Person:

Bass, Adam

Despite significant advances in diagnosis and treatment, the prognosis of esophageal adenocarcinoma remains poor highlighting the importance of early detection. Although white light (WL) upper endoscopy can be used for screening of the esophagus, it has limited sensitivity for early stage disease. Thus, development of new imaging technology to improve the diagnostic capabilities of upper GI endoscopy for early detection of esophageal adenocarcinoma is an important unmet need. The goal of this study was to develop a method for the detection of malignant lesions in the esophagus using WL upper endoscopy combined with near infrared (NIR) imaging with a protease activatable probe (Prosense750) selective for cathepsin B (CTSB). An orthotopic murine model for distal esophageal adenocarcinoma was generated through the implantation of OE-33 and OE-19 human esophageal adenocarcinoma lines in immunocompromised mice. The mice were imaged simultaneously for WL and NIR signal using a custom-built dual channel upper GI endoscope. The presence of tumor was confirmed by histology and target to background ratios (TBR) were compared for both WL and NIR imaging. NIR imaging with ProSense750 significantly improved upon the TBRs of esophageal tumor foci, with a TBR of 3.64(\pm)0.14 and 4.50(\pm)0.11 for the OE-33 and OE-19 tumors respectively, compared to 0.88(\pm)0.04 and 0.81(\pm)0.02 TBR for WL imaging. The combination of protease probes with novel imaging devices has the potential to improve esophageal tumor detection by fluorescently highlighting neoplastic regions.

The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a five-year survival rate of 15%, identification of new therapeutic targets for EAC is greatly important. We analyze the mutation spectra from whole exome sequencing of 149 EAC tumors/normal pairs, 15 of which have also been subjected to whole genome sequencing. We identify a mutational signature defined by a high prevalence of A to C transversions at AA dinucleotides. Statistical analysis of exome data identified significantly mutated 26 genes. Of these genes, four (TP53, CDKN2A, SMAD4, and PIK3CA) have been previously implicated in EAC. The novel significantly mutated genes include chromatin modifying factors and candidate contributors: SPG20, TLR4, ELMO1, and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 reveal increased cellular invasion. Therefore, we suggest a new hypothesis about the potential activation of the RAC1 pathway to be a contributor to EAC tumorigenesis.

It has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer.

ERBB2-directed therapy is now a routine component of therapy for ERBB2-amplified metastatic gastroesophageal adenocarcinomas. However, there is little knowledge of the mechanisms by which these tumors develop acquired resistance to ERBB2 inhibition. To investigate this question we sought to characterize cell line models of ERBB2-amplified gastroesophageal adenocarcinoma with acquired resistance to ERBB2 inhibition. We generated lapatinib-resistant (LR) subclones from an initially lapatinib-sensitive ERBB2-amplified esophageal adenocarcinoma cell line, OE19. We subsequently performed genomic characterization and functional analyses of resistant subclones with acquired lapatinib resistance. We identified a novel, acquired SrcE527K mutation in a subset of LR OE19 subclones. Cells with this mutant allele harbour increased Src phosphorylation. Genetic and pharmacologic inhibition of Src resensitized these subclones to lapatinib. Biochemically, Src mutations could activate both the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways in the lapatinib-treated LR OE19 cells. Ectopic expression of Src E527K mutation also was sufficient to induce lapatinib resistance in drug-naïve cells. These results indicate that pathologic activation of Src is a potential mechanism of acquired resistance to ERBB2 inhibition in ERBB2-amplified gastroesophageal cancer. Although Src mutation has not been described in primary tumor samples, we propose that the Src hyperactivation should be investigated in the settings of acquired resistance to ERBB2 inhibition in esophageal and gastric adenocarcinoma.

Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention.

Background: KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear. Methods: We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI. Results: KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation]. Conclusions: Tumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted.

Background: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. Findings: We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. Conclusion: The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy.

A more detailed understanding of the somatic genetic events that drive gastrointestinal adenocarcinomas is necessary to improve diagnosis and therapy. Using data from high-density genomic profiling arrays, we conducted an analysis of somatic copy-number aberrations (SCNAs) in 486 gastrointestinal adenocarcinomas including 296 esophageal and gastric cancers. Focal amplifications were substantially more prevalent in gastric/esophageal adenocarcinomas than colorectal tumors. We identified 64 regions of significant recurrent amplification and deletion, some shared and others unique to the adenocarcinoma types examined. Amplified genes were noted in 37% of gastric/esophageal tumors, including in therapeutically targetable kinases such as ERBB2, FGFR1, FGFR2, EGFR, and MET, suggesting the potential utility of genomic amplifications as biomarkers to guide therapy of gastric and esophageal cancers where targeted therapeutics have been less developed compared to colorectal cancers. Amplified loci implicated genes with known involvement in carcinogenesis but also pointed to regions harboring potentially novel cancer genes, including a recurrent deletion found in 15% of esophageal tumors where the Runt transcription factor subunit RUNX1 was implicated, including by functional experiments in tissue culture. Together, our results defined genomic features that were common and distinct to various gut-derived adenocarcinomas, potentially informing novel opportunities for targeted therapeutic interventions.

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial–mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically.

Background: Fibroblast growth factor receptor 2 (FGFR2) genetic alterations lead to tumor cell proliferation in various types of cancer. We hypothesized that FGFR2 amplification is associated with FGFR2 expression, resulting in tumor growth and poorer outcome in esophagogastric junction (EGJ) adenocarcinoma. Patients and Methods A total of 176 consecutive chemo-naive patients with EGJ adenocarcinoma were enrolled from two academic institutions. FGFR2 amplification was examined by real-time PCR (N = 140) and FGFR2 expression with immunohistochemical staining (N = 176), and compared against clinicopathological factors and patient outcomes. The effects of FGFR2 inhibition or overexpression on cell proliferation, cell cycle, and apoptosis assays were investigated in EGJ adenocarcinoma cell lines. Downstream FGFR2, AKT and ERK were also examined. Results: Based on the correlation between FGFR2 levels and FGFR2 overexpression in vitro, FGFR2 amplification was defined as copy number > 3.0. In clinical samples, FGFR2 amplification and FGFR2 IHC expression were 15% and 61%, respectively. Although these two statuses were significantly correlated (P < 0.05), only FGFR2 IHC expression was significantly associated with tumor depth (multivariate P < 0.001) and overall survival of patients (univariate P = 0.007). Supporting these findings, FGFR2 overexpression was associated with tumor cell proliferation, cell cycle progression, and anti-apoptosis. Selective inhibition of FGFR2 sufficiently suppressed tumor cell proliferation through de-phosphorylation of AKT and ERK. Conclusions: FGFR2 amplification was significantly associated with FGFR2 expression. FGFR2 expression (but not FGFR2 amplification) was associated with tumor growth and patient outcomes. Our findings support FGFR2 as a novel therapeutic target for EGJ adenocarcinoma.