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e15031 Background: Perioperative chemotherapy improves outcomes for pts with LRA ACA of the E/GEJ compared to surgery alone. Combination chemotherapy regimens are frequently employed, and toxicity ...

532 Background: Early mortality is a major problem in colorectal cancer and our understanding of risk factors and predictors is incomplete. A venous thromboembolism (VTE) Risk Score, comprising primary site, baseline hemoglobin, leukocyte and platelet counts, and body mass index [Khorana et al, Blood, 2008], has been shown to be associated with early mortality in solid tumors [Kuderer et al, 2008; Ay et al, 2013]. We evaluated the value of this Risk Score and other key prognostic variables in predicting early mortality for colorectal cancer (CRC) patients. Methods: CANTARISK was a prospective, non-interventional, global cohort study in patients with CRC and lung cancer initiating a new chemotherapy regimen. Clinical data were collected at 0, 2, 4, and 6 months. All data were compiled centrally and analyzed after the study closed. Risk score categories were as defined previously [Blood, 2008]. Statistically significant univariable associations and a priori prognostic variables were tested in multivariable models; adjusted odds ratios (OR) are presented. Results: A total of 1,789 CRC patients were enrolled from 2011-12; data on 1,533 evaluable patients are presented. Median age was 62 years; 71% were Caucasian. Geographic distribution was Europe 37%, North America 28%; Asia 23%; South America 12%. One-third (33%) had a rectal primary and 65% had metastatic disease. There were 184 deaths (10.3%) on study. For low, intermediate and high Risk Score, there were 8.1%, 11.2%, and 32.5% deaths, respectively. In multivariate analyses, the Risk Score remained an independent predictor of death (OR for high/intermediate vs. low score = 1.70, p = 0.0027), in addition to age (OR for each incremental year = 1.03, p = 0.0014), presence of metastatic disease (OR = 3.28, p < 0.0001), and ECOG performance status ≥2 (OR = 3.85, p < 0.0001). VTE itself was not associated with death. Conclusions: This prospective global cohort study demonstrates that the Risk Score is prognostic of early mortality in CRC patients, in addition to age, stage, and performance status. Intermediate or high-risk patients, as defined by the Risk Score, may benefit from additional interventions aimed at reducing early mortality.

558 Background: Bleeding is an important complication among patients with colorectal cancer but its prevalence and predictors are incompletely understood. We evaluated data from a global prospective cohort study to define the incidence, predictors and consequences of bleeding in patients undergoing adjuvant or palliative treatment for colorectal cancer. Methods: CANTARISK was a prospective, non-interventional, international cohort study in patients with lung and colorectal cancer on chemotherapy, the patients were not on long term anticoagulation. Clinical data were collected at baseline, and at 2, 4, and 6 months. All data were compiled centrally and analyzed after the study had closed. Statistically significant univariable associations and a priori variables were tested in multivariable models; adjusted odds ratios (OR) with confidence intervals (CIs) are presented. Results: A total of 1,789 patients with colorectal cancer were enrolled from 2011-12. Median age was 62 years; 61% were male; 71% were Caucasian and 18% were Asian; 37% were from Europe, 28% from North America, 23% from Asia, and 12% from South America. Ninety-two (5.14%) patients experienced at least one bleeding episode during antineoplastic treatment, ranging from 1 to 5 episodes each, with a total of 109 episodes. The gastrointestinal tract was the source in 49 (45%) episodes. Approximately one-third of episodes (n=34, 31%) necessitated transfusion, but only 1 (0.9%) was fatal. In multivariate analysis, independent predictors of bleeding included presence of metastases (OR = 2.13, 95% CI=1.25-3.65) and surgery within prior 6 months (OR = 3.66, 95% CI=1.78-7.47) but not intact primary tumor (OR = 1.57, 95% CI=0.99-2.51). Notably, age, baseline blood counts, ECOG performance score, and anticoagulant use during study were not associated with bleeding. Conclusions: Bleeding is prevalent amongst patients with colorectal cancer receiving systemic chemotherapy. Post-surgical patients and those with metastatic disease appear to be at highest risk. Resection of the primary tumor does not appear to significantly alter risk.

586 Background: Definitive resection of the primary is frequently part of the management of patients (pts) with stage IV rectal cancer with good performance status and low volume of systemic metastases. It is unclear whether delaying systemic therapy for up front surgical management of the primary compromises overall survival (OS). Methods: Pts with metastatic rectal adenocarcinoma who received definitive surgical resection between 1998-2011 were identified in an IRB approved registry. The sequencing of CT and surgery, and the use of perioperative radiation therapy (RT), was at the discretion of treating physicians. Preoperative chemotherapy (Pre-CT) regimens included 5-fluorouracil (5-FU) +/- leukovorin (LV), capecitabine, 5-FU/LV/oxaliplatin +/- avastin, or 5-FU/LV/irinocetan. RT dose was typically 50.4 Gy. OS was measured from the date of diagnosis. Baseline variables were compared using the Chi-square and unpaired t-tests. OS was calculated using the Kaplan Meier method. Univariate (UVA) and multivariate analysis (MVA) were performed using Cox proportional hazards regression to identify variables associated with OS. Results: In this study of 115 pts, 75 (65%) were treated with pre-CT, while 40 (35%) were treated with up front surgery. Of the pts who received surgery up front, 3 (8%) received RT and of the pts who received pre-CT, 62 (83%) received RT. The cohort was predominantly male (70%) with a median age of 57, median KPS of 80, and median follow-up of 24.1 months. 94% of pts had T3/T4 tumors, 80% had N+ disease, and 33% had poorly differentiated tumors. Liver directed therapy (LDT) was performed in 61% of pts. There was no significant difference in OS (32.3 vs. 32 months; p = 0.24) between pts treated with pre-CT and those who received surgery up front, respectively. UVA demonstrated that pre-CT was not associated with OS (HR 1.26; p = 0.544). MVA demonstrated that pts with poorly differentiated tumors (HR 2.04; p = 0.007) and those that did not undergo LDT (HR 2.45; p = 0.001) had inferior survival. Conclusions: Delaying systemic chemotherapy in order to achieve local control with surgical resection up front does not appear to impact OS in pts with stage IV rectal cancer.

