Joel B. Levine

Mutations in BRAF and KRAS Differentially Distinguish Serrated versus Non-Serrated Hyperplastic Aberrant Crypt Foci in Humans

We previously reported that colon carcinomas, adenomas, and hyperplastic polyps exhibiting a serrated histology were very likely to possess BRAF mutations, whereas when these same advanced colonic lesions exhibited non-serrated histology, they were wild type for BRAF; among hyperplastic polyps, KRAS mutations were found mainly in a non-serrated variant. On this basis, we predicted that hyperplastic aberrant crypt foci (ACF), a putative precancerous lesion found in the colon, exhibiting a serrated phenotype would also harbor BRAF mutations and that non-serrated ACF would not. In contrast, KRAS mutations would be found more often in the non-serrated ACF. We examined 55 ACF collected during screening colonoscopy from a total of 28 patients. Following laser capture microdissection, DNA was isolated, and mutations in BRAF and KRAS were determined by direct PCR sequencing. When hyperplastic lesions were further classified into serrated and non-serrated histologies, there was a strong inverse relationship between BRAF and KRAS mutations: a BRAF(V600E) mutation was identified in 10 of 16 serrated compared with 1 of 33 non-serrated lesions (P = 0.001); conversely, KRAS mutations were present in 3 of 16 serrated compared with 14 of 33 non-serrated lesions. Our finding of a strong association between BRAF mutations and serrated histology in hyperplastic ACF supports the idea that these lesions are an early, sentinel, or a potentially initiating step on the serrated pathway to colorectal carcinoma.

Activation of blood coagulation in Crohn's disease. Increased plasma fibrinopeptide A levels and enhanced generation of monocyte tissue factor activity.

We have examined the relationships among activation of blood coagulation, generation of monocyte procoagulant activity, and clinical activity in patients with Crohns disease. Subclinical activation of blood coagulation was measured using a radioimmunoassay for fibrinopeptide A. Fibrinopeptide A levels were strongly correlated with the level of disease activity as measured by the Crohns disease activity index. Patients with active disease who were successfully treated either medically or surgically demonstrated a reduction of fibrinopeptide A levels. Failure of fibrinopeptide A to return to the normal range predicted an early relapse. Monocyte tissue factor generation was assessed in both unstimulated and lipopolysaccharide-stimulated mononuclear cell cultures obtained from the peripheral blood of patients with Crohns disease. A strong correlation (r = 0.89) was observed between plasma fibrinopeptide A levels and monocyte tissue factor generation. These results suggest that monocyte procoagulant generation may contribute to the activation of blood coagulation in this inflammatory bowel disease. Moreover, fibrinopeptide A levels in Crohns disease may provide a useful quantitative measure of inflammatory activity.

Genetic signatures of High- and Low-Risk Aberrant Crypt Foci in a Mouse Model of Sporadic Colon Cancer

To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, β-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low- and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of β-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high- and low- risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.

Cytoplasmic Phospholipase A2 Levels Correlate with Apoptosis in Human Colon Tumorigenesis

Colon cancers often display perturbations in arachidonic acid metabolism, with elevated levels of cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) production frequently observed. Whereas COX-2 and PGE2 are associated with cancer cell survival and tumor angiogenesis, arachidonic acid itself is a strong apoptotic signal that may facilitate cancer cell death. To further explore how cancer cells exploit the progrowth actions of prostaglandins while suppressing the proapoptotic actions of intracellular arachidonic acid, we determined the cytoplasmic phospholipase A2 (cPLA2) and COX-2 expression levels in a panel of human colon tumors by immunohistochemistry. Although high levels of cPLA2 and COX-2 expression are predicted to facilitate maximal prostaglandin production, tumors frequently displayed a high-COX-2/low-cPLA2 phenotype. The least represented phenotype was the high expression of cPLA2, a characteristic predicted to generate the highest levels of intracellular arachidonic acid. The potential proapoptotic role of cPLA2 was supported by a higher frequency of terminal deoxynucleotidyl transferase–mediated nick end labeling staining in cPLA2-positive tumors. Moreover, analysis of preneoplastic aberrant crypt foci from high-risk patients suggests that acquisition of the high-COX-2/low-cPLA2 phenotype may arise at an early stage of colon carcinogenesis. We additionally inhibited cPLA2 in HT-29 cells using antisense oligonucleotides. Our results indicate that cPLA2 plays an important role in tumor necrosis factor α–induced apoptosis in human colon cancer cells. Our data further support the model in which colon cancer growth is favored when intracellular arachidonic acid levels are suppressed by inhibition of cPLA2 or by a high-COX-2/low-cPLA2 phenotype.

