Dror Berel

Genetic Predictors of Medically Refractory Ulcerative Colitis

Background: Acute severe ulcerative colitis (UC) remains a significant clinical challenge and the ability to predict, at an early stage, those individuals at risk of colectomy for medically refractory UC (MR‐UC) would be a major clinical advance. The aim of this study was to use a genome‐wide association study (GWAS) in a well‐characterized cohort of UC patients to identify genetic variation that contributes to MR‐UC. Methods: A GWAS comparing 324 MR‐UC patients with 537 non‐MR‐UC patients was analyzed using logistic regression and Cox proportional hazards methods. In addition, the MR‐UC patients were compared with 2601 healthy controls. Results: MR‐UC was associated with more extensive disease (P = 2.7 × 10−6) and a positive family history of UC (P = 0.004). A risk score based on the combination of 46 single nucleotide polymorphisms (SNPs) associated with MR‐UC explained 48% of the variance for colectomy risk in our cohort. Risk scores divided into quarters showed the risk of colectomy to be 0%, 17%, 74%, and 100% in the four groups. Comparison of the MR‐UC subjects with healthy controls confirmed the contribution of the major histocompatibility complex to severe UC (peak association: rs17207986, P = 1.4 × 10−16) and provided genome‐wide suggestive association at the TNFSF15 (TL1A) locus (peak association: rs11554257, P = 1.4 × 10−6). Conclusions: A SNP‐based risk scoring system, identified here by GWAS analyses, may provide a useful adjunct to clinical parameters for predicting the natural history of UC. Furthermore, discovery of genetic processes underlying disease severity may help to identify pathways for novel therapeutic intervention in severe UC. (Inflamm Bowel Dis 2010)

The impact of preoperative serum anti-TNFα therapy levels on early postoperative outcomes in inflammatory bowel disease surgery.

OBJECTIVE Assess the impact of preoperative serum antitumor necrosis factor-α (anti-TNFα) drug levels on 30-day postoperative morbidity in inflammatory bowel disease (IBD) patients. BACKGROUND Studies on the association of anti-TNFα drugs and postoperative outcomes in IBD are conflicting due to variable pharmacokinetics of anti-TNFα drugs. It remains to be seen whether preoperative serum anti-TNFα drug levels correlate with postoperative morbidity. METHODS Thirty-day postoperative outcomes of consecutive IBD surgical patients with serum drawn within 7 days preoperatively were studied. The total serum level of 3 anti-TNFα drugs (infliximab, adalimumab, and certolizumab) was measured, with ≥ 0.98 μg/mL considered as detected. Data were also reviewed according to a clinical cutoff value of 3 μg/mL. RESULTS A total of 217 patients [123 with Crohn disease (CD) and 94 with ulcerative colitis (UC)] were analyzed; 75 of 150 (50%) treated with anti-TNFα therapy did not have detected levels at the time of surgery. In the UC cohort, adverse postoperative outcome rates between the undetectable and detectable groups were similar when stratified according to type of UC surgery. In the CD cohort, there was a higher but statistically insignificant rate of adverse outcomes in the detectable versus undetectable groups. Using a cut off level of 3 μg/mL, postoperative morbidity (odds ratio [OR] = 2.5, P = 0.03) and infectious complications (OR = 3.0, P = 0.03) were significantly higher in the ≥ 3 μg/mL group. There were higher rates of postoperative morbidity (P = 0.047) and hospital readmissions (P = 0.04) in the ≥ 8 μg/mL compared with <3 μg/mL group. CONCLUSIONS Increasing preoperative serum anti-TNFα drug levels are associated with adverse postoperative outcomes in CD but not UC patients.

miR-409-3p/-5p promotes tumorigenesis, epithelial to mesenchymal transition and bone metastasis of human prostate cancer

Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells. Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 4636–46. ©2014 AACR.