144 Background: The benefit of adjuvant chemotherapy after preoperative chemoradiation and surgery is unclear in patients with locally advanced esophageal cancer. We studied the toxicities and clinical outcomes in patients treated with or without adjuvant chemotherapy (CTX) after trimodality therapy. Methods: Records of patients with T3+ or N+ esophageal cancer who received preoperative chemoradiation followed by surgical resection from 2003-2013 were reviewed. Patients with postoperative deaths or poor performance status within 3 months after surgery were excluded (n = 13). Tolerability and hematologic toxicities of adjuvant CTX were recorded. Clinical outcomes of patients treated with adjuvant CTX were compared with a cohort of patients who received no further therapy (NFT). Results: Of the 81 trimodality patients included in the study, 53 received CTX and 28 received NFT after surgery. Median follow-up time was 23 months. FOLFOX (34%), cisplatin/5-FU (15%), 5-FU/LV (15%), ECF (13%), and carboplatin/paclitaxel (9%) were the most commonly used adjuvant regimens. Multiple rationales for adjuvant CTX were cited, including pathologic nodal status (32%), favorable pathologic response (61%), and provider preference (51%). Grade III/IV hematologic toxicity occurred in 11% of the CTX group: leukopenia (8%/2%), neutropenia (4%/4%), and thrombocytopenia (2%/0%). Two patients in the CTX group did not complete their prescribed CTX, which was discontinued after 1 cycle. Patient and clinical characteristics between CTX and NFT patients were well-balanced, except for pathologic complete response (pCR) rates (CTX 25% vs. NFT 50%, p=0.03). Three-year OS and DFS were similar between CTX and NFT patients (74% vs 70%, 60% vs. 64%, respectively). In patients who achieved pCR (33% overall), adjuvant CTX was associated with an improved 3-yr OS (86% vs. 62%), but the difference did not reach statistical significance (p=0.22). Distant failures occurred in 11% of the CTX group and 18% of the NFT group. Conclusions: Adjuvant CTX after trimodality therapy in esophageal cancer is feasible and well-tolerated with encouraging clinical outcomes. Further studies are needed to define the role of adjuvant CTX in these patients.

e15000 Background: The role of epidermal growth factor receptor (EGFR) inhibition in resectable E/GEJ cancer is uncertain. We update and retrospectively compare results from two Cleveland Clinic trials of concurrent chemoradiotherapy (CCRT) and surgery; the second study differing only by the addition of gefitinib (G) to the treatment regimen. Methods: Eligibility required a diagnosis of E/GEJ squamous cell or adenocarcinoma (ACA), with an endoscopic ultrasound stage of at least T3, N1, or M1a (AJCC 6th). Patients (pts) in both trials received 5-FU (1000mg/m2/d) and cisplatin (20mg/m2/d) as continuous infusions over days 1-4 along with 30 Gy radiation at 1.5 Gy bid. Surgery followed in 4-6 weeks; identical CCRT was given 6-10 weeks later. The second trial added G, 250mg/d, on day 1 for 4 weeks, and again with postoperative CCRT for 2 years. Preliminary results and comparisons have been previously published. Results: Clinical characteristics were similar between the 80 pts on the G trial (2003-2006) and the 93 pts on the no-G trial (1999-2003): median age (58 vs. 59 years), male gender (91% vs. 86%), ACA (94% vs. 83%), and HER2 positivity (28% vs. 18%). Minimum follow-up for all pts was 5 years. Multivariable Cox analyses comparing the G vs. no-G pts and adjusting for statistically significant covariates demonstrated improved overall survival (HR 0.62, 95% CI 0.44-0.88, p=0.008), relapse-free survival (RFS) (HR 0.59, 95% CI 0.41-0.84, p=0.003), and distant metastatic recurrence (HR 0.64, 95% CI 0.43-0.96, p=0.03), but not locoregional recurrence. Further subgroup analyses demonstrated improved RFS with G in pts not experiencing a pathologic response (HR=0.59, 95% CI 0.38-0.92, p=0.021), with T4 or M1a disease at surgery (HR=0.33, 95% CI 0.13-0.87, p=0.024), or with HER2-negative tumors (HR=0.65, 95% CI 0.42-0.99, p=0.048). Conclusions: Although this retrospective comparison can only be considered exploratory, it suggests that gefitinib may improve clinical outcomes when combined with CCRT and surgery in the definitive treatment of E/GEJ cancer. Clinical benefit may be greatest in pts with a poor response to preoperative chemoradiation, and in those with HER2-negative tumors. Clinical trial information: NCT00258323.

