A B-Cell Gene Signature Correlates With the Extent of Gluten-Induced Intestinal Injury in Celiac Disease.

BACKGROUND & AIMS: Celiac disease (CeD) provides an opportunity to study autoimmunity and the transition in immune cells as dietary gluten induces small intestinal lesions. METHODS: Seventy-three celiac disease patients on a long-term, gluten-free diet ingested a known amount of gluten daily for 6 weeks. A peripheral blood sample and intestinal biopsy specimens were taken before and 6 weeks after initiating the gluten challenge. Biopsy results were reported on a continuous numeric scale that measured the villus-height-to-crypt-depth ratio to quantify gluten-induced intestinal injury. Pooled B and T cells were isolated from whole blood, and RNA was analyzed by DNA microarray looking for changes in peripheral B- and T-cell gene expression that correlated with changes in villus height to crypt depth, as patients maintained a relatively healthy intestinal mucosa or deteriorated in the face of a gluten challenge. RESULTS: Gluten-dependent intestinal damage from baseline to 6 weeks varied widely across all patients, ranging from no change to extensive damage. Genes differentially expressed in B cells correlated strongly with the extent of intestinal damage. A relative increase in B-cell gene expression correlated with a lack of sensitivity to gluten whereas their relative decrease correlated with gluten-induced mucosal injury. A core B-cell gene module, representing a subset of B-cell genes analyzed, accounted for the correlation with intestinal injury. CONCLUSIONS: Genes comprising the core B-cell module showed a net increase in expression from baseline to 6 weeks in patients with little to no intestinal damage, suggesting that these individuals may have mounted a B-cell immune response to maintain mucosal homeostasis and circumvent inflammation. DNA microarray data were deposited at the GEO repository (accession number: GSE87629; available: https://www.ncbi.nlm.nih.gov/geo/).

Safety, tolerability, and activity of ALV003: results from two phase 1 single, escalating-dose clinical trials.

BACKGROUND: Celiac disease is the most common hereditary autoimmune disease in humans. The only treatment option for non-refractory celiac disease patients is adherence to a strict life-long gluten-free diet, which often fails to normalize small bowel histology. ALV003 is a mixture of two proteases that degrades gluten and is in clinical development as an oral therapy for patients with celiac disease. AIMS: The safety, tolerability, and activity of ALV003 were assessed in two phase 1 clinical trials. METHODS: In study 1 (N = 28) the study drug was administered in the fasted state; in study 2 (N = 53) the study drug was administered together with a gluten-containing meal. Both studies were single-dose, single-blind, placebo-controlled, cross-over trials. ALV003 was dosed at escalating dose levels by cohort (100, 300, 900, and 1,800 mg) and gastric samples were aspirated using a nasogastric tube. Adverse events, serum drug levels, and anti-drug antibody titers were measured. Gastric samples were assessed for ALV003 enzymatic activity over time (gastric pharmacokinetics) and gluten degradation (gastric pharmacodynamics). RESULTS: All doses were well tolerated, and no serious adverse events or allergic reactions were observed. Gastric aspirates collected 30 min following a meal showed that 100 and 300 mg ALV003 degraded 75 ± 10% (N = 8) and 88 ± 5% (N = 8), respectively, of one gram of wheat bread gluten. CONCLUSIONS: ALV003 is an orally active protease that appears to be stable in the fed stomach and degrades dietary gluten in this compartment. Single doses of oral ALV003 were not associated with serious adverse reactions.

[Evaluation of p63 expression in lung cancer by use of complementary DNA and tissue microarray].

OBJECTIVE: To study p63 expression at mRNA transcript and protein levels in human lung cancers, including squamous cell lung carcinoma (SCC), adenocarcinoma, large cell lung carcinoma (LCLC) and small cell lung carcinoma (SCLC), or the corresponding metastatic foci. The relationship of p63 expression and alterations in p63 locus at chromosomal 3q27-29 was also determined. METHODS: p63 gene expression in 72 cases of SCC, adenocarcinoma, LCLC and SCLC was analyzed by cDNA microarray technology. Tissue microarray of specimens from 150 cases of primary lung cancer was prepared for immunohistochemical study for p63 protein. Possible chromosomal alterations at the p63 locus in 70 cases of primary lung cancer were studied by comparative genomic hybridization (CGH) technology. RESULTS: p63 mRNA transcript expression was significantly increased by more than 10-fold in SCC, as compared with that in other histologic subtypes including adenocarcinoma, LCLC and SCLC. p63 mRNA expression in metastatic foci was also remarkably higher than that in their primary tumors (P < 0.001). Immunostaining showed that p63 protein expression was observed in 94.64% of SCC, whereas only one lung adenocarcinoma (1.79%) was positive. Immunopositivity was also demonstrated in 2 of the 4 LCLC cases studied. None of the SCLC cases was positive. There was a statistically significant difference in p63 expression between pT1 and pT2 tumors (P < 0.05). The CGH results showed that overrepresentation of p63 locus at chromosomal 3q27-29 was a typical finding in SCC. p63 immunopositivity also correlated significantly with pronounced gains of p63 locus at chromosomal 3q27-q29 (P < 0.000 1), suggesting that strong expression of p63 in lung SCC was associated with increased gene amplification. CONCLUSION: p63 may play a role in oncogenesis of human lung squamous cell carcinoma and development of metastasis.