Randomized phase II trial of sulindac, atorvastatin, and prebiotic dietary fiber for colorectal cancer chemoprevention.

Sulindac, atorvastatin, or prebiotic dietary fiber may reduce colorectal cancer (CRC) risk. However, clinical trial data are currently limited. We conducted a randomized, phase II chemoprevention trial involving subjects 40 years or older, with previously resected colon cancer or multiple/advanced colorectal adenomas. Magnification chromoendoscopy (MCE) was performed to identify and characterize rectal aberrant crypt foci (ACF); eligibility criteria required five or more rectal ACFs at baseline. Intervention assignments were as follows: (a) atorvastatin 20 mg qd; (b) sulindac 150 mg bid; (c) oligofructose-enriched inulin (as ORAFTI®Synergy1) 6 gm bid; or (d) control (maltodextrin) 6 gm bid, for 6 months. Percent change in rectal ACF number (%ΔACF) within arm was the primary endpoint. Secondary endpoints included changes in proliferation (Ki67) and apoptosis (caspase-3), as measured from normal mucosa biopsy samples. Among 85 eligible randomized subjects, 76 (86%) completed the trial per protocol. The median (range) of rectal ACF was 9 (5–34) and 8 (0–37) at baseline and postintervention, respectively. The median (SD) for %ΔACF was 5.6 (−69% to 143%), −18.6 (−83% to 160%), −3.6 (−88% to 83%), and −10.0 (−100% to 117%) in the atorvastatin, sulindac, ORAFTI®Synergy1 and control arms, respectively. Neither within-arm (P = 0.12–0.59) nor between-arm (P = 0.30–0.92) comparisons of %ΔACF were statistically significant. The active and control interventions also seemed to have similar effects on mucosal proliferation and apoptosis (P > 0.05 for each comparison). Data from this multicenter, phase II trial do not provide convincing evidence of CRC risk reduction from 6-month interventions with atorvastatin, sulindac, or ORAFTI®Synergy1, although statistical power was limited by the relatively small sample size. Cancer Prev Res; 4(2); 259–69. ©2011 AACR.

HD Chromoendoscopy Coupled with DNA Mass Spectrometry Profiling Identifies Somatic Mutations in Microdissected Human Proximal Aberrant Crypt Foci

Despite increased implementation of screening colonoscopy, interval cancers in the proximal colon remain a major public health concern. This fact underscores the limitations of current screening paradigms and the need for developing advanced endoscopic techniques. The density of aberrant crypt foci (ACF), the earliest identifiable mucosal abnormality, may serve as a surrogate marker for colon cancer risk, but has rarely been studied in the proximal colon. To this end, high-definition (HD) chromoendoscopy was conducted to define the relevance of ACF in the proximal colon. In addition, due to limited ACF size, the development of a combinatorial approach was required to maximize data acquisition obtained from individual biopsy samples. Proximal and distal ACF samples were characterized for a total of 105 mutations across 22 known tumor suppressor and proto-oncogenes using high-throughput Sequenom MassARRAY analysis. From this profiling, a discrete number of somatic mutations were identified, including APCR876* and FLT3I836M, as well as a deletion within the EGFR gene. Combined, these data highlight the significance of ACF within the context of colon cancer pathogenesis, particularly in the proximal colon. Implications: The identification of cancer-related mutations in commonly overlooked mucosal lesions underscores the preventive benefit of implementing advanced endoscopic screening to larger patient populations, particularly in the proximal colon. Visual Overview: http://mcr.aacrjournals.org/content/early/2014/05/22/1541-7786.MCR-13-0624/F1.large.jpg. Mol Cancer Res; 12(6); 823–9. ©2014 AACR.