4087 Background: In pts with LRA ACA of the E/GEJ, CRT and surgery results in excellent locoregional control. Distant failure remains common, however, suggesting potential benefit from additional chemotherapy. This single arm phase II study investigated the addition of induction chemotherapy to surgery and post-operative CRT. Methods: Pts with an ultrasound-based clinical stage of T3, N1 or M1a (AJCC 6th) ACA of the E/GEJ were eligible. Induction chemotherapy with epirubicin 50mg/m2 d1, oxaliplatin 130mg/m2 d1, and flourouracil 200mg/m2/day continuous infusion for 3 weeks, was given every 21 days for 3 courses and was followed by surgical resection. Adjuvant CRT consisted of 50-55Gy at 1.8-2.0 Gy/d and 2 courses of cisplatin (20mg/m2/d) and flourouracil (1000mg/m2/d) given as 96 hour infusions during weeks 1 and 4 of radiotherapy. Results: Between 2/08 and 1/12, 60 evaluable pts enrolled; 95% male, 97% white and 78% with GEJ tumors. Resection was accomplished in 54 pts (90%) and adjuvant CRT in 48 (80%). Toxicity included 1 death during induction (2%), and 2 post-operative deaths (4%). Unplanned hospitalization was required in 18% of pts during induction and 19% during adjuvant CRT. Induction chemotherapy produced a symptomatic response in 79% of pts, a clinical (ultrasound) response in 48% and a pathologic response in 41% (5% complete). With a median follow-up of 31 months, the Kaplan-Meier 3-year projected locoregional control (LRC) is 84%, distant metastatic control (DMC) 44%, relapse-free survival (RFS) 39%, and overall survival (OS) 42%. Symptomatic response to induction and the percentage of remaining viable tumor at surgery proved the strongest predictors of DMC, RFS, and OS. Conclusions: Induction chemotherapy, surgery and adjuvant CRT is feasible and produces outcomes similar to other multimodality treatment schedules in LRA E/GEJ ACA. Despite excellent LRC, projected DMC and OS remain poor. A symptomatic response to induction and less residual viable tumor at surgery are associated with improved outcomes. Clinical trial information: NCT00601705.

The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow (BM) stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of BM stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, can potentially affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of BM stroma in MDS. To accomplish this aim, adherent non-hematopoietic cells were isolated from MDS patients and controls by immunomagnetic CD45 negative selection and then used for whole genome methylation studies using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR) . Global epigenetic profiling revealed that MDS stroma (n=7) was epigenetically distinct from normal BM stroma (n=3) (ANOVA, P In subsequent studies, we profiled stroma from another set of MDS patients treated with the DNMT inhibitor, 5-Azacytidine (5-Aza) (n=4). In contrast to untreated MDS patients, the 5-Aza treated MDS patients and healthy controls (p = NS) were epigenetically similar. These 5-Aza exposed stroma cells did not demonstrate increased cytosine methylation thus suggesting that these cells were also targeted by DNMT depletion by the drug. Finally, the ability of MDS and control stromal cells to support the growth of healthy CD34+ cells was tested by in-vitro colony formation assay. MDS stromal cell co-culture led to significant reduction in erythroid and myeloid colony formation, demonstrating the MDS stroma contributes to ineffective hematopoiesis seen in this disease. Strikingly, pretreatment of MDS stromal cells with 5-aza increased the colony formation seen in co-cultures, demonstrating again that DNMT inhibitors exert effects on the microenvironment. Thus our results reveal for the first time that MDS is characterized by widespread aberrant epigenetic changes in the BM microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, contributing in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire BM, including the microenvironment to improve our understanding of the pathophysiology of MDS and therapy. Citation Format: Tushar D. Bhagat, Matthias Bartenstein, Yiting Yu, A. Mario Marcondes, Davendra Sohal, John Greally, H Joachim Deeg, Amit Verma. Myelodysplasia bone marrow stromal cells have widespread aberrant hypermethylation that is targeted by treatment with DNMT inhibitors. [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 1503. doi:10.1158/1538-7445.AM2013-1503

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 naïve 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

4111 Background: Cholangiocarcinoma is an aggressive neoplasm. Current chemotherapy approaches achieve modest results. The epidermal growth factor receptor (EGFR) pathway appears to be associated with tumor stage, prognosis and response to therapy. This trial was designed to evaluate the tolerability and efficacy of the combination of panitumumab, a monoclonal anti-EGFR antibody, with gemcitabine and irinotecan, in patients with advanced cholangiocarcinoma. Molecular analysis of EGFR pathway genes was planned as well. Methods: Patients with advanced (unresectable or metastatic) cholangiocarcinoma, ECOG PS 0-2, and adequate liver, kidney and bone marrow function were treated with panitumumab (9 mg/kg) on day 1, and gemcitabine (1000 mg/m2) and irinotecan (100 mg/m2) on days 1 and 8 of a 21-day cycle. Tissue specimens were collected at diagnosis for correlative molecular analyses. Primary objective is to evaluate the 5-month progression-free survival (PFS) rate. Secondary objectives include overall response rate (ORR), overall survival (OS) and toxicity of the combination. Mutational analysis of EGFR (del 19; 858), KRAS (codons 12, 13) and BRAF (V600E) was done on samples with adequate material for testing. Results: There have been 26 (of planned 42) patients recruited to the study. A median of 6 (0-30) cycles were administered. There were no treatment related deaths. The most common gr 3 or higher toxicities were neutropenia (10 pts, 38%), thrombocytopenia (10 pts, 38%), skin rash (10 pts, 38%) and diarrhea (3 pts, 12%). During the study, there were 3 CR, 6 PR, 10 SD (disease control rate of 90%), and 2 PD (by RECIST) in 21 evaluable pts. Two pts went on to have surgical resection. Median OS is 12.7 months. Of 13 testable samples, no EGFR or BRAF mutations were identified; however, there were 7 KRAS mutations. Retrospective analysis showed no difference in OS by KRAS mutation status. Conclusions: Interim evaluation of this ongoing study showed encouraging tolerability and efficacy of this regimen. Several patients have KRAS mutations; there appears to be no association with response, however. The pre-specified efficacy criteria to continue enrollment were met.

