Aberrant DNA Methylation Is Associated With Poor Outcomes In Juvenile Myelomonocytic Leukemia

Abstract Recent studies suggest that aberrant methylation plays a fundamental role in the development of a variety of cancers, including myeloid malignancies. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloid neoplasm of early childhood that is characterized by both excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor. It is categorized as an overlap myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) according to the World Health Organization classification. We recently reported that somatic mutations in SETBP1 and JAK3 were identified in JMML patients and were associated with poor outcomes (Nat Genet 2013;45:937–41). The goal of this study was to clarify the clinical significance of aberrant DNA methylation in JMML. We studied 92 children (61 boys and 31 girls) who were diagnosed with JMML in institutions throughout Japan. A diagnosis of JMML was made based on internationally accepted criteria. We quantitatively evaluated the CpG methylation pattern in the promoter regions of 16 candidate genes (APC, BMP4, CALCA, CDH13, CDKN2A, CDKN2B, CHFR, DAPK, DMR-H19, ER, IGF2, MGMT, MLH1, RARB, RASSF1, TP73) from genomic DNA derived from bone marrow specimens at the time of diagnosis. This was accomplished by bisulfite conversion and the pryosequencing technique. We defined aberrant methylation as >3 standard deviations from the mean methylation level derived from 8 healthy individuals. The median age at diagnosis was 16 months (range, 0.3–160). By genetic analysis, PTPN11, NF1, NRAS, KRAS, and CBL mutations were found in 39 (42%), 7 (8%), 12 (13%), 13 (14%), and 11 (12%) patients, respectively. In addition, 16 patients had SETBP1 or JAK3 mutations. Karyotypic abnormalities were detected in 15 patients, including 8 with monosomy 7. The median monocyte count, percentage of hemoglobin F, and platelet count at the time of diagnosis were 4.6x109/L (range, 0.2–31.6), 21% (range, 0–68), and 61.0x109/L (range, 1.4–483), respectively. The median observation period was 18 months (range, 1–287). During observation, 56 of the 92 patients received allogeneic hematopoietic stem cell transplantation (HSCT), and 30 of 92 patients died. Outcomes were assessed according to transplantation-free survival (TFS), in which HSCT and death were censored, and overall survival (OS) by the Kaplan-Meier method. Aberrant methylation of BMP4, CALCA, CDKN2A, CDKN2B, DMR-H19 and RARB were detected, of which hypermethylation of BMP4, CALCA, CDKN2A, and RARB were associated with poor TFS according to univariate analyses (P<0.10). We integrated the number of aberrant methylation of these four genes to arrive at an aberrant methylation score (AMS). An AMS of 0, 1, 2, 3, and 4 was seen in 36, 29, 19, 7, and 1 of the 92 patients, respectively. The AMS was significantly higher in patients with SETBP1 or JAK3 mutations than in other patients (P=0.03): 1, 8, 3, 3, and 1 of the 16 patients showed an AMS of 0, 1, 2, 3, and 4, respectively. The probability of 5-year TFS was 42% in the AMS = 0 cohort and 4% in the AMS = 1 to 4 cohort, respectively (log-rank, P<0.001). Moreover, the probability of 5-year OS was 65% in the AMS = 0 to 2 cohort and 8% in the AMS = 3 and 4 cohort, respectively (log-rank, P=0.004). In multivariable analysis using the Cox-proportional hazard model, AMS = 1 to 4 (hazard ratio [HR], 2.6; 95% confidential interval [CI], 1.2–5.5; P=0.013), mutations of PTPN11 or NF1 (HR, 2.7; 95% CI, 1.3–5.5; P=0.010), and chromosomal aberration (HR, 3.3; 95% CI, 1.7–6.5; P=0.001) were independent predictors of poor TFS. Further, chromosomal aberration (HR, 4.4; 95% CI, 1.6–11.8; P=0.004) and platelet counts <33x109/L (HR, 2.8; 95% CI, 1.3–6.4; P=0.013) were independent predictors of poor OS. The present study shows that aberrant methylation of four genes (BMP4, CALCA, CDKN2A, and RARB) is associated with poor outcomes in JMML patients. Patients with SETBP1/JAK3 mutations frequently show the hypermethylation of these genes. Further, allogeneic HSCT is associated with improved outcomes for patients with AMS = 1 and 2. Therefore, these results suggest that examination of the methylation pattern of these four genes may help guide clinical decisions for the management of patients with JMML. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

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Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome

Blood ◽

10.1182/blood-2010-08-298968 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4871-4880 ◽

Cited By ~ 60

Author(s):

Christiane Olk-Batz ◽

Anna R. Poetsch ◽

Peter Nöllke ◽

Rainer Claus ◽

Manuela Zucknick ◽

...