[Study on relationship between p63 expression and 3q27-q29 alteration in non-small cell lung cancer].

BACKGROUND: To investigate the relationship of p63 expression and p63 locus at chromosomal 3q27-q29 in non-small cell lung cancer (NSCLC). METHODS: Chromosomal imbalance in 30 cases of squamous cell carcinoma (SCC) and 40 cases of adenocarcinoma of the lung were evaluated by comparative genomic hybridization (CGH) technology. A tissue microarray of specimens from 122 primary NSCLC specimens was employed and used for immunohistochemical detection of p63 protein expression. RESULTS: p63 positivity was found in 54 (44.26%) cases of NSCLC. p63 immunostaining was observed in 51 (86.44%) of 59 SCC, whereas only one adenocarcinoma (1.67%) showed immunoreactivity. Immunopositivity was seen in 2 (66.66%) of 3 large cell lung cancer (LCLC). No correlation existed between p63 protein expression and the age of patient, sex, tumor grading, tumor metastasis, prognosis (P > 0.05). The CGH results revealed that the gain of chromosome 3q27-q29 was identified in 32 (48.57%) of 70 NSCLC samples tested. Overrepresentation was detected in 24 cases of 30 SCC. In 40 adenocarcinoma, only 8 cases showed chromosome gain at chromosomal 3q27-q29. The comparison of p63 immunostaining with chromosomal alteration of 3q27-q29 demonstrated that pronounced gain was detected in 23 (95.83%) cases of 24 SCC with p63 immunopositivity. One case of adenocarcinoma that was p63 positive showed a chromosomal 3q27-q29 normal representation but not pronounced gain. CONCLUSIONS: The results suggest that p63 immuno-positivity correlates significantly with pronounced gains of the p63 locus at chromosomal 3q27-q29, and p63 gene amplification correlates with development and progression of lung SCC.

Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis.

The programmed cell death 4 gene (PDCD4), a newly identified transformation suppressor, was analysed in lung tumour cell lines and primary lung carcinomas. Reduced PDCD4 mRNA expression was observed in two immortalized lung cell lines and 18 cancer cell lines by northern blot analysis. In the survey of primary lung tumours, PDCD4 cDNA was poorly represented in 47 lung tumours compared with normal lung tissue by cDNA microarray analysis and this poor representation was significantly associated with high-grade (G3) adenocarcinomas (p = 0.012). Immunohistochemical analysis of 124 primary carcinomas comprising all subtypes demonstrated that PDCD4 protein expression was widely lost in tumour samples (83%) and was negatively related to poor prognosis (p = 0.013). The loss of PDCD4 expression correlated with higher grade and disease stage (p = 0.045 and 0.034, respectively), but not tumour size and nodal status. Similarly to the cDNA data, lack of PDCD4 expression was significantly linked to tumour grade in adenocarcinoma (n = 59, p = 0.048), while in squamous cell carcinoma (n = 58), no relationship between PDCD4 expression and clinicopathological parameters was established. These data suggest that the loss of PDCD4 expression is a prognostic factor in lung cancer and may correlate with tumour progression.

Nonparametric methods for identifying differentially expressed genes in microarray data.

MOTIVATION: Gene expression experiments provide a fast and systematic way to identify disease markers relevant to clinical care. In this study, we address the problem of robust identification of differentially expressed genes from microarray data. Differentially expressed genes, or discriminator genes, are genes with significantly different expression in two user-defined groups of microarray experiments. We compare three model-free approaches: (1). nonparametric t-test, (2). Wilcoxon (or Mann-Whitney) rank sum test, and (3). a heuristic method based on high Pearson correlation to a perfectly differentiating gene ('ideal discriminator method'). We systematically assess the performance of each method based on simulated and biological data under varying noise levels and p-value cutoffs. RESULTS: All methods exhibit very low false positive rates and identify a large fraction of the differentially expressed genes in simulated data sets with noise level similar to that of actual data. Overall, the rank sum test appears most conservative, which may be advantageous when the computationally identified genes need to be tested biologically. However, if a more inclusive list of markers is desired, a higher p-value cutoff or the nonparametric t-test may be appropriate. When applied to data from lung tumor and lymphoma data sets, the methods identify biologically relevant differentially expressed genes that allow clear separation of groups in question. Thus the methods described and evaluated here provide a convenient and robust way to identify differentially expressed genes for further biological and clinical analysis.