Allen Mo

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice

Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.

Distinct Transcriptional Changes and Epithelial–Stromal Interactions Are Altered in Early-Stage Colon Cancer Development

Although the progression of mutated colonic cells is dependent upon interactions between the initiated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here, the development of an ultrasensitive laser capture microdissection (LCM)/RNA-seq approach for studying the epithelial and stromal compartments of aberrant crypt foci (ACF) is described. ACF are the earliest identifiable preneoplastic lesion found within the human colon and are detected using high-definition endoscopy with contrast dye spray. The current analysis focused on the epithelium of ACF with somatic mutations to either KRAS, BRAF, or APC, and expression patterns compared with normal mucosa from each patient. By comparing gene expression patterns among groups, an increase in a number of proinflammatory NF-κB target genes was identified that was specific to ACF epithelium, including TIMP1, RELA, and RELB. Distinct transcriptional changes associated with each somatic mutation were observed and a subset of ACF display BRAFV600E-mediated senescence-associated transcriptome characterized by increased expression of CDKN2A. Finally, LCM-captured ACF-associated stroma was found to be transcriptionally distinct from normal-appearing stroma, with an upregulation of genes related to immune cell infiltration and fibroblast activation. Immunofluorescence confirmed increased CD3+ T cells within the stromal microenvironment of ACF and an abundance of activated fibroblasts. Collectively, these results provide new insight into the cellular interplay that occurs at the earliest stages of colonic neoplasia, highlighting the important role of NF-κB, activated stromal fibroblasts, and lymphocyte infiltration. Implications: Fibroblasts and immune cells in the stromal microenvironment play an important role during the earliest stages of colon carcinogenesis. Mol Cancer Res; 14(9); 795–804. ©2016 AACR.

Proximal Aberrant Crypt Foci Associate with Synchronous Neoplasia and Are Primed for Neoplastic Progression

Aberrant crypt foci (ACF) are the earliest morphologically identifiable lesion found within the human colon. Despite their relatively high frequency in the distal colon, few studies have examined the molecular characteristics of ACF within the proximal colon. In the following study, clinical participants (n = 184) were screened for ACF using high-definition chromoendoscopy with contrast dye-spray. Following pathologic confirmation, ACF biopsies were subjected to laser capture microdissection (LCM), and epithelial cells were evaluated for somatic mutations with a customized colorectal cancer mutation panel using DNA-mass spectrometry. Samples were further characterized for microsatellite instability (MSI). Logistic models were used to associate proximal ACF with synchronous (detected during the same procedure) neoplasia. Thirty-nine percent of participants had at least one histologically confirmed proximal ACF. Individuals with a proximal ACF were significantly more likely to present with a synchronous neoplasm (P = 0.001), and specifically, a proximal, tubular, or tubulovillous adenoma (multivariable OR = 2.69; 95% confidence interval, 1.12–6.47; P = 0.027). Proximal ACF were more likely to be dysplastic (52%) compared with distal ACF (13%; P < 0.0001). Somatic mutations to APC, BRAF, KRAS, NRAS, and ERBB2 were detected in 37% of proximal ACF. Hyperplastic ACF were more often MSI-high, but there were no differences in MSI status observed by colonic location. In summary, ACF are identified in the proximal colons of approximately 40% of individuals undergoing chromoendoscopy and more often in patients with synchronous proximal adenomas. Implications: This study provides the most complete set of data, to date, that ACF represent the earliest step in the adenoma–carcinoma sequence but remain below the detection limit of conventional endoscopy. Visual Overview: http//mcr.accrjournals.org/content/molcanres/16/3/486/F1.large.jpg. Mol Cancer Res; 16(3); 486–95. ©2017 AACR. Visual Overview

Targeted Transcriptional Profiling of Microdissected Biopsy Specimens Representing Early Colonic Neoplasia

Our incomplete understanding of the critical changes that accompany the earliest stages of tumor initiation provides a substantial hurdle for the development of novel intervention strategies for cancer prevention. Premalignant lesions are inherently difficult to characterize given their diminutive size, creating technical obstacles for accurate genetic profiling. Here, we describe an approach combining laser‐capture microdissection (LCM) with targeted RNA‐sequencing to study the transcriptional state of epithelial and stromal cells during the earliest detectable stage of human colorectal neoplasia, the aberrant crypt foci (ACF). We provide a robust and reproducible workflow for RNA isolation, library preparation, and expression profiling of laser‐captured cells from frozen OCT‐embedded tissue specimens. It is anticipated that the methodological approach outlined in this report will provide a framework for a broad range of microgenomics analyses that can be routinely applied to many other premalignant tissues. J. Cell. Biochem. 117: 2677–2681, 2016.