Keyword(s):

Dna Methylation ◽

Cox Regression ◽

Cpg Island ◽

Cpg Islands ◽

Prognostic Scores ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Prognostic Features ◽

Aberrant Dna Methylation ◽

Myelomonocytic Leukemia

Abstract Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

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Focusing PI and IMiD Resistance in Multiple Myeloma: Impact of DNA Methylation

Blood ◽

10.1182/blood-2018-99-118944 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 404-404

Author(s):

Larissa Haertle ◽

Max Bittrich ◽

Ramya Potabattula ◽

Matteo Claudio Da Via' ◽

Umair Munawar ◽

...

Keyword(s):

Dna Methylation ◽

Multiple Myeloma ◽

Drug Resistance ◽

Promoter Region ◽

Research Funding ◽

Methylation Pattern ◽

Aberrant Methylation ◽

Average Methylation ◽

Flanking Region ◽

Methylation Patterns

Abstract Immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI) are backbone agents in the treatment of Multiple Myeloma (MM). However, most patients develop drug resistance over time, with the underlying mechanisms poorly understood. Epigenetic modifications are effective modulatory mechanisms for gene silencing and known to be influenced by the exposure to environmental factors such as drug treatment. Dimopoulous et al. (Molecular Oncology, 2018) recently demonstrated that resistance to IMiDs is also associated with global changes in DNA methylation, although such changes were absent in the annotated promoter region of the CRBN gene. Similarly, DNA methylation of PSMD5 and other regulatory 19S proteasome subunit genes induces PI resistance in various cancer cell lines (Tsvetkov et al. PNAS 2017, eLive 2015). Deep bisulfite sequencing (DBS) is a targeted sequencing approach that allows to investigate CpG methylation at single DNA molecule resolution. In order to determine the role of promoter methylation in the development of drug resistance in MM, we analyzed primary tumor samples for a selected number of therapy-associated genes, i.eCRBN, PSMD5, TP53, MDM2 and cMYC. A total of 59 sequential time points (TP, CD138 purified MM cells) from 32 MM and 30 paired PBMCs were investigated. At least one TP for all the patients was in relapse to IMiDs and/or PIs. Five samples of (un)methylated DNA standards (0%, 25%, 50%, 75%, and 100%) were also included for every gene in order to confirm assay specificity. DBS primers for multiplexed libraries were designed using the PyroMark Assay Design 2.0 software (Qiagen). Per sample, 200ng of genomic DNA was bisulfite converted using the EpiTect® Fast 96 Bisulfite Conversion Kit (Qiagen) and the DBS libraries were sequenced with the Illumina MiSeq. FASTQ files were analyzed using the Amplikyzer2 pipeline. The median read coverage per sample and gene was 10,031X ± 6624 (SD). A locus was considered to be affected when the average methylation was higher than 15%. We confirmed the absence of methylation in the CRBN promoter region, as previously described, and found an unmethylated pattern in all analyzed samples (MM and PBMCs) for the tumor suppressor gene TP53 and the oncogenes MDM2 and cMYC. Strikingly, the CRBN promoter flanking region which lays 6,039bp downstream of the promoter, was identified to be highly methylated. 18 out of 28 IMiD resistant patients (64%) showed a hypermethylated state in this region (average methylation: 48.7%, range: 22.7%-83.3%), whereas none of the corresponding PBMCs was affected (5.7%; range, 2.6%-8.8%). Similarly, but to a lesser extent, PSMD5 hypermethylation was observed in 4 out of 27 (15%) PI resistant patients affected (MM cells; 31.3%, range 22.4%-50.8%, PMBCs; 2.0%, range; 0.8%-3.4%). Notably, in six patients that were previously exposed to IMiDs and PIs, an aberrant co-methylation of CRBN and PSMD5 was detected, with four of them being clearly resistant to both drug families. In three patients with longitudinal sampling, one was unmethylated at diagnosis in both genes. In PI- and IMiD-resistant relapse of the same patient four years later, methylation was detected in 27% of PSMD5 reads and 62% of the CRBN flanking region. The second patient showed 70% methylation in CRBN at primary diagnosis and 83% in IMiD-resistant relapse six years later, indicating a pre-existing condition. Of note this patient did not respond to Lenalidomide induction therapy. The third patient had no detectable methylation at diagnosis and relapse. In the remaining 12 patients with sequential sampling, we identified highly conserved methylation patterns in both genes, indicating certain stability of methylation pattern once selected. In addition, six patients with mutations in proteasome subunits and/or the CRL4/CRBN complex also showed methylation in CRBN or PSMD5. This proves that genomic mutations and aberrant methylation patterns are not mutually exclusive. Here we provide first evidence that the methylation of cancer driver genes like TP53 or cMYC, seem to be strictly regulated, whereas genes targeted by therapy like CRBN or PSMD5 have a dynamic methylation pattern. The accumulation of epimutations as a potential mechanism for drug resistance needs to be confirmed in larger cohorts, a recovering of the gene expression might provide a valid treatment strategy to explore. Figure. Figure. Disclosures Martinez Lopez: Celgene: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