A Comparison of Ku0063794, a Dual mTORC1 and mTORC2 Inhibitor, and Temsirolimus in Preclinical Renal Cell Carcinoma Models

Rapamycin analogs, temsirolimus and everolimus, are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). However, the mTOR kinase exists in two distinct multiprotein complexes, mTORC1 and mTORC2, and both complexes may be critical regulators of cell metabolism, growth and proliferation. Furthermore, it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus. We evaluated Ku0063794, which is a small molecule that inhibits both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling proteins downstream of both mTORC1 and mTORC2, including p70 S6K, 4E-BP1 and Akt. Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC cell lines, Caki-1 and 786-O, in vitro by inducing cell cycle arrest and autophagy, but not apoptosis. However, in a xenograft model there was no difference in the inhibition of tumor growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has additional effects on the tumor microenvironment. Consistent with this possibility, temsirolimus, but not Ku0063794, decreased tumor angiogenesis in vivo, and decreased the viability of HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant concentrations. Furthermore, expression levels of VEGF and PDGF were lower in Caki-1 and 786-O cells treated with temsirolimus than cells treated with Ku0063794.

Constitutive TL1A Expression under Colitogenic Conditions Modulates the Severity and Location of Gut Mucosal Inflammation and Induces Fibrostenosis

Intestinal fibrostenosis is a hallmark of severe Crohns disease and can lead to multiple surgeries. Patients with certain TNFSF15 variants overexpress TL1A. The aim of this study was to determine the effect of TL1A overexpression on intestinal inflammation and the development of fibrostenosis. We assessed the in vivo consequences of constitutive TL1A expression on gut mucosal inflammation and fibrostenosis using two murine models of chronic colitis. In the dextran sodium sulfate (DSS) and adoptive T-cell transfer models, there was proximal migration of colonic inflammation, worsened patchy intestinal inflammation, and long gross intestinal strictures in Tl1a transgenic compared to wild-type littermates. In the DSS model, myeloid- and T-cell-expressing Tl1a transgenic mice had increased T-cell activation markers and interleukin-17 expression compared to wild-type mice. In the T-cell transfer model, Rag1(-/-) mice receiving Tl1a transgenic T cells had increased interferon-γ expression but reduced T-helper 17 cells and IL-17 production. Narrowed ureters with hydronephrosis were found only in the Tl1a transgenic mice in all chronic colitis models. In human translational studies, Crohns disease patients with higher peripheral TL1A expression also exhibited intestinal fibrostenosis and worsened ileocecal inflammation with relative sparing of rectosigmoid inflammation. These data show that TL1A is an important cytokine that not only modulates the location and severity of mucosal inflammation, but also induces fibrostenosis.

Clinical, serologic, and genetic factors associated with pyoderma gangrenosum and erythema nodosum in inflammatory bowel disease patients

Background:Pyoderma gangrenosum (PG) and erythema nodosum (EN) are the most common cutaneous manifestations of inflammatory bowel disease (IBD) but little is known regarding their etiopathogenesis. Methods:We performed a case–control study comparing characteristics between IBD patients with a documented episode of PG (PG+) and/or EN (EN+) with those without PG (PG−) and EN (EN−). Data on clinical features were obtained by chart review. IBD-related serology was determined using enzyme-linked immunosorbent assay and genome-wide data generated using Illumina technology. Standard statistical tests for association were used. Results:We identified a total of 92 cases of PG and 103 cases of EN with genetic and clinical characteristics, of which 64 PG and 55 EN cases were available for serological analyses. Fewer male subjects were identified in the PG(+) (odds ratio 0.6, P = 0.009) and EN(+) groups (odds ratio 0.31, P = 0 < 0.0001). Colonic disease, previous IBD-related surgery, and noncutaneous extra-intestinal manifestations were more common among both PG(+) and EN(+) patients compared with controls. PG(+) was associated with anti-nuclear cytoplasmic antibody seropositivity (P = 0.03) and higher anti-nuclear cytoplasmic antibody level (P = 0.02) in Crohns disease. Genetic associations with PG included known IBD loci (IL8RA [P = 0.00003] and PRDM1 [0.03]) as well as with USP15 (4.8 × 10−6) and TIMP3 (5.6 ×10−7). Genetic associations with EN included known IBD susceptibility genes (PTGER4 [P = 8.8 × 10−4], ITGAL [0.03]) as well as SOCS5 (9.64 × 10−6), CD207 (3.14 × 10−6), ITGB3 (7.56 × 10−6), and rs6828740 (4q26) (P < 5.0 × 10−8). Multivariable models using clinical, serologic, and genetic parameters predicted PG (area under the curve = 0.8) and EN (area under the curve = 0.97). Conclusion:Cutaneous manifestations in IBD are associated with distinctive genetic characteristics and with the similar clinical characteristics, including the development of other extra-intestinal manifestations suggesting shared and distinct etiologies.

