Small intestinal permeability in patients with eosinophilic oesophagitis during active phase and remission.

BACKGROUND: Eosinophilic oesophagitis (EoE) is presumed to be an isolated oesophageal disease; yet other allergic diseases associated with eosinophilic infiltration of target tissues, such as asthma and eczema, show perturbed functions of other sites that may be involved in the diathesis of allergy modulation. AIM: To analyse small intestinal permeability in patients with active EoE and in a separate group of patients in remission. METHODS: Small bowel permeability was determined using a dual sugar method by calculating lactulose:mannitol (L:M) ratio in 17 patients who met consensus criteria for active EoE (>15 eos/HPF) and 8 patients in remission (<5 eos/HPF). Data from 28 healthy controls was used for comparison. RESULTS: Patients with active EoE had significantly higher L:M ratios when compared to controls (0.045 vs. 0.033, p<0.001) and to EoE in remission (0.041 vs. 0.027, p<.001). There was no significant difference in L:M between the group with EoEin remission and healthy controls. The current data show that L:M ratio of 0.033 also provides a reasonable cut-off that defined the active EoE group compared to patients in remission. The main component explaining the change in L:M ratio was increased absorption (and excretion) of lactulose ((1601 ± 106 ug) when compared to the EoE remission (969 ± 91 ug) and control (1043 ± 92 ug, p<.001) groups. CONCLUSIONS: Small bowel permeability is overall increased in patients with active EoE, and is normal in patients with EoE in remission when compared to healthy controls. The role of the small bowel in active EoE deserves further investigation.

Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19.

Nearly 10% of human gliomas are oligodendrogliomas. Deletion of chromosome arm 19q, often in conjunction with deletion of 1p, has been observed in 65-80% of these tumors. This has suggested the presence of a tumor suppressor gene located on the 19q arm. Chromosome 19 deletion is also of interest due to the better prognosis of patients with deletion, including longer survival and better response to chemotherapy, compared with patients without deletion. Two glioma cell lines with deletion of 19q were used for chromosome 19 microcell-mediated transfer, to assess the effect of replacing the deleted segment. Complementation with chromosome 19 significantly reduced the growth rate of the hybrid cells compared with the parental cell lines. Affymetrix U133 Plus 2.0 Gene Chip analysis was performed to measure and compare the expression of the chromosome 19 genes in the chromosome 19 hybrid cell lines to the parental cell line. Probes were considered significantly different when a P value <0.01 was seen in all of the cell line comparisons. Of 345 probes within the commonly deleted 19q region, seven genes (APOE, RCN3, FLJ10781, SAE1, STRN4, CCDC8, and BCL2L12) were identified as potential candidate genes. RT-PCR analysis of primary tumor specimens showed that several genes had significant differences when stratified by tumor morphology or deletion status. This suggests that one or more of these candidates may play a role in glioma formation or progression.

Molecular analysis of metaplastic breast carcinoma: high EGFR copy number via aneusomy.

Metaplastic breast carcinoma, a rare tumor composed of adenocarcinomatous and nonglandular growth patterns, is characterized by a propensity for distant metastases and resistance to standard anticancer therapies. We sought confirmation that this tumor is a basal-like breast cancer, expressing epidermal growth factor receptor (EGFR) and stem cell factor receptor (KIT). EGFR activating mutations and high copy number (associated with response to tyrosine kinase inhibitor gefitinib) and KIT activating mutations (associated with imatinib sensitivity) were then investigated. Seventy-seven metaplastic cases were identified (1976-2006); 38 with tumor blocks available underwent pathologic confirmation before EGFR and KIT immunohistochemical analyses. A tissue microarray of malignant glandular and metaplastic elements was constructed and analyzed immunohistochemically for cytokeratin 5/6, estrogen receptor, progesterone receptor, and p63, and by fluorescence in situ hybridization for EGFR and HER-2/neu. DNA isolated from individual elements was assessed for EGFR and KIT activating mutations. All assessable cases were negative for estrogen receptor, progesterone receptor, and (except one) HER2. The majority were positive for cytokeratin 5/6 (58%), p63 (59%), and EGFR overexpression (66%); 24% were KIT positive. No EGFR or KIT activating mutations were present; 26% of the primary metaplastic breast carcinomas were fluorescence in situ hybridization-positive, displaying high EGFR copy number secondary to aneusomy (22%) and amplification (4%). We report here that metaplastic breast carcinoma is a basal-like breast cancer lacking EGFR and KIT activating mutations but exhibiting high EGFR copy number (primarily via aneusomy), suggesting that EGFR tyrosine kinase inhibitors should be evaluated in this molecular subset of breast carcinomas.

