ALBERT

Description: Ectopic expression of c-Myc (Myc) in most primary cell types results in programmed cell death, and malignant transformation cannot occur without additional mutations that block apoptosis. The development of Myc-induced lymphoid tumors has been well studied and supports this model. Myc can be upregulated in acute myeloid leukemia (AML), but its exact role in myeloid leukemogenesis is unclear. To study its role in AML, we used a murine stem cell virus (MSCV) retroviral gene transfer/transplantation system to broadly express Myc in the bone marrow of mice either alone or in combination with antiapoptotic mutations. Myc expression in the context either of Arf/Ink4a loss or Bcl-2 coexpression induced a mixture of acute myeloid and acute lymphoid leukemias (AML+ALL). In the absence of antiapoptotic mutations however, all mice transplanted with MSCV-Myc (100%, n = 110) developed AML exclusively. MSCV-Myc-induced AML was polyclonal, readily transplantable, possessed an intact Arf-p53 pathway, and did not display cytogenetic abnormalities by spectral karyotyping (SKY) analysis. Lastly, we found that Myc preferentially stimulated the growth of myeloid progenitor cells in methylcellulose. These data provide the first direct evidence that Myc is a critical downstream effector of myeloid leukemogenesis and suggest that myeloid progenitors are intrinsically resistant to Myc-induced apoptosis. (Blood. 2005;106: 2452-2461)

Description: Expression of the constitutively activated TEL/PDGFβR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFβR activates multiple signal transduction pathways in cell-culture systems, and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB–mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5abnull/null) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably, these cell populations were maintained in Stat5abnull/null fetal livers and succumbed to transformation by c-Myc. Surprisingly, targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB–mediated transformation. Survival of TPiGFP→Stat5a−/− and TPiGFP→Stat5a+/− mice was significantly prolonged, demonstrating significant sensitivity of TEL-PDGFRB–induced MPD to the dosage of Stat5a. TEL-PDGFRB–mediated MPD was incompletely penetrant in TPiGFP→Stat5b−/− mice. In contrast, Src family kinases Lyn, Hck, and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together, these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB–induced myeloproliferation.

Description: Background: Follicular lymphoma (FL) is the most common indolent NHL (iNHL), exhibits a variable clinical course, and remains largely incurable. The pathogenesis of FL is complex and involves over expression of Bcl2 via t(14;18) translocation, as well as copy number alterations, recurrent somatic mutations, and changes in the tumor microenvironment. In line with recent publications, we hypothesized that recurrent somatic genomic mutations in FL will be present and may impact FL development, progression, transformation, and clinical outcomes. Methods: To address this, we performed exome sequencing (NimbleGen SeqCap EZ V2.0) of tumor and normal frozen tissue pairs from 24 patients in a discovery cohort with untreated FL (12), relapsed FL (6), or transformed FL/iNHL (6). We developed a custom capture assay (NimbleGen) that targets 7.05 MB corresponding to the coding, 5' and 3' UTR regions of 1717 genes. The custom capture genes included somatic mutations identified in our exome discovery cohort (898 genes) or somatic mutations previously published to be recurrently mutated in B cell NHL (819 genes). Instrument data from the discovery cohort exome and re-sequenced custom capture were combined and analyzed using the McDonnell Genome Institute (MGI) somatic caller pipeline (5 SNV callers, 3 indel callers), filtered (minimum 20X coverage, minimum 2.5% VAF, maximum 10% normal VAF) and manually reviewed. Additionally, the 1717 custom capture strategy was used to sequence an extension cohort consisting of FFPE tumor samples from 80 patients with FL, achieving 〉20x coverage for 〉75% of the targeted region. All discovery and extension samples have clinical annotations that include FLIPI prognostic score, treatment, and clinical outcomes. Results: Combined analysis of exome and custom capture data for the discovery cohort yielded a robust data set with good sequence coverage of 〉78% of the targeted regions with at least 20x depth in all samples and a mean depth of 89x. Based upon somatic mutations identified and manually reviewed using this approach, we conservatively estimate 0.98 mutations per MB in FL. 23 genes were recurrently mutated in 3 or more cases, and an additional 75 genes recurrently mutated in 2 cases in the discovery cohort. Consistent with recent publications (Li H et. al., Blood, 2014; Green MR, PNAS, 2015; Yildiz M et al, Blood, 2015) we confirmed a number of genes that were highly recurrently mutated in FL [TNFRSF14 (50%), Bcl2 (25%), IRF8 (13%), TP53 (13%)] including chromatin modifying genes consisting of histone methyl transferases [KMT2D/MLL2 (58%), EZH2 (13%)], histone acetyltransferases [CREBBP (42%), EP300 (17%)], histone linkers [HIST1H1C (13%), HIST1H1E (8%), HIST1H2BO (8%), HIST1H3G (8%), HIST2H2AC (8%); collectively 42%]. We also confirmed (ATP6V1B2, 13%) and found unreported (ATP6AP2, 8%; ATP6V0A1, 4%; ATP6V1F, 4%) mutations in vacuolar ATPase proton pump genes and P5 or Ca++ ATPase genes (ATP13A2, 4%; ATP13A4, 4%, ATP2B4, 4%;). We confirmed (CD79B, 13%; BCL10, 8%) and found unreported (CD22, 13%) mutations in components of the B cell receptor signaling pathway. The previously unreported recurrent mutations in CD22 were consistent with loss-of function (2 missense, 1 nonsense, 1 frame shift deletion). As a negative regulator of BCR signaling, mutation of CD22 may represent a strategy of to enhance BCR signals in malignant germinal center B cells. We also identified members of the SWI/SNF complex mutated in 33% of this FL cohort: ARID1B (8%), BCL11A (4%), SMARCB1 (4%) in addition to previously reported members BCL7A (12%), SMARCA4 (8%), ARID1A (4%). Somatic mutations were also identified in the Notch pathway: DTX1 (29%), Notch2 (4%), Notch3 (4%), Notch4 (4%). We identified several genes that have not been reported as highly recurrent in FL CXCR4 (42%, mutation calls primarily in RNA), DMD (13%), DNAH9 (13%), FLG (13%), GON4L (13%), PCDH7 (13%), RLTPR (13%), SCN7A (13%), ST6GAL1 (13%). Conclusions: FL genomes harbor a large number of recurrent mutations, consistent with a role in the development and progression of this malignancy. Analysis of the extension cohort and association of recurrently mutated genes and pathways with clinical outcomes is ongoing and will be presented. Disclosures Bartlett: Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding.