e13602 Background: MGCD265 is a multi-target oral tyrosine kinase receptor inhibitor that targets Met (wild type and clinically-relevant Met mutants), VEGFRs 1, 2, 3, Tie and Ron. Combining Met and EGFR inhibitors has been shown to be synergistic and can overcome resistance to EGFR inhibitors. MGCD265 in combination with erlotinib is being evaluated clinically for safety and efficacy. Methods: This is a phase I dose escalation trial of patients (pts) with advanced solid tumors, using the classic 3+3 design. MGCD265 and erlotinib were administered every day over a 21-day cycle. Safety evaluation included determination of dose limiting toxicities (DLTs) and the maximum tolerated dose of the combination. The pharmacokinetic (PK), pharmacodynamic (PD) profiles as well as anti-tumor activity, using RECIST 1.1, were also evaluated. Results: As ofJanuary 11, 2012, 45 pts were enrolled (median age: 58 years old; M/F: 27/18; ECOG 0/1: 20/25). MGCD265 was dose escalated from 96 mg/m2 QD to 162 mg/m2 BID, in combination with erlotinib (initially at 100 mg then at 150 mg QD). Diarrhea (n=6) was the only treatment-related ≥ grade 3 adverse event observed in ≥ 2 pts. The observed DLTs (all grade 3) were (n=1): diarrhea (96 mg/m2 QD MGCD265+100 mg erlotinib), rash and fatigue (144 mg/m2 QD MGCD265+150 mg erlotinib) and rhabdomyolysis (162 mg/m2 BID MGCD265+150 mg erlotinib). One out of 3 pts with NSCLC, who was positive for activating EGFR mutation, experienced a partial response (duration of response of 8 cycles). Seven pts with a variety of tumors experienced stable disease for 6 cycles or more. Three out of 8 pts with gastroesophageal cancer remained on study for ~12-26 cycles. The PD profile indicated a decrease in the plasma level of HGF at Cycle 1 Day 8 compared to baseline in some patients. Conclusions: MGCD265, at the doses tested, was well tolerated in combination with full-dose erlotinib. The activity of MGCD265 combined with erlotinib supports phase II development of the combination.

328 Background: Cholangiocarcinoma is an aggressive neoplasm. Current chemotherapy approaches suggest that combinations may be superior to single agents in this disease. Over-expression of epidermal growth factor receptor (EGFR) is associated with tumor stage and prognosis. This study was designed to evaluate the efficacy and tolerability of the combination of panitumumab, a monoclonal anti-EGFR antibody, with gemcitabine and irinotecan in patients with advanced and metastatic cholangiocarcinoma. Methods: Pts with advanced unresectable or metastatic cholangiocarcinoma, ECOG PS 0-2, and adequate liver, kidney and bone marrow function were treated with panitumumab (9 mg/kg) on day 1, gemcitabine (1000 mg/m2/100 min) iv and irinotecan (100 mg/m2) iv on days 1 and 8 of a 21-day cycle. Tissue specimens were collected based on availability for future biomarker analyses. The primary objective was to evaluate the 5-month progression-free survival (PFS) rate. The secondary objectives include overall response rate (ORR), overall survival rate (OS) and toxicity of the combination. Results: There have been 24 (of planned 42) pts recruited to the study. Toxicity of the combination was assessed in all enrolled pts. There were no treatment related deaths. The most common gr 3 or higher toxicity was were neutropenia (10 pts [40%]), thrombocytopenia (6 pts, [25%]), skin rash (4 pts [17%]) and diarrhea (3 pts, [12%]). One pt developed a gr 2 infusion reaction, which was considered related to panitumumab. Efficacy was evaluated in 20 pts. Among them, there were 2 CR, 6 PR, and 10 SD (with disease control rate of 90%), and 2 PD (assessed by RECIST criteria). Two pts went on to have surgical resection. Eight pts had ≥ 10 cycles of the therapy, and dose modification/interruption were needed for some of these pts. Conclusions: The data of this on-going study showed encouraging results of the combination of panitumumab with gemcitabine and irinotecan in both tolerability and efficacy. The pre-specified efficacy criteria to continue enrollment were met. Further analysis by biomarker status (KRAS/BRAF/EGFR) is forthcoming.

The preoperative management of locally advanced rectal cancer has evolved over the years to establish fluoropyrimidine-based chemoradiation as the usual standard of care. With the advent of newer agents – chemotherapeutic and biologic – for treatment of colorectal cancer, their role in this setting is being evaluated as well. This review is focusing on up-to-date data and studies regarding preoperative treatment of locally advanced rectal cancer.

Materials/Methods:We reviewed the records of patientswho underwent pancreaticoduodenectomyand chemoradiation for pancreatic and duodenal cancers at the Hospital of the University of Pennsylvania between 1982 and 2006. A total of 116 patients had data available to evaluate for patterns of failure. Time was calculated from the date of surgery to first documented failure, death without failure, or last contact without failure. A cumulative incidence analysis was performed to estimate failure rates by site of first failure.

3083 Background: MGCD265 targets wild type Met, as well as clinically relevant Met mutants, VEGFR-1,-2 and -3, Ron and Tie-2. MGCD265 combinability with erlotinib is being evaluated in patients (pt...

Abstract 737 Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-beta is a hematopoietic inhibitory cytokine that has been indirectly linked to the pathogenesis of some subsets of MDS and acute leukemias. We have shown (Blood, 112(8):3434; 2008) that smad2, a component of the TGF-beta signaling pathway, is constitutively activated and upregulated in MDS progenitors. Since there is conflicting data about upregulation of TGF-beta levels in MDS, we next sought to determine the molecular basis of TGF-beta receptor-I (TBRI) overactivation and subsequent smad2 phosphorylation / activation in this disease. We observed that smad-7, a negative regulator of TBRI kinase, is markedly down regulated in a meta-analysis of gene expression studies from 89 MDS bone marrow derived CD34+ cells when compared to 61 normal controls (Adjusted P Disclosures: No relevant conflicts of interest to declare.