Low Serum Vitamin D: A Surrogate Marker for Advanced Colon Adenoma?

Aims: To examine the association between low 25-OH Vitamin D levels and prevalence of advanced adenomas (AAs) in screening/surveillance colonoscopy patients. Rationale: Low serum 25-OH Vitamin D has been associated with an increased risk for colon cancer. In the Adenoma-Carcinoma pathway, a subset of colon polyps (AA) have been regarded as high-risk precursor lesions. We used a retrospective case-control design to examine the association between Vitamin D deficiency and the prevalence of AA in a high-risk population. Materials and Methods: We examined a total of 354 patients who presented for initial screening or surveillance colonoscopy at our Colon Cancer Prevention Program. Our main exposure variable was serum Vitamin D levels and the outcome was AAs defined as those adenomas that were large (≥1 cm) or had advanced pathology (>25% villous components or high-grade dysplasia). Known risk factors were also collected from the patients’ charts including gender, age, smoking, and family history. Bivariate and multivariate analyses were performed to examine the relationship between serum 25-OH Vitamin D levels and AAs. A total of 354 patients [(males, 188; females, 166); average age, 61 y] charts were reviewed. Vitamin D levels ranged between 4 and 70 ng/mL, with a mean of 25 ng/mL (clinical laboratory normal>30 ng/mL). There was no significant association between serum levels and time of the year of blood draw. Risk for tubular adenoma and AA increased as Vitamin D levels decreased to <30 ng/mL (P=0.002). In total, 80% of AAs were detected in patients whose levels were below this value (odds ratio, 3.36; 95% confidence interval, 1.40-8.03; P=0.007). Bivariate analysis also showed a positive association between smokers with AA as well as those with a family history of colon cancer (P=0.011) and low Vitamin D levels (P=0.001). A multivariate analysis using quintiles of Vitamin D levels demonstrated an increased risk of AAs for patients with levels in the second quintile (33 ng/mL) (odds ratio, 4.3; P=0.01) Main Conclusions: Most patients presenting in our Colon Cancer Prevention Program have low levels of serum 25-OH Vitamin D. Analysis of the results of both screening and surveillance colonoscopies demonstrated an inverse relation between serum 25-OH Vitamin D level and AAs.

Pancreatic ascites: Management by caudal pancreatectomy and side-to-side pancreaticojejunostomy

A patient with pancreatic ascites is presented who had neither a pseudocyst nor demonstrable pancreatic duct disruption, despite the presence of both calculi and strictures in a dilated duct of Wirsung. Concurrently, the patient exhibited intractable abdominal pain characteristic of end-stage chronic alcoholic pancreatitis. The pancreatic ascites responded only briefly to nonoperative management with hyperalimentation. Side-to-side pancreticojejunostomy with caudal pancreatectomy relieved the patient of both pain and ascites, suggesting that this more direct approach may be worthy of consideration in patients with similar findings.

Detection of Antigen-Specific Human Serum Proteins Related to the T-Cell Receptor in Infectious Disease and in an Immune Response to Milk Proteins or Chemicals

A monoclonal IgG2 antibody, MG3C9-1 A12, was prepared by immunization of mice with human serum Cohn Fraction III proteins enriched for TCR Ca+ proteins. MG3C9-1 A12 bound to Mr 28,000, antigen-specific TCR Ca+, beta-, and TCR Ca+, beta+ serum proteins associated with TGF-beta1, 2. The IgG2 monoclonal antibody also bound to T-lymphocyte proteins but did not bind to B lymphocyte proteins, human albumin, IgM, IgG, IgA, or TGF-beta1, 2, 3 immunogenic peptides. Monoclonal MG3C9-1 A12 detected TCR-related proteins specific for filarial extract, milk proteins, or benzoic acid in the sera of individuals with chronic or asymptomatic filariasis, milk intolerance, or sensitivity to toluene, respectively. TCR-related serum proteins were also detected intracellularly in mononuclear cells in frozen sections of ileum from a patient with milk intolerance and reactive mesenteric lymph nodes from a patient with a gastric ulcer. The results suggest that antigen-specific TCR-related serum proteins may be elevated during an immune response to oral, environmental, or infectious stimuli.

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