A t(1;19)(q10;p10) mediates the combined deletions of 1p and 19q and predicts a better prognosis of patients with oligodendroglioma.

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms, suggesting a t(1;19)(q10;p10). Using stem cell medium, we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture, one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas, the prevalence of fusion was 81%. Among NCCTG patients, CEP1/19p12 fusion prevalence was 55%, 47%, and 0% among the oligodendrogliomas, mixed oligoastrocytomas, and astrocytomas, respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P < 0.001, chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion, the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients.

YKL-40 expression is associated with poorer response to radiation and shorter overall survival in glioblastoma.

PURPOSE: YKL-40 is a secreted protein that has been reported to be overexpressed in epithelial cancers and gliomas, although its function is unknown. Previous data in a smaller sample set suggested that YKL-40 was a marker associated with a poorer clinical outcome and a genetically defined subgroup of glioblastoma. Here we test these findings in a larger series of patients with glioblastoma, and in particular, determine if tumor YKL-40 expression is associated with radiation response. EXPERIMENTAL DESIGN: Patients (n=147) with subtotal resections were studied for imaging-assessed changes in tumor size in serial studies following radiation therapy. An additional set (n=140) of glioblastoma patients who underwent a gross-total resection was tested to validate the survival association and extend them to patients with minimal residual disease. RESULTS: In the subtotal resection group, higher YKL-40 expression was significantly associated with poorer radiation response, shorter time to progression and shorter overall survival. The association of higher YKL-40 expression with poorer survival was validated in the gross-total resection group. In multivariate analysis with both groups combined (n = 287), YKL-40 was an independent predictor of survival after adjusting for patient age, performance status, and extent of resection. YKL-40 expression was also compared with genetically defined subsets of glioblastoma by assessing epidermal growth factor receptor amplification and loss at chromosome 10q, two of the common recurring aberrations in these tumors, using fluorescent in situ hybridization. YKL-40 was significantly associated with 10q loss. CONCLUSIONS: The findings implicate YKL-40 as an important marker of therapeutic response and genetic subtype in glioblastomas and suggest that it may play an oncogenic role in these tumors.

Fluorescence in situ hybridization detection of cytogenetic abnormalities in B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma.

Routine cytogenetic analysis of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) frequently fails to identify an abnormal clone due to the low rate of spontaneous mitoses and poor response to mitogen stimulation. Recent studies utilizing interphase fluorescence in situ hybridization (FISH) suggest that prognostically significant chromosomal abnormalities occur more frequently in B-CLL/SLL than has been previously recognized. The purpose of this study was to compare the chromosomal abnormalities detected by karyotyping and FISH in cases of B-CLL/SLL, and to correlate these with clinical features and survival. Seventy-two cases were studied for chromosome 3, 12 or 18 aneuploidy, and for rearrangements involving 11q13, 11q23, 13q14, 14q32 and 17p13. The median age of the patients was 54 years (range, 30-87 years). Clinical staging of B-CLL patients showed that 70% of the patients were Rai stage 0, 1, or 2, and 30% stage 3 or 4. Karyotyping identified chromosomal abnormalities in 31% of the cases, whereas FISH studies were abnormal in 72% of cases including 64% of the cases with normal karyotypes. The most common abnormalities were deletion 13q14 (46%), trisomy 12 (21%), and 14q32 rearrangements (21%). At diagnosis, patients with trisomy 12 were more likely to have a high LDH (P = 0.04), but no other significant differences in the clinical or laboratory features, Rai stage, or survival were found among patients with normal cytogenetics vs. those with chromosomal abnormalities. Univariate analysis showed that B-symptoms (P = 0.044), anemia (P = 0.0006), absolute lymphocytosis > or = 30,000/mm3 (P = 0.029), and Rai stage 3 or 4 (P = 0.0038) at initial presentation were associated with an increased risk of death, but only Rai stage 3 or 4 (P = 0.0038) was significant in multivariate analysis. Interphase FISH studies improve the cytogenetic diagnosis when performed in conjunction with karyotyping in B-CLL/SLL, but the prognostic relevance of various abnormalities could not be confirmed in this study.

Hyperandrogenism induces a proinflammatory TNFα response to glucose ingestion in a receptor-dependent fashion.

