Immune microenvironment profiling of normal appearing colorectal mucosa biopsied over repeat patient visits reproducibly separates lynch syndrome patients based on their history of colon cancer.

Introduction: Lynch syndrome (LS) is the most common hereditary cause of colorectal cancer (CRC), increasing lifetime risk of CRC by up to 70%. Despite this higher lifetime risk, disease penetrance in LS patients is highly variable and most LS patients undergoing CRC surveillance will not develop CRC. Therefore, biomarkers that can correctly and consistently predict CRC risk in LS patients are needed to both optimize LS patient surveillance and help identify better prevention strategies that reduce risk of CRC development in the subset of high-risk LS patients. Methods: Normal-appearing colorectal tissue biopsies were obtained during repeat surveillance colonoscopies of LS patients with and without a history of CRC, healthy controls (HC), and patients with a history of sporadic CRC. Biopsies were cultured in an ex-vivo explant system and their supernatants were assayed via multiplexed ELISA to profile the local immune signaling microenvironment. High quality cytokines were identified using the rxCOV fidelity metric. These cytokines were used to perform elastic-net penalized logistic regression-based biomarker selection by computing a new measure - overall selection probability - that quantifies the ability of each marker to discriminate between patient cohorts being compared. Results: Our study demonstrated that cytokine based local immune microenvironment profiling was reproducible over repeat visits and sensitive to patient LS-status and CRC history. Furthermore, we identified sets of cytokines whose differential expression was predictive of LS-status in patients when compared to sporadic CRC patients and in identifying those LS patients with or without a history of CRC. Enrichment analysis based on these biomarkers revealed an LS and CRC status dependent constitutive inflammatory state of the normal appearing colonic mucosa. Discussion: This prospective pilot study demonstrated that immune profiling of normal appearing colonic mucosa discriminates LS patients with a prior history of CRC from those without it, as well as patients with a history of sporadic CRC from HC. Importantly, it suggests the existence of immune signatures specific to LS-status and CRC history. We anticipate that our findings have the potential to assess CRC risk in individuals with LS and help in preemptively mitigating it by optimizing surveillance and identifying candidate prevention targets. Further studies are required to validate our findings in an independent cohort of LS patients over multiple visits.

Immune Microenvironment Profiling of Normal Appearing Colorectal Mucosa Biopsied Over Repeat Patient Visits Reproduciably Separates Lynch Syndrome Patients Based on Their History of Colon Cancer.

Introduction: Lynch syndrome (LS) is the most common hereditary cause of colorectal cancer (CRC), increasing lifetime risk of CRC by up to 70%. Despite this higher lifetime risk, disease penetrance in LS patients is highly variable and most LS patients undergoing CRC surveillance will not develop CRC. Therefore, biomarkers that can correctly and consistently predict CRC risk in LS patients are needed to both optimize LS patient surveillance and help identify better prevention strategies that reduce risk of CRC development in the subset of high-risk LS patients. Methods: Normal-appearing colorectal tissue biopsies were obtained during repeat surveillance colonoscopies of LS patients with and without a history of CRC, healthy controls (HC), and patients with a history of sporadic CRC. Biopsies were cultured in an ex-vivo explant system and their supernatants were assayed via multiplexed ELISA to profile the local immune signaling microenvironment. High quality cytokine signatures were identified using rx COV fidelity metric. These signatures were used to perform biomarker selection by computing their selection probability based on penalized logistic regression. Results: Our study demonstrated that cytokine based local immune microenvironment profiling was reproducible over repeat visits and sensitive to patient LS-status and CRC history. Furthermore, we identified sets of biomarkers whose differential expression was predictive of LS-status in patients when compared to sporadic CRC patients and in identifying those LS patients with or without a history of CRC. Enrichment analysis based on these biomarkers revealed an LS and CRC status dependent constitutive inflammatory state of the normal appearing colonic mucosa. Discussion: This prospective pilot study demonstrated that immune profiling of normal appearing colonic mucosa discriminates LS patients with a prior history of CRC from those without it, as well as patients with a history of sporadic CRC from HC. Importantly, it suggests existence of immune signatures specific to LS-status and CRC history. We anticipate that our findings have the potential to assess CRC risk in individuals with LS and help in preemptively mitigating it by optimizing surveillance and identifying candidate prevention targets. Further studies are required to validate our findings in an independent cohort of LS patients over multiple visits.