Colorectal cancer screening and surveillance in Crohn's colitis

AIMS To assess colonoscopic screening and surveillance for detecting neoplasia in patients with long-standing colonic Crohns disease (CD). PATIENTS AND METHODS Colonoscopy and biopsy records from patients with colonic CD were evaluated at the Cedars-Sinai Inflammatory Bowel Disease Center during a 17-year period (1992-2009). RESULTS Overall, 904 screening and surveillance examinations were performed on 411 patients with Crohns colitis (mean 2.2 examinations per patient). The screening and surveillance examinations detected neoplasia in 5.6% of the patient population; 2.7% had low-grade dysplasia (LGD) (n=11), 0.7% had high-grade dysplasia (HGD) (n=3), and 2.2% had carcinoma (anal carcinoma n=3; rectal carcinoma n=6). Mean age of CD diagnosis was 25.6±0.8 years in those with normal examinations, compared to 17.7±2.7 years (p<0.001) in those with HGD, 36.85±1.43 in those with LGD (p=0.021) and 28.32±3.24 years in those with any dysplasia/cancer (p=0.034). Disease duration in patients with normal examinations was 19.1±0.5 years, compared to 36.8±4.4 years (p<0.001) in HGD, 16.88±2.59 in those with LGD (p=0.253) and 30.68±4.03 years in those with any dysplasia/cancer (p=0.152). The mean interval between examinations was higher in HGD (31.5±9.4 months) compared to those with normal colonoscopies (12.92±1.250 months; p=0.002). CONCLUSIONS We detected cancer or dysplasia in 5.6% of patients with long-standing Crohns colitis enrolled in a screening and surveillance program. Younger age at diagnosis of CD, longer disease course, and greater interval between exams were risk factors for the development of dysplasia.

Markers of intestinal inflammation in patients with ankylosing spondylitis: a pilot study

IntroductionInflammatory bowel disease (IBD) and ankylosing spondylitis (AS) are similar chronic inflammatory diseases whose definitive etiology is unknown. Following recent clinical and genetic evidence supporting an intertwined pathogenic relationship, we conducted a pilot study to measure fecal calprotectin (fCAL) and IBD-related serologies in AS patients.MethodsConsecutive AS patients were recruited from a long-term prospectively collected longitudinal AS cohort at Cedars-Sinai Medical Center. Controls were recruited from Cedars-Sinai Medical Center employees or spouses of patients with AS. Sera were tested by ELISA for IBD-associated serologies (antineutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisiae antibody IgG and IgA, anti-I2, anti-OmpC, and anti-CBir1). The Bath Ankylosing Spondylitis Disease Activity Index, the Bath Ankylosing Spondylitis Functional Index, and the Bath Ankylosing Spondylitis Radiology Index were completed for AS patients.ResultsA total of 81 subjects (39 AS patients and 42 controls) were included for analysis. The average age of AS patients was 47 years and the average disease duration was 22 years. AS patients were predominantly male; 76% were HLA-B27-positive. Median fCAL levels were 42 μg/g and 17 μg/g in the AS group and controls, respectively (P < 0.001). When using the manufacturers recommended cutoff value for positivity of 50 μg/g, stool samples of 41% of AS patients and 10% of controls were positive for fCAL (P = 0.0016). With the exception of ANCA, there were no significant differences in antibody levels between patients and controls. Median ANCA was 6.9 ELISA units in AS patients and 4.3 ELISA units in the controls. Among AS patients stratified by fCAL level, there were statistically significant differences between patients and controls for multiple IBD-associated antibodies.ConclusionCalprotectin levels were elevated in 41% of patients with AS with a cutoff value for positivity of 50 μg/g. fCAL-positive AS patients displayed higher medians of most IBD-specific antibodies when compared with healthy controls or fCAL-negative AS patients. Further studies are needed to determine whether fCAL can be used to identify and characterize a subgroup of AS patients whose disease might be driven by subclinical bowel inflammation.