The del(5q) is the most commonly reported deletion in de novo MDS and is found in 10–15% of all patients. Our group demonstrated haploinsufficiency for the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region in patients with the 5q- syndrome (Boultwood et al, Br J Haematol2007, 139:578–89). Haploinsufficiency of RPS14 has been shown to be the mechanism underlying the erythroid defect in this disorder (Ebert et al, Nature2008, 451:335–9). We have recently shown that haploinsufficiency of RPS14 in patients with the 5q- syndrome is associated with deregulated expression of ribosomal- and translation-related genes, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis (Pellagatti et al, Br J Haematol2008, 142:57–64). The del(5q) in the 5q-syndrome is cytogenetically indistinguishable from the del(5q) found in other MDS and in the vast majority of these patients the CDR of the 5q- syndrome will be deleted (and therefore one allele of RPS14 will be lost). We are investigating the hypothesis that haploinsufficiency of RPS14 and consequent deregulated ribosome biogenesis may also play a role in the pathogenesis of non-5q- syndrome MDS patients with del(5q). Using Affymetrix U133 Plus2.0 arrays, we have studied the expression profiles of a group of 579 ribosomal- and translation-related genes in the CD34+ cells of 21 non-5q- syndrome MDS patients with del(5q) and 95 MDS patients without del(5q). 168 of 579 ribosomal-and translation-related probe sets were found to be significantly differentially expressed between these two groups, with approximately 90% of these showing lower expression levels in patients with del(5q). Hierarchical clustering using this set of 168 genes gave a good separation between patients with and without the del(5q). RPS14 was one of the most significant differentially expressed genes, with lower expression levels in patients with del(5q) confirming its haploinsufficient status in these patients. Other significant differentially expressed genes include the ribosomal protein RPL22L1, and the translation initiation factors EIF4EBP3 and EIF4B. Interestingly, when samples from 16 patients with 5q- syndrome were included in the analysis, hierarchical clustering using significantly differentially expressed ribosomal- and translation-related genes showed that most patients with 5q- syndrome and most patients with del(5q) clustered together. We are currently using polysome profile analysis on bone marrow cells to examine the levels of the 40S ribosomal subunit in patients with del(5q) and without del(5q). Our results support the hypothesis that haploinsufficiency of RPS14 and deregulation of ribosomal- and translation-related genes contribute to disease pathogenesis in MDS patients with del(5q). An exciting possibility is that other MDS with the del(5q) and the 5q- syndrome share a related molecular basis in that they are all disorders of defective ribosomal biogenesis.

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though global epigenetic alterations in MDS have not been well described. The transmission of these epigenetic marks during hematopoietic differentiation and their role in disease pathophysiology is also unknown. We first compared global methylation profiles of 8 bone marrow samples with peripheral leucocytes by using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. We observed a high correlation (r=0.89–0.96) of epigenetic marks between bone marrow and peripheral blood samples suggesting that a majority of epigenetic marks can be also be seen in differentiated cells. We subsequently compared peripheral blood leucocytes from 20 patients with MDS with 10 age-matched normal and anemic controls. Parallel gene expression analysis was performed using 37K oligo maskless arrays on cDNA from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Epigenetic profiling demonstrated two clusters of MDS based on similarity of aberrant epigenetic changes. Overall, there was a trend towards hypermethylation in MDS, albeit not statistically significant given the large number of relatively unchanged genes. Detailed analysis revealed several novel differentially methylated genes that had corresponding changes in gene expression, when MDS samples were compared to the controls with a low false discovery rate of analysis. Interesting genes getting hypomethylated and overexpressed included TNF superfamily member 9, granulocyte pep A, microsomal glutathione S-transferase, homeo box B4, mitochondrial RPL11, and others. Similarly, the set of genes that were getting hypermethylated with associated decrease in gene expression included Evi-1, DAPK, HOXB3, Protein Phosphatase 1, CEBPB, mutated in colorectal cancer (MCC), myeloid-lymphoid or mixed-lineage leukemia 5 (MLL5), plasminogen-related protein B, ovarian cancer related protein 1 (ORP1), and others. In addition, we did array-based comparative genome hybridization (aCGH) to look at exact genome copy number changes in these samples. We found changes that were not detectable by conventional karyotyping in all samples. Commonly seen alterations were del(14q11), del(20q11), del(5q13), del(8p23), amp(1q42), amp(5q11), amp(17q12), amp(19q13) and amp(7q22). Integrative analysis revealed sets of genes that were either silenced by methylation or deletion in different patients. Thus, our data demonstrates that promoter DNA methylation changes are an important phenomenon in MDS evolution, and are associated with changes in expression of genes playing important roles in cancer development and/or progression. We also show that previously unrecognizable changes in copy number exist in most patients with MDS. In addition, our work shows that whole genome methylation assays, even when done on peripheral blood leukocytes, can be used for potential biomarker studies in the diagnosis of MDS.

The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of bone marrow stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of bone marrow stroma in this disease. To accomplish this aim, bone marrow mononuclear cells from MDS patients and controls were grown to form adherent cell layers and then depleted for hematopoietic elements by immunomagnetic CD45 negative selection. CD45 negative adherent cells were subsequently expanded and then used for whole genome methylation studies using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) which uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantify individual promoter CpG island methylation. A custom whole genome human promoter array (Roche-Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Global epigenetic profiling revealed that MDS stroma (n=6) was epigenetically distinct from normal bone marrow stroma (n=4) (ANOVA, P In subsequent studies, we profiled stroma from another set of MDS patients who had been treated with the DNMT inhibitor, 5-Azacytidine (n=4). In contrast to untreated MDS patients, there were no significant epigenetic differences between these 5-Azacytidine treated MDS patients and healthy controls (p = NS). These 5-Azacytidine exposed stroma cells did not demonstrate global hypomethylation (as hypothesized after DNMT inhibitor treatment) and were characterized by both hyper- and hypo-methylated loci similar to healthy controls. Thus our results reveal that MDS is characterized by widespread aberrant epigenetic changes in the bone marrow microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, and we hypothesize that those stroma effects contribute in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire bone marrow, including the microenvironment, if we are to improve our understanding of the pathophysiology of MDS and further improve therapy.