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Aberrant DNA Methylation Is Associated with a Poor Outcome in Juvenile Myelomonocytic Leukemia

PLoS ONE ◽

10.1371/journal.pone.0145394 ◽

2015 ◽

Vol 10 (12) ◽

pp. e0145394 ◽

Cited By ~ 17

Author(s):

Hirotoshi Sakaguchi ◽

Hideki Muramatsu ◽

Yusuke Okuno ◽

Hideki Makishima ◽

Yinyan Xu ◽

...

Keyword(s):

Dna Methylation ◽

Juvenile Myelomonocytic Leukemia ◽

Poor Outcome ◽

Aberrant Dna Methylation ◽

Myelomonocytic Leukemia

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Aberrant DNA Methylation Characterizes a Subtype of Juvenile Myelomonocytic Leukemia (JMML) with Poor Outcome.

Blood ◽

10.1182/blood.v114.22.828.828 ◽

2009 ◽

Vol 114 (22) ◽

pp. 828-828

Author(s):

Christiane Batz ◽

Inga Sandrock ◽

Peter Nöllke ◽

Brigitte Strahm ◽

Henrik Hasle ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Platelet Count ◽

Juvenile Myelomonocytic Leukemia ◽

Significant Prognostic Factor ◽

Dna Hypermethylation ◽

Poor Outcome ◽

Aberrant Dna Methylation ◽

Promoter Dna ◽

Myelomonocytic Leukemia

Abstract Abstract 828 Promoter DNA hypermethylation contributes to the malignant phenotype in cancer including myeloproliferative neoplasms and myeloid leukemia. We hypothesized that aberrant DNA methylation also occurs in juvenile myelomonocytic leukemia (JMML) and asked whether it is associated with clinical, hematologic or prognostic features of the disease. Denaturing liquid chromatography was used to analyze peripheral blood or bone marrow samples from 87 children with JMML and 17 healthy control subjects for changes in DNA methylation at 14 candidate gene loci (BMP4, CALCA, CDKN1C, CDKN2B, DAPK1, MGMT, MLH1, PAWR, RARB, RASA1, RASSF1, RECK, SOCS1, TP73). We identified 4 genes with promoter DNA hypermethylation in JMML: BMP4 (34% of cases), CALCA (30%), CDKN2B (28%) and RARB (23%); the other 10 loci were unmethylated. The pattern of hypermethylation of the 4 genes allowed the categorization of JMML cases into three groups: no methylation (40/87 patients), intermediate methylation (1 or 2 genes; 29/87 patients) or high methylation (3 or 4 genes; 18/87 patients). Lineage-specific cell sorting demonstrated that aberrant methylation was restricted to clonal cell populations and could be traced back to the CD34+ JMML progenitor cell compartment. This observation supports the concept that DNA methylation is associated with early pathogenetic events in JMML. A correlative analysis of methylation groups with clinical or hematologic features showed that high methylation was strongly associated with higher age and increased hemoglobin F level at diagnosis (both p<0.01). By contrast, there was no significant association with leukocyte count, absolute monocytes, platelet count, blast percentage in blood or bone marrow, spleen size, karyotype or mutational category (PTPN11, RAS, CBL, NF1). Importantly, the presence of hypermethylation at diagnosis predicted cases with poor outcome; the 5-year overall survival (OS) in the no / intermediate / high methylation groups was 0.63 [0.47–0.79] / 0.52 [0.30–0.74] / 0.24 [0.04–0.44] (p<0.01). Among patients receiving hematopoietic stem cell transplantation, hypermethylation characterized cases with significantly increased probability of relapse: the 5-year relapse incidence in the no / intermediate / high methylation groups was 0.22 [0.11–0.45] / 0.21 [0.09–0.50] / 0.69 [0.49–0.96] (p<0.01). The predictive power of high methylation was also reflected in a multivariate Cox model for OS that included age at diagnosis, sex, platelet count and mutational category as other variables; here methylation was the only significant prognostic factor. Furthermore, longitudinal analyses indicated that some cases had acquired a higher methylation phenotype at relapse. In summary, we report aberrant DNA methylation as the most important molecular predictor of outcome in JMML. We suggest that a high-methylation phenotype characterizes an aggressive biologic variant of this leukemia. Disclosures: No relevant conflicts of interest to declare.