CONTEXT: Hyperandrogenism and inflammation are related in polycystic ovary syndrome (PCOS). Hyperandrogenemia can induce inflammation in reproductive-age women, but the mechanism for this phenomenon is unclear. OBJECTIVE: We examined the in vivo and in vitro effects of hyperandrogenism on mononuclear cell (MNC)-derived androgen receptor (AR) status and TNFα release. DESIGN: This study combined a randomized, controlled, double-blind protocol with laboratory-based cell culture experiments. SETTING: This work was performed in an academic medical center. PARTICIPANTS: Lean, healthy, reproductive-age women were treated with 130 mg of dehydroepiandrosterone (DHEA) or placebo (n = 8 subjects each) for 5 days and also provided untreated fasting blood samples (n = 12 subjects) for cell culture experiments. MAIN OUTCOME MEASURES: AR mRNA content and TNFα release were measured before and after DHEA administration in the fasting state and 2 hours after glucose ingestion. TNFα release in the fasting state was also measured in cultured MNCs exposed to androgens with or without flutamide preincubation. RESULTS: At baseline, subjects receiving DHEA or placebo exhibited no significant difference in androgens and TNFα release from MNCs before and after glucose ingestion. Compared with placebo, DHEA administration raised levels of T, androstenedione, and DHEA sulfate, and increased MNC-derived AR mRNA content and TNFα release in the fasting state and in response to glucose ingestion. Compared with MNC exposure to baseline concentrations of DHEA (175 ng/dL) or T (50 ng/dL), the absolute change in TNFα release increased after exposure to T concentrations of 125 and 250 ng/dL and a DHEA concentration of 1750 ng/dL. Preincubation with flutamide reduced the TNFα response by ≥ 60% across all T concentrations. CONCLUSION: Androgen excess in vivo and in vitro comparable to what is present in PCOS increases TNFα release from MNCs of lean healthy reproductive-age women in a receptor-dependent fashion. Hyperandrogenemia activates and sensitizes MNCs to glucose in this population.

Hyperandrogenism exerts an anti-inflammatory effect in obese women with polycystic ovary syndrome.

We determined the effect of chronic androgen suppression on inflammation in women with polycystic ovary syndrome (PCOS) compared to weight-matched controls. We performed a pilot project using samples from previous prospective, controlled studies. Nine women with PCOS (5 obese, 4 lean) and 9 ovulatory controls (5 obese, 4 lean) participated in the study. Androgens, C-reactive protein (CRP), interleukin-6 (IL-6), free fatty acids (FFA) and body weight were measured before and after 3 and 6 months of gonadotropin-releasing hormone (GnRH) agonist administration. GnRH agonist treatment decreased estradiol, testosterone and androstenedione to similar levels in all subjects. CRP and IL-6 increased in obese women with PCOS, was unaltered in lean women with PCOS and obese controls, and decreased in lean controls after 6 months of treatment. FFA decreased and body weight increased in obese women with PCOS, but did not change significantly in lean women with PCOS and in either control group after 6 months of treatment. The testosterone reduction was related to increases in weight and IL-6. The fall in FFA was related to the rise in CRP. The increases in weight and IL-6 were related to the rise in CRP. We propose that hyperandrogenism in PCOS may exert an anti-inflammatory effect when obesity is present, but may not promote inflammation in the disorder; and that circulating androgens have a pleiotropic effect on inflammation depending on the combination of PCOS and weight status in a given individual.

Hyperandrogenism sensitizes leukocytes to hyperglycemia to promote oxidative stress in lean reproductive-age women.

CONTEXT: Hyperandrogenism and oxidative stress are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate oxidative stress. OBJECTIVE: The purpose of this study was to determine the effect of oral androgen administration on fasting and glucose-stimulated leukocytic reactive oxygen species (ROS) generation, reduced nicotinamide adenine dinucleotide phosphate oxidase p47(phox) subunit gene expression, and plasma thiobarbituric acid-reactive substances (TBARS) in lean healthy reproductive-age women. PARTICIPANTS, DESIGN, AND SETTING: Sixteen lean healthy ovulatory reproductive-age women were treated with 130 mg dehydroepiandrosterone (DHEA) or placebo (n = 8 each) for 5 d in this randomized, controlled, double-blind study that was performed at an an academic medical center. MAIN OUTCOME MEASURES: Leukocytic ROS generation, p47(phox) gene expression, and plasma TBARS were quantified in the fasting state and 2 h after glucose ingestion, before and after treatment. RESULTS: Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any prooxidant markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-sulfate, increased the percent change in glucose-challenged p47(phox) RNA content, and increased the percent change in fasting and glucose-challenged ROS generation from mononuclear cells and polymorphonuclear cells, p47(phox) protein content, and plasma TBARS. CONCLUSION: Elevation of circulating androgens comparable to what is present in PCOS increases leukocytic ROS generation, p47(phox) gene expression, and plasma TBARS to promote oxidative stress in lean healthy reproductive-age women. Thus, hyperandrogenemia activates and sensitizes leukocytes to glucose in this population.

Interphase cytogenetics for 1p19q and t(1;19)(q10;p10) may distinguish prognostically relevant subgroups in extraventricular neurocytoma.