Recent work (Ebert et al, Nature 2008, Jan 17:335-9) has shown that ribosomal protein S14 (RPS14) is underexpressed in myelodysplasia (MDS) with deletion of chromosome 5q and plays a role in its pathogenesis. The role of ribosomal proteins in other more common subtypes of MDS is unknown. We conducted a meta-analytical comparison of gene expression profiles of 60 cases of non 5q- MDS CD34+ cells with 52 normal CD34+ profiles. These datasets were obtained from seven independent studies from NCBI’s GEO database. The data was integrated based on UniGene IDs and were quantile normalized to ensure cross-study comparability. (Based on our previous approach; Sohal et al PLOS One, 2008, Zhou et al, Blood, 2008). Using significance analysis of microarrays (SAM) and a false discovery rate (FDR) of just 0.04%, we found that ribosomal protein were the class of genes that were most significantly altered in MDS. We observed that RPL35a, RPS9, RPL10, RPL22, RPS14, RPS10, RPS15a, RPS24, RPL24, RPL36, RPL21, RPL23 were strikingly downregulated in non-5q- MDS CD34+ cells. To determine if these alterations were a result of changes in DNA copy numbers of these genes, we examined 20 MDS samples by high resolution array comparative genome hybridization (aCGH) performed on Nimblegen whole genome tiling arrays. aCGH at 6kb resolution revealed deletions in RPL14, RPL22, RPL36, RPS10, RPS5 and even RPS14 in distinct selected cases of non 5q- MDS. These small deletions, which were not identifiable by traditional karyotyping methods, may be putative mechanisms of ribosomal protein downregulation and ultimately, in MDS development and/or progression. Since MDS is also characterized by aberrant epigenetic silencing of genes, we next examined the methylation status of ribosomal gene promoters by high resolution global DNA methylation profiling by using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)). This assay uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. While there were sporadic changes in some patients, we did not observe any consistently significant changes in methylation of these ribosomal gene promoters on comparison with normal anemic controls. In summary, we show novel widespread alterations in ribosomal protein expression in MDS, at least some of which are associated with genomic deletions. These findings illustrate the applicability of meta-analytical genomic approaches in a heterogeneous disease such as MDS. Most importantly, our data points to the dramatic role of alteration in the protein translational machinery in pathogenesis of MDS.

Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of aberrant DNA methylation in the pathobiology of this disease. Whole genome methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 9 patients with IMF were compared to 9 age-matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays using cDNA from the same samples. Methylation analysis showed that myelofibrosis samples clustered separately from normal and anemic controls when grouped by unsupervised clustering based on Pearson’s correlation coefficient. On the other hand, global gene expression demonstrated no clear cut differences between myelofibrosis and control samples suggesting that methylation profiling has greater discriminatory power when compared to conventional gene expression profiling. A high correlation (r=0.88–0.96) was observed between whole genome methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can act as a valid surrogates for epigenomic analysis. Further analysis showed that genes aberrantly methylated in all myelofibrosis samples included v-myc, histone 2A, TNF, TNF Receptor1, FGF14 and others. Functional pathway analysis by Ingenuity showed that pathways involved in Inflammation and Cell signaling were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes for chemokine CXCL13, APC, IL-3, STAT2 and others. The pathways most activated by hypomethylation were involved in hematopoiesis and cell growth and proliferation demonstrating the biological validity of our analysis. Thus, our data demonstrates that myelofibrosis is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and lead to translational studies with agents targeting DNA methylation.

Microarray based studies of global Gene Expression (GE) have led to dramatic advances in our understanding of various biological processes and have resulted in a large amount of data in public repositories, like the Gene Expression Omnibus (GEO). Metaanalysis of this data has the potential to yield important biological information, but is hampered by technical issues due to different platforms and gene annotations used in various studies. In an attempt to conduct a metaanalysis, a total of 69 individual normal hematopoietic stem cell (HSC) GE datasets (9 whole bone marrow, 57 CD34+ cell studies) were identified in GEO. These had been done on 3 microarray platforms (Affymetrix U95, U133 A/B and U133 Plus 2.0). Since the probe identifiers and complementary cDNAs were different on these platforms, we integrated the data using both Unigene and RefSeq protein IDs and obtained a total of 8598 common Unigene and 8345 RefSeq probes after removing missing values. Unsupervised clustering of normalized GE values demonstrated that experimental conditions, lab where the experiments were performed and different microarray platforms can result in variability in GE patterns from similar sources of cells. To determine the degree of dissimilarity of these datasets from those obtained from biologically distinct tissues, GE profiles from various human tissues (brain, heart, kidney, etc.) were obtained from GEO and compared with hematopoietic stem cells. Unsupervised clustering showed that samples from the same tissue of origin clustered together despite different platforms/labs, demonstrating that our approach can group biologically distinct tissues together in spite of experimental and platform variability. To further test the discriminatory ability of the metaanalysis, we took datasets from hematologic malignancies and normal hematopoietic and non-hematopoietic tissues analyzed with the same platform (U133). We observed greater similarity between leukemias, myelodysplasia (MDS) and normal HSCs when compared to non-hematopoietic tissues, again validating the discriminatory power of this metaanalysis. In fact, some datasets from bone marrow samples from MDS were very similar to normal CD34+ cells and clustered within their groups. We believe this was a strong validation of our analysis as MDS is a preleukemic disorder with varying levels of pathology and can have cases that are genetically very similar to normal hematopoietic stems. We next attempted to search for a gene expression signature characteristic of HSCs by finding genes that were uniformly enriched in HSC datasets and at the same time differentially expressed when compared to normal non-hematopoietic tissues. We found 46 such “stemness” genes in our dataset. Functional pathway analysis by Ingenuity revealed that these genes were part of cell cycle and hematopoiesis pathways, thus decreasing the likelihood of our findings to be due to chance. In addition to known genes such as Gata2, Myb, Lyn kinase and Stat5A; several novel functional genes like SWI/SNF family member SMARCE1, Bone marrow stromal antigen 2, Septin 6, Topoisomerase II and H2A histone proteins were found to be enriched in HSCs by our analysis. Thus, we demonstrate a feasible and valid approach for metaanalysis of publicly available gene expression data that can yield further insights into human physiology and disease.