A phase IIa randomized, double-blind trial of erlotinib in inhibiting epidermal growth factor receptor signaling in aberrant crypt foci of the colorectum.

Colorectal cancer progresses through multiple distinct stages that are potentially amenable to chemopreventative intervention. Epidermal growth factor receptor (EGFR) inhibitors are efficacious in advanced tumors including colorectal cancer. There is significant evidence that EGFR also plays important roles in colorectal cancer initiation, and that EGFR inhibitors block tumorigenesis. We performed a double-blind randomized clinical trial to test whether the EGFR inhibitor erlotinib given for up to 30 days had an acceptable safety and efficacy profile to reduce EGFR signaling biomarkers in colorectal aberrant crypt foci (ACF), a subset of which progress to colorectal cancer, and normal rectal tissue. A total of 45 patients were randomized to one of three erlotinib doses (25, 50, and 100 mg) with randomization stratified by nonsteroidal anti-inflammatory drug (NSAID) use. There were no unanticipated adverse events with erlotinib therapy. Erlotinib was detected in both normal rectal mucosa and ACFs. Colorectal ACF phosphorylated ERK (pERK), phosphorylated EGFR (pEGFR), and total EGFR signaling changes from baseline were modest and there was no dose response. Overall, this trial did not meet is primary efficacy endpoint. Colorectal EGFR signaling inhibition by erlotinib is therefore likely insufficient to merit further studies without additional prescreening stratification or potentially longer duration of use. Cancer Prev Res; 8(3); 222–30. ©2015 AACR.

Abstract 3240: Proximal human aberrant crypt foci as surrogate markers of colorectal cancer risk

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite effective use of screening colonoscopy, colorectal cancer (CRC) remains the second leading cause of cancer-related deaths. ‘Interval’ cancers, or those occurring between screening colonoscopy procedures, are often found within the proximal (right) colon, underscoring the need for advanced screening approaches using high-definition chromoendoscopy. To better define the colonic mucosa at-risk, we have focused considerable effort on identification and molecular analysis of aberrant crypt foci (ACF), an early macroscopically detectable lesion commonly found in the distal colon. ACF may serve as a surrogate marker of cancer risk, but have rarely been studied within context of the right colon. We hypothesize that proximal ACF may associate with risk factors for CRC and act as a surrogate marker for CRC risk. While distal ACF are frequent (approximately 13 per patient) proximal ACF are not (<1 per patient). We report that subjects with at least one proximal ACF are significantly more likely to have higher ACF multiplicity (p = 0.0002) and more importantly, are more likely to harbor synchronous colonic neoplasia (OR=2.38 [1.24-4.59], p = 0.0087). Histologically, proximal ACF are more frequently dysplastic (41%) compared to distal colon ACF (8%). ACF (n=49) were analyzed for a panel of oncogenes and tumor suppressors using mass spectrometry (MS)-based genotyping (Sequenom). Among the 112 targets analyzed, after KRAS (20%) and BRAF (22%), APC mutations (12%) were most common and specifically associated with dysplasia. Due to their diminutive size and flat morphology, proximal ACF are almost certainly missed during routine colonoscopy. However, our findings suggest that the identification, removal and analysis of proximal ACF may be especially important during screening colonoscopy of high-risk individuals. Citation Format: David A. Drew, Matthew P. Hanley, Allen Mo, Gyuhyeong Goh, Nicole A. Horelik, Thomas J. Devers, Joel Levine, Richard G. Stevens, James J. Grady, Daniel W. Rosenberg. Proximal human aberrant crypt foci as surrogate markers of colorectal cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2014-3240

CDKN2A Copy Number Loss Is an Independent Prognostic Factor in HPV-Negative Head and Neck Squamous Cell Carcinoma