Non-steroidal anti-inflammatory drugs induce immunogenic cell death in suppressing colorectal tumorigenesis.

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer (CRC). However, the mechanism by which NSAIDs suppress colorectal tumorigenesis remains unclear. We previously showed that NSAIDs selectively kill emerging tumor cells via death receptor (DR) signaling and a synthetic lethal interaction mediated by the proapoptotic Bcl-2 family protein BID. In this study, we found NSAIDs induce endoplasmic reticulum (ER) stress to activate DR signaling and BID in tumor suppression. Importantly, our results unveiled an ER stress- and BID-dependent immunogenic effect of NSAIDs, which may be critical for tumor suppression. NSAID treatment induced hallmarks of immunogenic cell death (ICD) in CRC cells and colonic epithelial cells upon loss of APC tumor suppressor, and elevated tumor-infiltrating lymphocytes (TILs) in the polyps of APCMin/+ mice. ER stress inhibition or BID deletion abrogated the antitumor and immunogenic effects of NSAIDs. Furthermore, increased ER stress and TILs were detected in human advanced adenomas from NSAID-treated patients. Together, our results suggest that NSAIDs induce ER stress- and BID-mediated ICD to restore immunosurveillance and suppress colorectal tumor formation.

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis.

Genomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the in-situ higher-order folding of chromatin structure during malignant transformation remains largely unknown. Here, using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncover a gradual decompaction and fragmentation of higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, and prior to tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal functional consequences in enhanced transcription activities and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin disruption to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.

Smooth muscle tumors of the gastrointestinal tract: an analysis of prognostic features in 407 cases.

Smooth muscle tumors represent the second most common mural mesenchymal neoplasm in the gastrointestinal tract, but established criteria for prognostic assessment of these tumors are lacking. A large cohort of surgically resected intramural gastrointestinal smooth muscle tumors from 31 institutions was analyzed to identify potential prognostic features. Pathologic features were assessed by expert gastrointestinal and/or soft tissue pathologists at each center. Immunohistochemical confirmation was required. A total of 407 cases from the esophagus (n = 97, 24%), stomach (n = 180, 44%), small bowel (n = 74, 18%), and colorectum (n = 56, 14%) were identified. Patients ranged in age from 19 to 92 years (mean 55 years), with a slight female predominance (57%). Mean tumor size was 5.4 cm, with the largest tumor measuring 29 cm. Disease progression following surgery, defined as local recurrence, metastasis, or disease-related death, occurred in 56 patients (14%). Colorectal tumors were most likely to progress, followed by small bowel and gastric tumors. None of the esophageal tumors in this series progressed. Receiver operator characteristic analysis identified optimal cutoffs of 9.8 cm and 3 mitoses/5 mm2 for discriminating between progressive and non-progressive tumors. Histologic features strongly associated with progression by univariate analysis included moderate-to-severe atypia, high cellularity, abnormal differentiation (defined as differentiation not closely resembling that of normal smooth muscle), tumor necrosis, mucosal ulceration, lamina propria involvement, and serosal involvement (P < 0.0001 for all features). Age, sex, and margin status were not significantly associated with progression (P = 0.23, 0.82, and 0.07, respectively). A risk assessment table was created based on tumor site, size, and mitotic count, and Kaplan-Meier plots of progression-free survival for each subgroup revealed progression-based tiers. Based on our findings, it appears that nonesophageal gastrointestinal smooth muscle tumors measuring >10 cm and/or showing ≥3 mitoses/5 mm2 may behave aggressively, and therefore close clinical follow-up is recommended in these cases.

Complete biochemical response after pulmonary metastasectomy in prostate adenocarcinoma.

BACKGROUND: Prostate cancer most commonly metastasizes to bones or lymph nodes, and metastatic prostate cancer is suggestive of disseminated disease. Metastatic disease is usually not amenable to surgery. CASE PRESENTATION: The current report presents a unique case of in which the excision of a solitary pulmonary metastasis resulted in undetectable prostate-specific antigen. CONCLUSION: This case and others suggest metastasectomy could be considered in cases with solitary metastasis, and physicians should have a careful discussion regarding risks and possible benefits from surgical excision.