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. MDS is characterized by large mono-allelic deletions that lead to loss of heterozygosity. It is unclear how the loss of one allele affects the expression of genes on the remaining allele and the role epigenetic silencing plays in this process. To study this directly, we performed whole genome methylation analysis on 3 selected MDS cases with deletions of chromosomes 5q, 7q and 20q. DNA methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction enzyme digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of genes by calculating HpaII/MspI cut fragment intensity ratio. Array Comparative Genomic Hybridization (aCGH) was used to map the exact breakpoints in peripheral blood leucocytes obtained from these 3 MDS cases. Gene expression analysis of the cDNA from the same cells was performed using 37K oligo maskless arrays and integrated with methylation analysis. Promoter methylation status and gene expression of corresponding genes of six age-matched healthy and anemic controls were used as controls. Our results showed that the 5q14.2–31.3 deletion encoded for a total of 248 genes. On comparison with healthy controls, 73 gene promoters were hypomethylated and 143 were methylated. Promoters with lower methylation levels were associated with a greater proportion of genes with higher expression values, thus suggesting that epigenetic modification is a regulator of their transcription. On the other hand, hypermethylation of the remaining allele led to underexpression of important genes such as APC, Cyclin H, CD14 antigen, protein phosphatase 2 (PPP2CA), heat shock 70kDa protein 4, H2A histone family members and others. Similarly, the 7q11.2–36.3 deletion encoded for a total of 497 genes. Out of these, 130 promoters were methylated and resulted in lower expression of genes not previously implicated in MDS such as Alpha 2 Type I Collagen, Schwachman-Bodian-Diamond syndrome protein, MEF3 and others. Lastly, the 20q11.21–13.13 segment encoded for 173 genes out of which 67 were methylated when compared to controls. In these large deleted regions, only 8–25% of the genes had lower expression when compared to controls; suggesting that large segmental mono-allelic deletions themselves do not result in decreased gene expression. Most importantly, epigenetic modifications of the remaining allele seems to be a very important regulator of gene expression in these cases and can be a potential target for therapeutic strategies.

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though the genes affected by epigenetic alterations are not well known. Whole genome DNA methylation was analyzed by a recently described novel method, The HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 13 patients with MDS were compared to 9 age matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays on cDNA obtained from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Unsupervised clustering based on epigenetic profiling demonstrated that only two cases of early MDS clustered with normals as compared to absolutely no separation between MDS and normals with clustering based on gene expression patterns. A high correlation (r=0.88–0.96) was observed between global methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can be a valid surrogate for epigenomic analysis. Further analysis showed that genes consistently aberrantly methylated in MDS included Syk kinase, HOXB3, several histone acetyltranferases and others. Functional analysis by Ingenuity showed that cancer and cell signaling pathways were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes from Ras oncogene family, the CDC42 GTPase, various methyl binding proteins and other proteins mainly encoding for cancer and hematopoiesis functional pathways, thus biologically validating our analysis. Therefore, our data demonstrates that MDS is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and can potentially uncover a new set of therapeutic gene targets.

In addition to genetic alterations, epigenetic events have been recently proposed to play a role in the pathogenesis and progression of various malignancies. We wanted to study the role of both global genetic and epigenetic changes during leukemic progression by combining Whole genome methylation, Gene expression and DNA copy number alteration analyses. We used a cell line model of LGL derived from the same patient at two different stages of disease (Daibata, Int J Cancer.2004;108(6):845) for comparison. The first cell line, MOTN1, is IL-2 dependant and was derived during indolent course of LGL, while PLT-2 typifies the aggressive phenotype and is IL-2 independent in vitro. Whole genome methylation was analyzed by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Gene expression analysis was performed using 37K oligo maskless arrays and high density array comparative genomic hybridization (aCGH) was performed at 6Kb resolution (Nimblegen). aCGH showed significantly higher sensitivity when compared to conventional cytogenetic analysis and revealed that a total of 548 genes were deleted and 635 amplified during leukemic progression. Gene amplification led to significantly increased mean expression of important genes such as CDK6, Fyn kinase, DICER, MAP3K9 and deletion led to decreased gene expression of genes implicated in many cancer pathways by Ingenuity functional analysis. Methylation analysis showed that there was a trend towards hypomethylation with disease progression. The mean change in the HpaII/MspI (HELP) Log (2) ratio was 0.67, where a negative value indicates methylation and positive value indicates hypomethylation. Several genes implicated in hematologic diseases were found to be hypomethylated and overexpressed, including CDK8, IGF binding protein 5, HDAC4 and MAPK14. Methylation and suppression of various genes belonging to the suppressive TGF-β and TNF pathways was observed during leukemic evolution. A comparison of primary LGL samples with normal NK cells and CD4 lymphocytes demonstrated a consistent trend towards global hypomethylation. Significantly, many genes hypomethylated in the cell lines were found to be hypomethylated in 2 primary patient samples of NK and T LGL as well; these included members of RAS oncogene family, and many cell cycle regulators. These results show that interplay of both genetic and epigenetic events may be responsible for transformation of malignancies. For example, in our model we observed that pro-proliferative cyclin dependant protein kinases can be overactivated by both mechanisms, CDK6 by gene amplification and CDK8 by DNA hypomethylation. These findings strongly suggest that progression of LGL from the stable to the aggressive phase is associated with significant epigenetic changes, in addition to cytogenetic alterations.

Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of chromosomal alterations and DNA methylation in the pathobiology of this disease. We used a combination of gene expression analysis, high density array based comparative genomic hybridization (aCGH) and genome wide methylation analysis to perform an integrated genomic analysis of MF. Gene expression analysis was performed using 37K oligo maskless arrays and high density aCGH was performed at 6Kb resolution using Nimblegen platform. Whole genome methylation was analyzed by a recently described novel method ( Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific digestion by HpaII and MspI followed by pcr amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. aCGH revealed a very high number of microdeletions (range 622–1148, mean ± SD 555±144 ) and amplifications (range 463–770, mean ± SD 781±246) in a pilot study conducted in 4 patients with MF. Twenty three common regions were found to amplified in all patients which included regions of chr3p25, chr8p21, chr12q24, chr14q32, chr17p13 and chr17q12, which code for a novel set of genes including B-cell CLL/lymphoma 7A, GTPase activating Rap, BRF1 and others. Five regions were found to be commonly deleted in all samples (chr1q31, chr5q12, chr20p13, chrXq21, chrXq28). Thirty eight DNA segments were found to be deleted and 142 amplified in 75% of the samples. Several potential pathogenic genes encoding for transcription factors, cytokines and cytokine receptors were found to be coded by these segments. A custom human oligo array was used to determine methylation by calculating HpaII/MspI cut fragment intensity ratio. All patient samples had a very high level of methylation (range 64–82%, mean 72% ± 8.6%). Expression was found to be significantly decreased for the genes that were methylated (p

The myelodysplastic syndromes (MDS) are collections of heterogeneous hematologic diseases characterized by clonal hematopoietic defects. Even though gene expression studies have tried to differentiate between prognostic subgroups, there are concerns with standardization and validity of these studies. Decitabine and 5-azacytidine have shown activity in MDS, but it is unclear if hypomethylation can explain their efficacy. To address these issues we have developed a novel platform that uses a combination of gene expression analysis, high density array based comparative genomic hybridization (aCGH) and genome wide methylation analysis to perform an integrated high throughput epigenomic analysis of MDS. Using this approach, we conducted a pilot study in 5 patients with MDS. Gene expression analysis was performed using 37K oligo maskless arrays and high density aCGH was performed at 6Kb resolution using the Nimblegen platform. Whole genome methylation was analyzed by a recently described novel method ( Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific digestion by HpaII and MspI followed by pcr amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. aCGH revealed a very high number of deletions (range 515–2722, mean 1073±937) and amplifications (range 614–1247, mean 843±254) not visualized by conventional cytogenetic analysis. These were also seen on peripheral blood mononuclear cells and correlated with those seen in the bone marrow. 22 common regions were found to amplified and 15 regions commonly deleted in 80% of the samples and contained genes involved in transcription and signaling. A custom human oligo array was used to determine methylation by calculating HpaII/MspI cut fragment intensity ratio. MDS samples demonstrated a very high level of methylation (range 70–84%). Expression was found to be significantly decreased for the genes that were methylated (p

While microarray analysis of global gene expression yields enormous amounts of data, there are concerns about standardization and validity of findings. Consequently, we wanted to determine the variability in gene expression studies of human bone marrow in the literature and study the factors that account for these differences. We also wanted to determine if certain genes were consistently and differentially enriched in human bone marrow stem cells. A total of 64 individual datasets were collected from gene expression omnimbus (GEO) database for our analysis (2001–2006). Most of the datasets had been used as controls in studies of hematological malignancies. 13 datasets were hybridized to the Affymetrix U95 chip, 38 analyzed by the Affymetrix human U133A chip and 13 by the U133 plus 2.0 platform. RNA for these studies was derived from purified normal CD34+ cells in 48 cases and from unsorted normal bone marrow mononuclear cells in 16 cases. To merge data from different platforms, we converted individual probe Sequence_ids to RefSeq gene IDs and analyzed them by SAS (SAS Institute, Cary, NC) and Arrayassist software package (Stratagene©). A total of 23686 unique gene IDs were obtained for analysis after the data were normalized, and a KNN algorithm was used to fill the gaps in the data. Our results reveal that there is marked variability in gene expression patterns in this cohort. The data sets clustered together primarily on the basis of the laboratory that performed the assays. (Hierarchical clustering based on average Euclidean distances). Clustering was further defined by the type of chip/platform used for the analysis. Interestingly, the similarity between CD34+ sorted and ununsorted whole BM samples was greater than interplatform similarity between the same phenotypes of cells examined. Notwithstanding the variability in gene expression, there were a novel set of genes that were differentially enriched in all 64 samples. These genes included transcription factors (Kruppel like factor 6), translational proteins (eukaryotic translation initiation factor 4A, isoform 1, ribosomal proteins) and other proteins not previously implicated in hematopoeisis (guanine nucleotide binding protein (GNAS), Calnexin, HLA associated proteins, dUTP pryophosphatase etc.) Mouse homologues of several of these proteins were found to be overexpressed in a previous well respected study of mouse hematopoeitic stem cells (Ramalho-Santos et al, Science2002;298(5593)). To further validate these findings, we performed gene expression array analysis on primary bone marrow cells using a completely different platform (Nimblegen 37K arrays) and demonstrated enrichment of majority of these genes. Thus, we provide a blueprint for conducting similar meta-analysis across various microarray platforms and our findings disclose tremendous platform and lab dependant differences in microarray gene expression patterns. In spite of this variability, data mining of discrete datasets can be a useful tool for gene discovery. Finally, we are in the process of constructing a publicly searchable database of normal human bone marrow gene expression which may serve as a source of controls for gene expression studies of hematopoeitic malignancies by various investigators.