro, Chin-Tung Chen, Esther Babady, Michael F. Berger, Ahmet Zehir, Marc Ladanyi, Jaclyn F. Hechtman, Zsofia K. Stadler, Jinru Shia, Martin R. Weiser. Unique tumor microbiome in microsatellite instability high (MSI-H) colon carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6095.

192 Background: Rectal cancers are clinically different from colon cancers and have not yet been molecularly characterized due to the scarcity of pretreatment specimens. Discovery of molecular determinants of response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II and III) are needed to select those who can avoid surgery and benefit from watch-and-wait strategies. Methods: We profiled 371 pre-treatment specimens using targeted-exome sequencing of 468 cancer genes (n = 325), whole-exome sequencing (n = 100), and RNA-sequencing (n = 113). The targeted-sequencing cohort included patients with stage I (n = 44), II (n = 41), III (n = 176), and IV (n = 64) disease. Primary tumors were divided into lower (LR: 0-4 cm to anal verge, n = 62), middle (MR: 4-8 cm, n = 115), and upper rectum (UR: 8-12 cm, n = 107). We examined molecular determinants of complete response (CR) and relapse free survival (RFS) in LARC patients treated with chemoradiotherapy only (CRT: n = 39), induction chemotherapy + CRT (INCT: n = 87) and consolidation chemotherapy after CRT (CCNT: n = 63). Results: Among MSS cases, oncogenic gene and signaling pathway alterations did not vary by clinical stage. WNT pathway alterations, driven by APC mutations, were more frequent in the UR (89% UR v 86% MR v 60% LR, p < 0.001) while RTK/RAS alterations were more frequent in the LR (54% UR v 69% MR v 72% LR, p < 0.03). A set of genes enriched in mTOR signaling, G2M checkpoint, EMT transition, and DNA repair were overexpressed in the UR (FDR < 0.1). The 5-yr RFS rate for LARC was 75% (CI: 68%-82%) and 24% of the cases had a CR (n = 45). MSI cases had a higher rate of CR compared to MSS cases (50% v 23%, p = 0.07) and none relapsed (n = 8). KRAS-altered MSS tumors exhibited worse RFS in cases treated with CNCT (5-yr: 74% v 97%, p = 0.01), but not in cases treated with INCT (5-yr: 67% v 72%, p = 0.7). Conclusions: WNT alterations are more frequent in the UR while RTK/RAS alterations are more frequent in the LR, suggesting differences in tumor biology between proximal and distal rectal cancer. Further, we report correlations between distinct molecular profiles and response to treatment paradigms that could guide the design of future clinical trials.

This paper proposes an integrated model for investigating how physicians' perceived individual and technology characteristics affect their technological stress (technostress) that is derived from using mobile electronic medical records (MEMRs). Individual characteristics comprise constructs of mobile self-efficacy and technology dependence, whereas perceived technology characteristics comprise constructs of perceived usefulness, complexity, and reliability. We employed the survey method to collect 158 valid questionnaires from physicians working at three branch hospitals to determine perceptions regarding MEMRs, yielding a response rate of 33.62%. Partial least squares, a structural equation modeling technique, was used for model examination and hypothesis testing. The results show that physicians have a low perception of MEMR dependence and technostress. Furthermore, physicians' perceived MEMR technology dependency, mobile self-efficacy, and complexity were proven to significantly affect physician technostress when using MEMRs, whereas perceived usefulness and reliability were not. The explanatory power of the research model reached 67.8%. The results of this study provide valuable insights and significant knowledge for technostress in health care, particularly from academic and practical perspectives.

Background Sorafenib is the standard systemic therapy for unresectable or recurrent hepatocellular carcinoma (HCC) but adds minimal increase in survival. Therefore, there is a great need to develop novel therapies for advanced or recurrent HCC. One emerging field of cancer treatment involves oncolytic viruses that specifically infect, replicate within, and kill cancer cells. In this study, we examined the ability of GLV-1h68, a recombinant vaccinia virus derived from the vaccine strain that was used to eradicate smallpox, to kill sorafenib-resistant (SR) HCC cell lines. Methods Four SR HCC cell lines were generated by repeated passage in the presence of sorafenib. Median inhibitory concentration was determined for all cell lines. The infectivity, viral replication, and cytotoxicity of GLV-1h68 were assayed for both parental and SR HCC cells. Results Infectivity increased in a time and concentration-dependent manner in all cell lines. All cell lines supported efficient replication of virus. No difference between the rates of cell death between the parental and SR cell lines was observed. Conclusion Our results demonstrate that the oncolytic vaccinia virus GLV-1h68 kills both parental and SR HCC cell lines efficiently. This study indicates that patients who have failed treatment with sorafenib remain viable candidates for oncolytic therapy.