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Myelodysplastic/Myeloproliferative Neoplasms

Hematology ◽

10.1182/asheducation-2011.1.264 ◽

2011 ◽

Vol 2011 (1) ◽

pp. 264-272 ◽

Cited By ~ 29

Author(s):

Mario Cazzola ◽

Luca Malcovati ◽

Rosangela Invernizzi

Keyword(s):

Clinical Laboratory ◽

Myeloproliferative Neoplasms ◽

Chronic Myelomonocytic Leukemia ◽

World Health ◽

Refractory Anemia ◽

Molecular Diagnostic ◽

Juvenile Myelomonocytic Leukemia ◽

Lymphoid Tissues ◽

Ring Sideroblasts ◽

Myelomonocytic Leukemia

Abstract According to the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, myelodysplastic/myeloproliferative neoplasms are clonal myeloid neoplasms that have some clinical, laboratory, or morphologic findings that support a diagnosis of myelodysplastic syndrome, and other findings that are more consistent with myeloproliferative neoplasms. These disorders include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable. The best characterized of these latter unclassifiable conditions is the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis. This article focuses on myelodysplastic/myeloproliferative neoplasms of adulthood, with particular emphasis on chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts associated with marked thrombocytosis. Recent studies have partly clarified the molecular basis of these disorders, laying the groundwork for the development of molecular diagnostic and prognostic tools. It is hoped that these advances will soon translate into improved therapeutic approaches.

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CRISPR-mediated modification of DNA methylation pattern in the new era of cancer therapy

Epigenomics ◽

10.2217/epi-2020-0110 ◽

2020 ◽

Vol 12 (20) ◽

pp. 1845-1859

Author(s):

Faezeh Maroufi ◽

Amirhosein Maali ◽

Meghdad Abdollahpour-Alitappeh ◽

Mohammad Hossein Ahmadi ◽

Mehdi Azad

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cancer Progression ◽

Methylation Pattern ◽

Epigenetic Therapy ◽

Special Importance ◽

New Era ◽

Major Mechanism ◽

Aberrant Dna Methylation ◽

Normal Status

In the last 2 decades, a wide variety of studies have been conducted on epigenetics and its role in various cancers. A major mechanism of epigenetic regulation is DNA methylation, including aberrant DNA methylation variations such as hypermethylation and hypomethylation in the promoters of critical genes, which are commonly detected in tumors and mark the early stages of cancer development. Therefore, epigenetic therapy has been of special importance in the last decade for cancer treatment. In epigenetic therapy, all efforts are made to modulate gene expression to the normal status. Importantly, recent studies have shown that epigenetic therapy is focusing on the new gene editing technology, CRISPR-Cas9. This tool was found to be able to effectively modulate gene expression and alter almost any sequence in the genome of cells, resulting in events such as a change in acetylation, methylation, or histone modifications. Of note, the CRISPR-Cas9 system can be used for the treatment of cancers caused by epigenetic alterations. The CRISPR-Cas9 system has greater advantages than other available methods, including potent activity, easy design and high velocity as well as the ability to target any DNA or RNA site. In this review, we described epigenetic modulators, which can be used in the CRISPR-Cas9 system, as well as their functions in gene expression alterations that lead to cancer initiation and progression. In addition, we surveyed various species of CRISPR-dead Cas9 (dCas9) systems, a mutant version of Cas9 with no endonuclease activity. Such systems are applicable in epigenetic therapy for gene expression modulation through chemical group editing on nucleosomes and chromatin remodeling, which finally return the cell to the normal status and prevent cancer progression.

