Potential role of nuclear appearance in pathologic recognition and delimitation of sessile serrated polyps of the colon: a karyometric study

OBJECTIVE: To estimate the extent to which surgical pathologists can rely on abnormal nuclear appearance to recognize sessile serrated adenoma (SSP) and to define its extent. STUDY DESIGN: Digitized images of nuclei of superficial crypt cells from SSPs, banal hyperplastic polyps (BHPs), tubular adenomas (TAs) and normal colonic mucosa (N) in surgical pathology specimens were analyzed for size, shape, area, optical density (summed and average) and 22 Markovian texture characteristics. RESULTS: Statistical classification functions correctly distinguished TA from N nuclear image profiles in 93.3% of cases, SSP from N in 70.0% and BHP from N in 74.1%. SSP nuclear feature vectors were less effectively separated from N than BHP and TA on discriminant analysis of the combined data set, and correct classification was achieved in 79.6% of TAs, 53.5% of SSPs and N and 64.2% of BHPs. CONCLUSION: Karyometry distinguished stained nuclei of TAs, but had limited ability to separate nuclei of SSP from those of N and BHP. This suggests that the pathologist attempting to diagnose or delimit the margins of an SSP will find nuclear appearances less helpful than when examining a TA.     

Immunohistochemical staining patterns of MUC1, MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum.

We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyperplastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyperplastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum.     

Near-infrared autofluorescence spectroscopy for in vivo identification of hyperplastic and adenomatous polyps in the colon

This study reports the implementation of an endoscope-based near-infrared (NIR) autofluorescence (AF) spectroscopy technique for in vivo differentiation of normal, hyperplastic and adenomatous colonic polyps during clinical colonoscopic examination. A total of 198 in vivo NIR AF spectra in the range of 810–1050 nm were acquired from colonic tissues (normal (n = 116); hyperplastic (n = 48); and adenomatous polyps (n = 34)) of 96 patients undergoing colonoscopic screening. Significant differences (p < 0.001, one-way analysis of variance (ANOVA)) in in vivo NIR AF intensity among normal, hyperplastic, and adenomatous polyps are observed. Multivariate statistical techniques, including principal components analysis (PCA) and linear discriminate analysis (LDA) together with the leave-one tissue site-out, cross-validation, were used to develop diagnostic algorithms for distinguishing adenomatous polyps from normal and hyperplastic colonic polyps based on NIR AF spectral features. The PCA–LDA modeling on in vivo colonic NIR AF dataset yields diagnostic sensitivities of 83.6%, 77.1%, and 88.2%; and specificities of 96.3%, 88.0%, and 92.1%, respectively, for classification of normal, hyperplastic and adenomatous colonic polyps. This work suggests that NIR AF spectroscopy associated with PCA–LDA algorithms has potential for in vivo diagnosis and detection of colonic precancer at colonoscopy.     

Nasal gene expression differentiates COPD from controls and overlaps bronchial gene expression

Background

Nasal gene expression profiling is a promising method to characterize COPD non-invasively. We aimed to identify a nasal gene expression profile to distinguish COPD patients from healthy controls. We investigated whether this COPD-associated gene expression profile in nasal epithelium is comparable with the profile observed in bronchial epithelium.

Methods

Genome wide gene expression analysis was performed on nasal epithelial brushes of 31 severe COPD patients and 22 controls, all current smokers, using Affymetrix Human Gene 1.0 ST Arrays. We repeated the gene expression analysis on bronchial epithelial brushes in 2 independent cohorts of mild-to-moderate COPD patients and controls.

Results

In nasal epithelium, 135 genes were significantly differentially expressed between severe COPD patients and controls, 21 being up- and 114 downregulated in COPD (false discovery rate?<?0.01). Gene Set Enrichment Analysis (GSEA) showed significant concordant enrichment of COPD-associated nasal and bronchial gene expression in both independent cohorts (FDR_GSEA <?0.001).

Conclusion

We identified a nasal gene expression profile that differentiates severe COPD patients from controls. Of interest, part of the nasal gene expression changes in COPD mimics differentially expressed genes in the bronchus. These findings indicate that nasal gene expression profiling is potentially useful as a non-invasive biomarker in COPD.