Prevalence of Anal Dysplasia in Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Although the prevalence of anal dysplasia is higher in some immunosuppressed populations, the prevalence in patients with inflammatory bowel disease (IBD) is unknown. We examined the prevalence of abnormal anal cytology among IBD patients, and its relation to the human papilloma virus (HPV). METHODS: Adults with IBD and age-matched healthy controls (HC) were recruited. IBD patients were categorized as nonimmunosuppressed (IBD-N) or immunosuppressed (IBD-I). Anal Papanicolaou tests were performed for HPV testing and classification by a cytopathologist as follows: negative, atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, cancer, or unsatisfactory. RESULTS: A total of 270 subjects (100 IBD-I, 94 IBD-N, and 76 HC) were recruited. ASC-US were detected in 19 subjects, with a trend toward a higher prevalence among IBD subjects compared with HC (8.8% vs 2.6%; P = .10). The prevalence did not differ with respect to immunosuppression. Crohn's disease (CD) subjects had a higher prevalence of ASC-US compared with others with IBD (P = .02). Among those with CD, female sex and disease duration longer than 10 years were risk factors. There were no cases of low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion, or anal cancer in the cohort. HPV was present in 5.3% and 1.5% of subjects with and without ASC-US, respectively (P = .26). CONCLUSIONS: Although there was a trend toward abnormal anal Papanicolaou tests in IBD subjects compared with HC, there was no difference based on immunosuppression. The presence of HPV did not correlate with abnormal anal cytology. Risk factors associated with this increased trend include female CD subjects and those with a longer duration of CD. ClinicalTrials.gov number: NCT01860963; https://clinicaltrials.gov/ct2/show/NCT01860963.

LEF-1 is frequently expressed in colorectal carcinoma and not in other gastrointestinal tract adenocarcinomas: an immunohistochemical survey of 602 gastrointestinal tract neoplasms.

LEF-1 is a DNA-binding protein that interacts with ß-catenin and activates Wnt-responsive target genes. We analyzed the immunohistochemical expression of LEF-1 in 602 gastrointestinal and pancreatobiliary neoplasms in an attempt to (1) investigate the utility of LEF-1 immunohistochemistry as an ancillary marker in gastrointestinal/pancreatobiliary neoplasia, and (2) to perform a clinicopathologic and survival analysis of colorectal carcinoma stratified by LEF-1 expression. LEF-1 nuclear positivity was frequently identified in colorectal carcinoma (89/241, 37%) and only infrequently identified in other neoplasms: 11% esophagus/esophagogastric adenocarcinomas, 7% gastric adenocarcinomas, 1% pancreatic ductal adenocarcinomas, 4% pancreatic intraductal papillary mucinous neoplasms, and in no cases of appendiceal mucinous neoplasms or pancreatic mucinous cystic neoplasms. LEF-1 expression was identified in 35% of colorectal carcinomas that lacked CK20 and CDX2 expression. In colorectal carcinomas, LEF-1-positive tumors more frequently harbored KRAS mutations compared with LEF-1-negative tumors (39% vs. 16%, P=0.005). Patients with moderate/strong LEF-1-positive colorectal carcinoma had a trend of worse overall survival compared with patients with colorectal carcinomas with weak/negative LEF-1 expression (5 y overall survival, 31% vs. 47%, P=0.15). In conclusion, LEF-1 is most commonly expressed in colorectal carcinoma and infrequently observed in the upper gastrointestinal tract and pancreatic adenocarcinoma. LEF-1 Immunohistochemistry may be especially useful as an ancillary diagnostic marker in colorectal carcinomas, which lack the expression of both CK20 and CDX2. LEF-1 expression is associated with the presence of KRAS mutations and may have prognostic value as a trend of worse overall survival is seen in patients with LEF-1-positive colorectal carcinoma.

Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR.

For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffin embedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is oftentimes degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. Using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 gene (FGFR2) that is amplified in a gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas.