Davendra Sohal

Cerebral Protection Devices Reduce Periprocedural Strokes during Carotid Angioplasty and Stenting: A Systematic Review of the Current Literature:

Purpose: To compare through a systematic review of published literature the stroke outcomes in protected and unprotected carotid artery stenting (CAS). Methods: PubMed and Cochrane electronic databases were queried to identify peer-reviewed publications from 1995 to 2007 meeting our pre-defined criteria for inclusion (English language, human only, at least 20 patients reported) and exclusion (procedures performed for the treatment of total occlusion, dissection, or aneurysmal disease; urgently performed procedures; use of covered stents; access other than transfemoral). Information was collected on a standardized data abstraction form for pooled analysis of total strokes within 30 days of procedure in all patients and in symptomatic and asymptomatic subgroups. A random effects meta-analysis of studies with concurrently reported data on protected and unprotected CAS was performed. Results: Initial database query resulted in 2485 articles, of which 134 were included in the final analyses (12,263 protected CAS patients and 11,198 unprotected CAS patients). Twenty-four studies included data on both protected and unprotected CAS. Using pooled analysis of all 134 reports, the relative risk (RR) for stroke was 0.62 (95% CI 0.54 to 0.72) in favor of protected CAS. Subgroup analysis revealed a significant benefit for protected CAS in both symptomatic (RR 0.67; 95% CI 0.52 to 0.56) and asymptomatic (RR 0.61; 95% CI 0.41 to 0.90) patients (p<0.05). Meta-analysis of the 24 studies reporting data on both protected and unprotected stenting demonstrated a relative risk of 0.59 (95% CI 0.47 to 0.73) for stroke, again favoring protected CAS (p<0.001). Conclusion: Our systematic review indicated that the use of cerebral protection devices decreased the risk of perioperative stroke with CAS. A well designed randomized trial can further confirm our findings and possibly indicate the device with the best outcomes.

Inhibition of the TGF-β receptor I kinase promotes hematopoiesis in MDS

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells, providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-beta-mediated gene activation in BM stromal cells, and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

Reduced SMAD7 Leads to Overactivation of TGF-β Signaling in MDS that Can Be Reversed by a Specific Inhibitor of TGF-β Receptor I Kinase

Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS.

Methylome Profiling Reveals Distinct Alterations in Phenotypic and Mutational Subgroups of Myeloproliferative Neoplasms

Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34+ stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

Meta-Analysis of Microarray Studies Reveals a Novel Hematopoietic Progenitor Cell Signature and Demonstrates Feasibility of Inter-Platform Data Integration

Microarray-based studies of global gene expression (GE) have resulted in a large amount of data that can be mined for further insights into disease and physiology. Meta-analysis of these data is hampered by technical limitations due to many different platforms, gene annotations and probes used in different studies. We tested the feasibility of conducting a meta-analysis of GE studies to determine a transcriptional signature of hematopoietic progenitor and stem cells. Data from studies that used normal bone marrow-derived hematopoietic progenitors was integrated using both RefSeq and UniGene identifiers. We observed that in spite of variability introduced by experimental conditions and different microarray platforms, our meta-analytical approach can distinguish biologically distinct normal tissues by clustering them based on their cell of origin. When studied in terms of disease states, GE studies of leukemias and myelodysplasia progenitors tend to cluster with normal progenitors and remain distinct from other normal tissues, further validating the discriminatory power of this meta-analysis. Furthermore, analysis of 57 normal hematopoietic stem and progenitor cell GE samples was used to determine a gene expression signature characteristic of these cells. Genes that were most uniformly expressed in progenitors and at the same time differentially expressed when compared to other normal tissues were found to be involved in important biological processes such as cell cycle regulation and hematopoiesis. Validation studies using a different microarray platform demonstrated the enrichment of several genes such as SMARCE, Septin 6 and others not previously implicated in hematopoiesis. Most interestingly, alpha-integrin, the only common stemness gene discovered in a recent comparative murine analysis (Science 302(5644):393) was also enriched in our dataset, demonstrating the usefulness of this analytical approach.

Epigenetically Aberrant Stroma in MDS Propagates Disease via Wnt/β-Catenin Activation

The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow-derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine-treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98-HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846-57. ©2017 AACR.

A practical guide to the management of dysphagia in patients with metastatic esophageal cancer

In patients with esophageal cancer, dysphagia can be a distressing symptom, subsequently resulting in weight loss and malnutrition. While luminal obstruction is a common complication, the optimal management approach remains undefined. Given the high morbidity and mortality of esophagectomy and esophageal bypass, surgical palliation has long fallen out of favor. Instead, various other palliative approaches are available, all of which are relatively effective, but differ in regards to time to benefit, durability and toxicity. The available literature is relatively limited, and decisions regarding how to manage dysphagia, and when to appropriately employ these various therapies, are left to individual judgment. Using a tertiary center experience and a review of the current literature, we would typically recommend either systemic chemotherapy or palliative external beam radiotherapy (EBRT) as initial measures. The benefits of chemotherapy include the high likelihood of symptomatic improvement, avoidance of delays in systemic therapy, and minimal loco-regional toxicity. Radiotherapy would similarly be an effective initial approach and may be best suited for patients with low volume (oligo-metastatic) disease. For patients who develop progressive dysphagia later in their disease course, radiotherapy or SEMS placement are the main therapy options. We recommend against “double palliation,” in which patients receive two palliative therapies simultaneously. Overall, decisions regarding management of malignant dysphagia should be individualized to consider the severity of the obstruction, the need for systemic therapy, prior therapy received, and finally the patient’s life expectancy and personal wishes.

Abstract 1504: Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The microenvironment where a tumor originates plays an important role in its initiation, growth, progression and metastatic capability. Since in most cancers the microenvironment is not derived from the malignant clone and does not contain oncogenic mutations, it is likely that the tumor microenvironment is reprogrammed epigenetically to support the growth of the tumor. To test this hypothesis, Fibroblasts associated with primary pancreatic adenocarcinomas (N=7) & healthy fibroblast controls (N=4) were analyzed for genome wide alterations in DNA cytosine methylation by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR). Methylome profiling revealed widespread aberrant cytosine methylation in Pancreatic cancer associated fibroblasts (CAFs) with demethylation of many important gene promoters as the predominant epigenetic event. In addition to loss of methylation, aberrant hypermethylation of GALNTS, JAZF1, MTCH1, SP8, GRB14 was also seen in CAFs. In addition to epigenetic differences, pancreatic CAFs were also found to have widespread transcriptmoic differences as seen by parallel gene expression profiling. Next, we analyzed tumor mediated reprogramming of microenvironment at a higher resolution by a Massive parallel sequencing based methylation analysis (HELP-Tagging) on CAFs differentiated from mesenchymal stem cells (MSCs) in the presence of pancreatic cancer cell conditioned medium. HELP-tagging showed widespread epigenetic reprogramming with 11,100 hypomethylated and 1709 hypermethylated loci. Comparison of in-vitro reprogramed loci with the aberrantly methylated loci from primary CAFs showed a core set of 140 loci that were commonly differentially methylated in both samples. The chemokine receptor CXCR4 was observed to overexpressed & demethylated in both cohorts & was found to be expressed on the surface of primary pancreatic CAFs by immunohistochemistry. Functional studies demonstrated that co-culture of pancreatic cancer cells with CAFs (from MSCs) led to significant increase in malignant cell invasion when compared to co-culture with naive MSCs. This increased invasion was abrogated by blockade of CXCR4 by AMD-3100 and by knockdown of CXCR4 by siRNAs in orthotopically derived CAFs; demonstrating a critical role for this receptor in regulating tumor promoting abilities of the microenvironment. Thus our results reveal for the first time that pancreatic CAFs are characterized by widespread epigenomic reprogramming that includes loss of methylation at many important loci. Validation of an aberrantly demethylated target, CXCR4, shows that inhibition of this receptor can abrogate the ability of CAFs in promoting cancer cell invasion. These results also provide a preclinical rationale for the use of clinically relevant CXCR4 antagonist AMD-3100 (plerixafor) in pancreatic cancer. Citation Format: Tushar D. Bhagat, Yanique Rattigan, Brijesh Patel, Strepell Mirte, Yiting Yu, Davendra Sohal, Matthias Bartenstein, Orsolya Giricz, Shanisha AK Gordon, Nishanth Vallumsetla, Rahul Polineni, Meher Walia, Paraic Kenny, John Greally, Debabrata Banerjee, Anirban Maitra, Amit Verma. Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2013-1504