Trial registration

ClinicalTrials.gov registration number NCT01351792 (registration date May 10, 2011), ClinicalTrials.gov registration number NCT00848406 (registration date February 19, 2009), ClinicalTrials.gov registration number NCT00807469 (registration date December 11, 2008).

     

Molecular characterization of sessile serrated adenoma/polyps with dysplasia/carcinoma based on immunohistochemistry,next-generation sequencing,and microsatellite instability testing: a case series study

Background

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are considered early precursor lesions in the serrated neoplasia pathway. Recent studies have shown associations of SSA/Ps with lost MLH1 expression, a CpG island methylator phenotype, and BRAF mutations. However, the molecular biological features of SSA/Ps with early neoplastic progression have not yet been fully elucidated, owing to the rarity of cases of SSA/P with advanced histology such as cytologic dysplasia or invasive carcinoma. In this study, we aimed to elucidate the molecular biological features of SSA/Ps with dysplasia/carcinoma, representing relatively early stages of the serrated neoplasia pathway.

Methods

We performed immunostaining for β-catenin, MLH1, and mucins (e.g., MUC2, MUC5AC, MUC6, and CD10); targeted next-generation sequencing; and microsatellite instability (MSI) testing in 8 SSA/P lesions comprised of 4 SSA/Ps with high-grade dysplasia and 4 SSA/Ps with submucosal carcinoma.

Results

Lost MLH1 expression was found in 5 cases. All lesions studied were positive for nuclear β-catenin expression. Regarding phenotypic mucin expression, all lesions were positive for MUC2, but negative for CD10. MUC5AC and MUC6 positivity was observed in 7 cases. Genetically, the most frequently mutated gene was BRAF (7 cases), and other mutations were detected in FBXW7 (3 cases); TP53 (2 cases), and KIT, PTEN, SMAD4, and SMARCB1 (1 case each). Furthermore, 4 of 8 lesions were MSI-high and the remaining 4 lesions were microsatellite-stable (MSS). Interestingly, all 4 MSI-high lesions displayed MLH1 loss, 3 of which harbored a FBXW7 mutation, but not a TP53 mutation. However, 2 MSS lesions harbored a TP53 mutation, although none harbored a FBXW7 mutation.

Conclusions

SSA/Ps with dysplasia/carcinoma frequently harbored BRAF mutations. Activation of the WNT/β-catenin signaling pathway may facilitate the development of dysplasia in SSA/Ps and progression to carcinoma. Furthermore, our results suggested that these lesions might be associated with both MSI-high and MSS colorectal cancer, which might be distinguished by distinct molecular biological features such as lost MLH1 expression, FBXW7 mutations, and TP53 mutations.

     

miRNA expression in colon polyps provides evidence for a multihit model of colon cancer

Changes in miRNA expression are a common feature in colon cancer. Those changes occurring in the transition from normal to adenoma and from adenoma to carcinoma, however, have not been well defined. Additionally, miRNA changes among tumor subgroups of colon cancer have also not been adequately evaluated. In this study, we examined the global miRNA expression in 315 samples that included 52 normal colonic mucosa, 41 tubulovillous adenomas, 158 adenocarcinomas with proficient DNA mismatch repair (pMMR) selected for stage and age of onset, and 64 adenocarcinomas with defective DNA mismatch repair (dMMR) selected for sporadic (n = 53) and inherited colon cancer (n = 11). Sporadic dMMR tumors all had MLH1 inactivation due to promoter hypermethylation. Unsupervised PCA and cluster analysis demonstrated that normal colon tissue, adenomas, pMMR carcinomas and dMMR carcinomas were all clearly discernable. The majority of miRNAs that were differentially expressed between normal and polyp were also differentially expressed with a similar magnitude in the comparison of normal to both the pMMR and dMMR tumor groups, suggesting a stepwise progression for transformation from normal colon to carcinoma. Among the miRNAs demonstrating the largest fold up- or down-regulated changes (≥4), four novel (miR-31, miR-1, miR-9 and miR-99a) and two previously reported (miR-137 and miR-135b) miRNAs were identified in the normal/adenoma comparison. All but one of these (miR-99a) demonstrated similar expression differences in the two normal/carcinoma comparisons, suggesting that these early tumor changes are important in both the pMMR- and dMMR-derived cancers. The comparison between pMMR and dMMR tumors identified four miRNAs (miR-31, miR-552, miR-592 and miR-224) with statistically significant expression differences (≥2-fold change).     