Co-deletion of chromosome arms 1p and 19q, characteristic of oligodendroglial tumors, was recently found to be mediated by t(1;19)(q10;p10). To evaluate the prevalence of 1p19q co-deletion and t(1;19) in extraventricular neurocytomas (EVN), we studied tumors from 23 patients, including 13 females and 10 males (median age at diagnosis 34 years, range 2-76 years). Fluorescence in situ hybridization (FISH) studies were performed with probes targeting 1p36/1q25 and 19q13/19p13 to assess for 1p19q co-deletion, as well as chromosome 1 alpha-satellite and 19p12 to detect t(1;19)(q10;p10). FISH was successful in 21 (91%) cases and demonstrated 1p19q co-deletion in five cases (24%) or isolated 1p loss in two cases (10%). Evidence for t(1;19) was found in four (of five) cases with 1p19q co-deletion. Three tumors with 1p19q loss and t(1;19) demonstrated atypical histologic features, compared with one (of 17) tumors without 1p19q co-deletion (P = 0.01, Fisher exact test). In addition, tumors with t(1;19) showed increased mitotic activity compared with tumors without t(1;19) (P = 0.045; Wilcoxon rank sum test). The four patients with t(1;19) developed tumor recurrence (n = 3), or expired (n = 2) 3.5 to 5.5 years after first resection. These results suggest that 1p19q loss and t(1;19) occur in a subset of EVN, and may be associated with aggressive histology in these tumors.

Functional and clinical significance of variants localized to 8q24 in colon cancer.

Multiple genome-wide association studies have identified several susceptibility variants for colon cancer at 8q24. However, the functional roles of these variants have yet to be elucidated. Here, we evaluated the potential role of these markers in tumor progression and examined association with commonly observed structural abnormalities in this region, c-MYC amplification and chromosome fragility at FRA8C and FRA8D. We first replicated the previously reported association by testing 1,178 cases and 1,009 clinic-based controls with eight markers localized to three specific regions at 8q24. We observed significant associations with colon cancer risk with markers rs13254738 (ordinal odds ratio, 0.82; 95% confidence interval, 0.072-0.94; P(trend) = 0.0037) and rs6983267 (ordinal odds ratio, 1.17; 95% confidence interval, 1.03-1.32, P(trend) = 0.013). Survival analysis was done using a separate set of 460 cases to evaluate the clinical significance of these markers. Overall, univariate analysis did not detect survival differences for any of the markers. We also tested a subset of the 460 cases (n = 380) for structural abnormalities at or near the c-MYC locus using fluorescence in situ hybridization analysis. Furthermore, we evaluated a small number of cases homozygous for the rs6983267 alleles to test for differences in fragile site induction. None of the 8q markers correlated with amplification at the c-MYC locus as detected by fluorescence in situ hybridization, and no clear pattern of breakage was observed at the FRA8C and FRA8D sites. In this study, we confirm the association for several single nucleotide polymorphisms at 8q24 in colon cancer but have not detected any structural role relating to c-MYC amplification or chromosomal fragility. Finally, these risk alleles do not seem to be associated with survival.

The prognostic impact of histology and 1p/19q status in anaplastic oligodendroglial tumors.

BACKGROUND: It has been reported previously that the combined loss of chromosomal arms 1p and 19q is a significant predictor of outcome for patients with anaplastic oligodendroglial (AO) tumors and that such chromosomal loss correlates with classic histology in AO. The authors sought to determine whether histology was an equivalent or superior predictor of outcome compared with 1p/19q status in 131 patients with AO tumors. METHODS: The status of 1p and 19q was determined using real-time, quantitative polymerase chain reaction analysis and/or fluorescence in situ hybridization. Clinical features (response to adjuvant therapy and tumor location) and molecular genetic abnormalities (9p and 10q deletions, overexpression of p53 and epidermal growth factor receptor) were determined on available specimens. Histologic assessments for classic oligodendroglial features were performed by five neuropathologists. RESULTS: Classic histology was associated closely with 1p/19q loss, as reported previously. Patients who had tumors that were considered classic by at least four of the five neuropathologists showed significantly increased progression-free and overall survival compared with the patients who had less classic tumors. The authors also tested the correlation between 1p/19q status and outcome in subsets of patients stratified according to classic tumor features. The association of 1p/19q status with survival was related closely to the presence of classic histology. Loss of 1p/19q was predictive of improved outcome only among patients who had tumors with classic histologic features. CONCLUSIONS: The current results suggested that, in addition to 1p/19q status, histologic features contribute information to the prediction of outcome in patients with AO. Loss of 1p and 19q appeared to be a prognostic marker only in the subset of patients who had AO tumors with classic histologic features.