A noted clinical application for liquid biopsy is as a non-invasive method of detecting and monitoring of minimal residual disease (MRD) in patients with cancer. The low concentration of circulating tumor DNA in blood, especially in early stage cancers, however, complicates the detection of tumor-derived cell-free DNA. To address this challenge, we have developed MSK-ACCESS (Analysis of Circulating cfDNA to Examine Somatic Status), a custom NGS assay covering selected exons from 129 cancer related genes for high-sensitivity detection of somatic mutations from plasma. Using ultra-high depth sequencing, with duplex unique molecular indexing (UMI), unique dual sample barcodes, and background error suppression, MSK-ACCESS is able to detect low-frequency (0.1%) variants with high confidence. The design of MSK-ACCESS leverages our dataset of more than 30,000 tumors profiled by our institutional tumor sequencing assay, MSK-IMPACT, ensuring that the majority of patients harbor multiple mutations that can be tracked in plasma. We have validated MSK-ACCESS using plasma samples collected from 40 healthy individuals and 70 cancer patients harboring a range of somatic mutations in 11 genes. Greater than 95% of mutations at allele fractions >0.1% were empirically detected, and we established the performance characteristics of the assay through intra- and inter-assay reproducibility tests and dilution experiments. We have initiated clinical trials in multiple tumor types to evaluate the benefit of early therapeutic intervention in patients where MSK-ACCESS can detect circulating tumor DNA following surgery. Tumor mutations revealed by MSK-IMPACT in surgically resected specimens will be monitored at regular intervals as evidence of MRD. As a proof of concept, we have applied MSK-ACCESS to monitor variants known from tissue tumor sequencing in pre- and post-surgical cfDNA samples from 9 colon adenocarcinoma patients. All samples were sequenced to an average total depth of approximately 20,000X coverage and subsequently collapsed to consensus sequences exhibiting an average noise level less than 0.0006%. Circulating tumor DNA was detected in 66%(6/9) of the pre-surgical samples. Of these samples, ctDNA was also detected in 50% (3/6) of the post-surgical samples. Overall, this study shows that MSK-ACCESS can be used to successfully detect MRD. Citation Format: Maysun M. Hasan, Juber Patel, Ian Johnson, Fanli Meng, Grittney K. Tam, Xiaohong Jing, Julie L. Yang, A. Rose Brannon, Jayakumaran Gowtham, Dennis P. Stephens, Monica Diosdado, Ryma Benayed, Ahmet Zehir, Chin-Tung Chen, Martin R. Weiser, Dana Tsui, Brian Houck-Loomis, Michael Berger. Tracking minimal residual disease in post-operative cell-free DNA using MSK-ACCESS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1387.

602 Background: In liquid biopsy, analyses of circulating tumor DNA (ctDNA) in blood have been used as a tool in cancer screening, identifying tumor mutations and monitoring treatment responses/recurrences. Aberrant methylation of some genes have been associated with cancer. Two genes, BCAT1 and IKZF1, are highly methylated and specific to colorectal cancer (CRC) in blood. Detection of these two methylated genes in post-surgery samples also correlates with higher chance of recurrence. Here we investigated the presence of methylated BCAT1 and IKZF1 at multiple time-points for each patient. Methods: Plasma was extracted from blood within 4 hours of the blood collection. ctDNA were extracted from plasma and bisulphite converted using commercially available kits. Real-time qPCR was used to analyze the converted DNA in three replicates and deemed positive if at least one replicate detected either methylated BCAT1 or IKZF1. Quantification of both genes were determined using separate gene-specific standard curves. Results: Nine Stage II/III rectal cancer (RC) patients was enrolled into the study, with one pre-treatment blood draw and several subsequent draws collected throughout the treatment cycles. five patients tested positive for at least one of the methylated genes prior to treatment. Four of these five patients showed an immediate drop in detection for the assayed genes after one cycle of chemotherapy and either remains at low concentration or is undetectable through the rest of the treatment cycles. These patients either have partial or complete response to the treatment regimen. One patient with continuous high levels of methylated BCAT1 and IKZF1 post-treatment have recurred two months after surgery. Conclusions: Methylated BCAT1 and/or IKZF1 were detected in over 50% of the Stage II/III RC patients pre-treatment. This assay is sensitive to the effect of chemotherapy regimen. A significant number of these patients showed a partial/complete response to their treatment regimen and/or reduction in tumor burden, which is reflected in the loss of detection of these two genes. This study shows that this assay may be a viable tool to pair with Imaging for assessment of patient response to treatment regimen.

BackgroundThe response of rectal cancers to neoadjuvant chemoradiation (CRT) is variable, but tools to predict response remain lacking. We evaluated whether KRAS and TP53 mutations are associated with pathologic complete response (pCR) and lymph node metastasis after adjusting for neoadjuvant regimen.MethodsRetrospective analysis of 229 pretreatment biopsies from patients with stage II/III rectal cancer was performed. All patients received CRT. Patients received 0-8 cycles of FOLFOX either before or after CRT, but prior to surgical excision. A subset was analyzed to assess concordance between mutation calls by Sanger Sequencing and a next-generation assay.ResultsA total of 96 tumors (42%) had KRAS mutation, 150 had TP53 mutation (66%), and 59 (26%) had both. Following neoadjuvant therapy, 59 patients (26%) achieved pCR. Of 133 KRAS wild-type tumors, 45 (34%) had pCR, compared with 14 of 96 (15%) KRAS mutant tumors (p=.001). KRAS mutation remained independently associated with a lower pCR rate on multivariable analysis after adjusting for clinical stage, CRT-to-surgery interval and cycles of FOLFOX (OR 0.34; 95% CI 0.17-0.66, p&nbsp

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

HER2 amplification is found in more than 15% of gastric cancers and is associated with poor clinical outcome. Lapatinib, a dual HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has shown promising in vitro results in treating HER2+ cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here, we investigated whether activated MET can confer resistance to lapatinib inhibition of gastric cancer cells. A panel of gastric cancer cell lines was treated with lapatinib, and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT, and extracellular signal-regulated kinase was inhibited by lapatinib and presumably led to cell-cycle arrest as observed with flow cytometry. Hepatocyte growth factor (HGF) activation of MET receptors rescued cells from lapatinib-induced growth inhibition by restimulating the downstream pathways and restoring normal cell-cycle progression. This rescue effect could be abrogated by inhibiting MET with PHA-665752 (a highly specific MET inhibitor) or downregulating MET expression with short interfering RNA. No synergy in growth inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like growth factor 1 and fibroblast growth factor 3 could not uniformly rescue the lapatinib-treated gastric cancer cells. In conclusion, HGF/MET–mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in gastric cancer cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation. Mol Cancer Ther; 11(3); 660–9. ©2012 AACR.

Tumor cells with genomic amplification of MET display constitutive activation of the MET tyrosine kinase, which renders them highly sensitive to MET inhibition. Several MET inhibitors have recently entered clinical trials; however, as with other molecularly targeted agents, resistance is likely to develop. Therefore, elucidating possible mechanisms of resistance is of clinical interest. We hypothesized that collateral growth factor receptor pathway activation can overcome the effects of MET inhibition in MET-amplified cancer cells by reactivating key survival pathways. Treatment of MET-amplified GTL-16 and MKN-45 gastric cancer cells with the highly selective MET inhibitor PHA-665752 abrogated MEK/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling, resulting in cyclin D1 loss and G1 arrest. PHA-665752 also inhibited baseline phosphorylation of epidermal growth factor receptor (EGFR) and HER-3, which are transactivated via MET-driven receptor cross-talk in these cells. However, MET-independent HER kinase activation using EGF (which binds to and activates EGFR) or heregulin-β1 (which binds to and activates HER-3) was able to overcome the growth-inhibitory effects of MET inhibition by restimulating MEK/MAPK and/or PI3K/AKT signaling, suggesting a possible escape mechanism. Importantly, dual inhibition of MET and HER kinase signaling using PHA-665752 in combination with the EGFR inhibitor gefitinib or in combination with inhibitors of MEK and AKT prevented the above rescue effects. Our results illustrate that highly targeted MET tyrosine kinase inhibition leaves MET oncogene-“addicted” cancer cells vulnerable to HER kinase-mediated reactivation of the MEK/MAPK and PI3K/AKT pathways, providing a rationale for combined inhibition of MET and HER kinase signaling in MET-amplified tumors that coexpress EGFR and/or HER-3. [Mol Cancer Ther 2008;7(11):3499–508]