Dissection of a metastatic gene expression signature into distinct components

Background  

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

Immunohistochemical expression of p53 and Ki67 in colorectal adenomas and prediction of malignancy and development of new polyps

The aim of this study was to investigate the immunohistochemical expression of p53 and Ki67 in colorectal adenomas in order to clarify their significance as indicators of malignancy and development of new polyps. Seventy-eight polyps were removed from 51 patients and examined. Twenty-nine patients (56.9%) had adenomas with low-grade atypia (13 of them developed new polyps at 3-year follow-up) and 22 (43.1%) had adenomas with high-grade atypia (6 of them developed new polyps at 3-year follow-up). We tested the association between p53 and Ki67 expression and various clinicopathological variables, and regression analysis was performed to identify the risk factors for malignancy and development of new adenomas. A significant correlation between the grade of atypia and p53 immunoreactivity was observed. Ki67 expression was not related to atypia and no correlation was found between p53 and Ki67 immunoreactivity. Regression analysis showed that size (p=0.0002) and p53 staining (p=0.0111) were the selected factors related to malignant transformation, whereas the number of synchronous primary polyps emerged as the only predictive factor of development of new adenomas, although without statistical significance. The expression of biological markers may be in future added to the currently examined features of polyps; however, further studies are needed to better define their predictive value.     

Generalized dermal perifollicular fibromas with polyps of the colon

Summary The cases of 2 siblings with a rare, generalized genodermatosis have been reported, the most conspicuous feature being multiple perifollicular fibromas. In this type of dermal hamartomas, an apparent autosomal dominant trait was noted as well as the association with a small number of colon polyps in one of the patients. The author assumes a distinct dermo-intestinal syndrome not reported hitherto, which can be differentiated from Gardner's syndrome.     

Identification of a global gene expression signature of B-chronic lymphocytic leukemia

B-chronic lymphocytic leukemia (B-CLL) is an adult-onset leukemia characterized by significant accumulation of apoptosis-resistant monoclonal B lymphocytes. In this study, we performed gene expression profiling on B cells obtained from 10 healthy age-matched individuals and CLL B cells from 38 B-CLL patients to identify key genetic differences between CLL and normal B cells. In addition, we leveraged recent independent studies to assess the reproducibility of our molecular B-CLL signature. We used a novel combination of several methods of data analysis including our own software and identified 70 previously unreported genes that differentiate leukemic cells from normal B cells, as well as confirmed recently reported B-CLL specific expression levels of an additional 10 genes. Importantly, many of these genes have previously been linked with other cancers, thus lending further support to their importance as candidate genes leading to B-CLL pathogenesis. We have also validated a subset of these genes using independent methodologies. Moreover, we show that our genes can be used to create a diagnostics signature that performs with perfect sensitivity and specificity in an independent cohort of 21 B-CLL and 20 normal subjects, thus strongly validating the informative nature of our set of genes. Finally, we identified a group of 31 genes that distinguish between low (Rai stage 0) and high (Rai stage 4) risk patients, suggesting that there may also be a gene expression signature that associates with disease progression.     

Identification of dilated cardiomyopathy signature genes through gene expression and network data integration

Dilated cardiomyopathy (DCM) is a leading cause of heart failure (HF) and cardiac transplantations in Western countries. Single-source gene expression analysis studies have identified potential disease biomarkers and drug targets. However, because of the diversity of experimental settings and relative lack of data, concerns have been raised about the robustness and reproducibility of the predictions. This study presents the identification of robust and reproducible DCM signature genes based on the integration of several independent data sets and functional network information. Gene expression profiles from three public data sets containing DCM and non-DCM samples were integrated and analyzed, which allowed the implementation of clinical diagnostic models. Differentially expressed genes were evaluated in the context of a global protein–protein interaction network, constructed as part of this study. Potential associations with HF were identified by searching the scientific literature. From these analyses, classification models were built and their effectiveness in differentiating between DCM and non-DCM samples was estimated. The main outcome was a set of integrated, potentially novel DCM signature genes, which may be used as reliable disease biomarkers. An empirical demonstration of the power of the integrative classification models against single-source models is also given.