Background HPV infection is associated with high p16 expression and good prognosis in head and neck squamous cell carcinomas (HNSCCs). Analysis of CDKN2A, the gene encoding p16, may further elucidate the association between p16 expression and prognosis. We sought to determine whether CDKN2A copy number loss was associated with poor survival in HPV-negative HNSCCs. Methods The Cancer Genome Atlas HNSCC clinical and genomic data were obtained and integrated. Patients <80 years old with a primary tumor in the oral cavity, oropharynx, hypopharynx, or larynx were included. Stratifying by copy number loss status, CDKN2A mRNA and p16 protein expression levels were examined and overall survival (OS) and disease-free survival (DFS) were evaluated. Results 401 patients with HPV-negative HNSCC were identified. 146 patients demonstrated CDKN2A copy number loss. The CDKN2A copy number loss group expressed significantly lower levels of CDKN2A mRNA and p16 protein than did the non-copy number loss group. Median OS for patients with and without CDKN2A copy number loss was 16.5 and 46.6 months, respectively (p = 0.007). Median DFS for both groups was 11.6 and 19.2 months, respectively (p = 0.03). In both univariate and multivariable analyses, stage IV designation, receipt of chemotherapy and CDKN2A copy number loss were predictive of OS. Conclusion CDKN2A copy number loss predicted poor survival independently of other patient and treatment factors and may be a clinically useful prognostic factor.

Associations of dietary fat with risk of early neoplasia in the proximal colon in a population-based case–control study

PurposeExcess dietary fat consumption is strongly associated with the risk of colorectal cancer, but less is known about its role in the earliest stages of carcinogenesis, particularly within the proximal colon. In the following case–control study, we evaluated the relationship between the intake of dietary fats and the frequency of early proximal neoplasia [aberrant crypt foci (ACF) or polyps], detectable by high-definition colonoscopy with contrast dye-spray.MethodsAverage-risk screening individuals underwent a high-definition colonoscopy procedure as part of larger ongoing clinical study of precancerous lesions in the proximal colon. Dietary fat intake was assessed using the Block Brief Food Frequency Questionnaire, which estimates average dietary intake based on 70 food items. The diets of individuals with no endoscopically identifiable lesions (n = 36) were compared to those with either ACF or polyps detected in the proximal colon.ResultsIn multivariate analysis, high dietary intake of total polyunsaturated fatty acids (PUFAs) and intake of omega-6 and omega-3 fatty acids were positively associated with neoplastic lesions in the proximal colon. When comparing ACF and polyp groups separately, a positive association was observed for both proximal polyps (OR 2.28; CI 1.16–7.09) and ACF (OR 2.86; CI 1.16–7.09) for total PUFA intake. Furthermore, the prevalence of proximal ACF was increased with higher intake of omega-6 (OR 3.54; CI 1.32–9.47) and omega-3 fatty acids (OR 2.29; CI 1.02–5.13), although there was no discernible difference in the omega-6/omega-3 ratio.ConclusionsThese results suggest that dietary PUFAs may be positively associated with risk of early neoplasia in the proximal colon. This study provides further evidence that dietary PUFA composition may play an important role in altering the microenvironment within the human colon.

Abstract 4082: Epithelial-stromal interactions are altered at the earliest stages of colon cancer development

While progression of nonmalignant colonic cells carrying somatic mutations to early malignancy is dependent upon interactions between mutated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here we report the development of an ultra-sensitive laser capture microdissection/RNA-seq approach for profiling the epithelial and stromal compartments of mutated aberrant crypt foci (ACF). ACF are the earliest detectable pre-neoplastic lesion found in human colon and were identified using high-definition endoscopy with contrast dye-spray. We focused on epithelial and stromal cells of ACF carrying somatic mutations to either KRAS, BRAF or APC, and compared these to control samples from each patient. By comparing the gene expression from each group, we identified an increase in a number of pro-inflammatory NF-κB target genes that are specific to ACF epithelium (including TIMP1, RELA and RELB). We confirmed distinct transcriptional changes associated with each somatic mutation and demonstrate that a subset of ACF display a BRAFV600E-mediated senescence-associated transcriptome, characterized by increased expression of CDKN2A (p16). Furthermore, ACF-associated stroma is transcriptionally distinct from adjacent normal stroma and genes related to immune cell infiltration and activation of fibroblasts are up-regulated. Immunofluorescence analysis confirms the abundance of activated fibroblasts in ACF stroma regardless of mutational status. These results provide new insight into the cellular interplay that occurs at the very early stages of colon cancer development, highlighting the role of activated stromal fibroblasts and inflammation. Citation Format: Allen Mo, Stephen Jackson, Kamini Varma, Alan Carpino, Charles Giardina, Thomas J. Devers, Daniel W. Rosenberg. Epithelial-stromal interactions are altered at the earliest stages of colon cancer development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4082.