Distinct IFNG methylation in a subset of ulcerative colitis patients based on reactivity to microbial antigens

Background: High antibody reactivity toward microbial antigens in Crohns disease (CD) patients is predictive of a more aggressive disease course. However, few ulcerative colitis (UC) patients exhibit serologic reactivity toward microbial antigens. Mucosal expression of IFN‐&ggr; plays a pivotal role in inflammatory bowel disease (IBD) pathogenesis. Recent genome‐wide association studies (GWAS) surprisingly link UC, but not CD, risk loci to IFNG. We recently demonstrated that mucosal T cells from IBD patients exhibit distinct patterns of IFNG methylation compared to controls. This study evaluated the relationship between IFNG methylation and serologic and clinical profiles in peripheral T cells from IBD patients. Methods: DNA from peripheral T cells of 163 IBD patients (91 CD and 64 UC) and 42 controls was analyzed for methylation of eight IFNG sites. Serum markers ASCA, OmpC, I2, CBir, and pANCA were measured by enzyme‐linked immunosorbent assay (ELISA). IFN‐&ggr; secretion was measured by ELISA. Results: IBD patients requiring surgery exhibited reduced IFNG methylation compared to nonsurgical patients (P < 0.02). Enhancement of IFN‐&ggr; secretion (P < 0.003), along with high antibody responses toward multiple microbial antigens (P < 0.017) in UC, but not CD, patients was correlated with decreased IFNG methylation. pANCA levels were not correlated with IFNG methylation. Conclusions: Levels of IFNG methylation were correlated with immune response to microbial components and expression of IFN‐&ggr; in UC patients. Serological and epigenetic markers identify a subset of UC patients with an expression profile of a key TH1 pathogenic cytokine. These data may provide a useful tool to classify a more homogeneous subset of UC patients, allowing for improved diagnostics and targeted therapeutics. (Inflamm Bowel Dis 2011;)

A prospective analysis of clinical variables, serologic factors, and outcome of ileal pouch-anal anastomosis in patients with backwash ileitis.

PURPOSE: The outcome of ileal pouch-anal anastomosis in patients with backwash ileitis is controversial. We prospectively compared the outcomes of ileal pouch-anal anastomosis in colitis patients with backwash ileitis and colitis patients without backwash ileitis. METHODS: Consecutive colitis patients undergoing ileal pouch-anal anastomosis were reviewed. All patients were classified after surgery as being either backwash ileitis-positive or backwash ileitis-negative. Serum drawn preoperatively was assayed, using enzyme-linked immunosorbent assay, for anti-Saccharomyces cerevisiae, anti-outer membrane of porin C, anti-CBir1, anti-I2, and perinuclear anti-neutrophil cytoplasmic antibody. Outcomes included acute pouchitis (antibiotic responsive), chronic pouchitis (antibiotic dependent or refractory), or de novo Crohns disease (small inflammation above the pouch inlet or pouch fistula). RESULTS: Out of 334 patients, 39 (12%) were backwash ileitis-positive. Compared with backwash ileitis-negative patients, backwash ileitis-positive patients had a higher incidence of pancolitis (100% vs 74%; P = .0001), primary sclerosing cholangitis (15% vs 2%; P = .001) and high-level (>100 enzyme-linked immunosorbent assay units/ml) perinuclear anti-neutrophil cytoplasmic antibody expression (29% vs 9%; P = .001). After a median follow-up of 26 months, 53 patients (16%) developed acute pouchitis, 37 (11%) developed chronic pouchitis, and 40 (12%) developed de novo Crohns disease. There was no significant difference between the backwash ileitis-positive and backwash ileitis-negative patient groups in the incidence of acute pouchitis, chronic pouchitis, or de novo Crohns disease. CONCLUSION: There was a significantly higher incidence of pancolitis, primary sclerosing cholangitis, and high-level perinuclear anti-neutrophil cytoplasmic antibody expression in backwash ileitis-positive patients than in backwash ileitis-negative patients. The incidence of acute pouchitis, chronic pouchitis, and de novo Crohns disease after ileal pouch-anal anastomosis does not differ significantly between backwash ileitis-positive and backwash ileitis-negative patients.