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Short Survival and High Rates of Transformation Prevail in Chronic Myelomonocytic Leukemia Despite Hypomethylating Agent Therapy.

Blood ◽

10.1182/blood.v120.21.2800.2800 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2800-2800

Author(s):

Emily J. Vannorsdall ◽

Vu H. Duong ◽

Xinyi Ng ◽

Dan P. Zandberg ◽

Michael L. Tidwell ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Trials ◽

Research Funding ◽

Response Rates ◽

Chronic Myelomonocytic Leukemia ◽

World Health ◽

Free Survival ◽

Entire Cohort ◽

Myelomonocytic Leukemia

Abstract Abstract 2800 Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder categorized as a mixed myeloproliferative/myelodysplastic disorder in the World Health Organization classification system. Diagnostic criteria include a persistent peripheral blood monocytosis >1 × 109/L and bone marrow dysplasia. Our recent review of SEER Medicare data (ASH 2011 abstract 2784) demonstrated that CMML has a shorter overall survival (OS) and more frequent progression to acute myeloid leukemia (AML), compared to myelodysplastic syndromes (MDS). Due to the heterogeneity of this disease and its differences from MDS, efforts to identify prognostic factors have been ongoing. The MD Anderson prognostic score was previously validated, but was derived from patients treated prior to the availability of the hypomethylating agents (HMAs) azacitidine and decitabine. HMAs have now emerged as standard therapy, with reported response rates of 37–69%, but their impact on survival and AML transformation is unclear. The OS of CMML patients has been reported at 12–18 months and transformation rates have varied between 15–52%. We reviewed our own single-center experience with CMML over the past 12 years. Methods: We conducted a retrospective review of CMML patients evaluated at the University of Maryland Greenebaum Cancer Center between January 2000 and August 2012. Patient and disease characteristics, treatments, complications, progression to AML, and OS were recorded and analyzed. Descriptive statistics were used for baseline characteristics and Kaplan-Meier analysis was performed for all time-to-event data. Statistical analyses were performed using SPSS version 20.0. Results: We identified 35 patients with CMML, 71% were male and 71% white, with a median age of 69 (range 34–86) years; 75% had <10% bone marrow (BM) blasts and 68% had low-risk cytogenetic findings (normal karyotype or -Y). Most patients treated prior to 2005 received hydroxyurea and/or erythropoiesis-stimulating agents or were enrolled on clinical trials, while patients treated since 2005 received HMAs as primary therapy. The median OS of the entire cohort was 19.5 months, with 49% of patients progressing to AML with a median time to progression (TTP) of 16.9 months. Of the entire cohort, patients with <10% and ≥10% BM blasts had an estimated OS of 19.4 and 11.7 months respectively (p=.021). Patients with low-, intermediate-, and high-risk (complex karyotype, +8, or chromosome 7 abnormalities) cytogenetic findings had an estimated OS of 23.3, 16.5, and 12.0 months respectively (p<0.001). Twenty-two patients received HMAs. Their estimated OS was 16.5 months, compared to 23.0 months for patients who did not receive HMAs (p =.683); 50% of patients treated with HMAs had known progression to AML, with TTP varying from 3–28 months. AML-free-survival was 16 months in patients receiving HMAs, compared to 14 months in patients not treated with HMAs (p=0.960). The majority of patients receiving HMA therapy (63%) were treated with ≥ 6 cycles; 57% of these patients transformed to AML despite initial response, often in a sudden and unpredictable manner. Conclusions: Published trials using HMAs in CMML have been limited by small patient numbers, short median follow-up, and paucity of data on AML transformation. Our study had a median follow-up period of 41.1 months. We found a high rate of AML transformation and short OS even in patients who received HMAs. HMA treatment had no statistically significant impact on AML-free survival or OS. Although the results may be confounded by some selection bias, treatment with HMAs was largely based on the date of diagnosis rather than prognostic variables or performance status. Therefore, the favorable response rates previously reported with these agents, and also seen in our patients, do not appear to translate into an OS or AML-free-survival advantage. Our study underscores the continued need for novel agents and the need to prioritize clinical trials for this group of patients. Additionally, based on our data, early bone marrow transplantation should be strongly considered for CMML patients when feasible. Disclosures: Davidoff: Novartis: Research Funding; Celgene: Research Funding; GlaskoSmithKline: Research Funding. Baer:Novartis, Inc.: Research Funding; Celgene, Inc.: Research Funding.

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