Description: Introduction Detection of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) has become an important clinical tool in the treatment of patients with acute leukemia. At our institution, we routinely employ Children's Oncology Group (COG) validated B-lymphoblastic leukemia (B-ALL) MFC-MRD analysis for the detection of MRD in pediatric patients after induction therapy for B-ALL. We have extended this COG-validated assay to the adult B-ALL population. This technique relies on the identification of aberrant immunophenotypes expressed by residual bone marrow B-lymphoblasts compared to the normal pattern of maturing hematogones using a 6-color, 3-tube MFC. This assay was initially designed and validated for the pediatric B-ALL population, but the majority of patients with B-ALL at our institution represent adults. Therefore, we characterized the frequency of abnormal cell surface antigen expression of B-ALL MRD with the COG-validated method in the adult B-ALL population and compared our findings with those of our pediatric population. Methods This study was approved by the Institutional Review Board (IRB) of Washington University School of Medicine in St Louis, MO. A total number of 289 bone marrow specimens were submitted for B-ALL MRD analysis over a time period of 14 months. Antibodies employed in the COG-validated method were CD19, CD20, CD10, CD38, CD9, CD45, CD58, CD13/CD33, CD34, CD71 and CD3. At least 1 million cell events per tube (or as many as possible) were acquired using Beckman Coulter Navios flow cytometers, and Kaluza software was used for analysis. Leukemia-associated immunophenotypic aberrancies were identified, and the proportion of aberrant leukemia cells was calculated as percentage of mononuclear cells. We defined any case with an aberrant immunophenotype that encompassed ≤5% of mononuclear cells as an MRD case, and it was included in subsequent analyses. For each case of B-ALL MRD, we examined side scatter along with the above-mentioned COG-validated markers to asses for aberrancies. Aberrant expression was defined as either over- or under-expression for an antigen when compared to the expression pattern of normal hematogones (CD45, CD19, CD10, CD38, CD58, CD9, CD34) or aberrant positivity/negativity (CD20, CD13/33). Side scatter was considered abnormal if higher or lower than normal hematogones. Fisher's Exact test to compare proportions was used for all statistical analyses. Results Of the 289 cases, 64 cases (22%) from a total of 46 patients showed positivity for an aberrant B-lymphoblast population, of which 32 cases (50%) represented MRD ≤5%. Of these 32 cases, 22 were from adults and 10 from children. The MRD levels ranged from 0.010% to 3.8%. Next, we assessed the frequency at which each cell surface antigen was aberrantly expressed and compared results between adult and pediatric patients. For the majority of both adult and pediatric patients with B-ALL MRD, we observed concordant abnormal expression between adult and pediatric cases (listed respectively) of CD10 expression (95% vs. 90%, p=0.534), CD38 expression (68% vs. 70%, p=1.0), CD58 expression (64% vs. 60%, p=1.0), CD9 expression (91% vs. 90%, p=1.0), CD34 expression (86% vs. 70%, p= 0.346), and CD45 expression (55% vs. 80%, p= 0.248). Abnormal expression was less frequent for CD19 (27% vs. 40%, p= 0.683) and CD13/CD33 (18% vs. 20%, p=1.0), as well as side scatter changes (45% vs. 10%, p=0.106). Over-expression was the most common immunophenotypic aberrancy for CD10, CD58, and CD34; under-expression was more common for CD38, CD9, and CD45. Adult and pediatric patients had no significant differences in abnormal expression patterns, with the exception of CD20. In adult patients, CD20 expression was abnormal (negative) in 64% of cases, while only 10% of pediatric patients were negative (p=0.0052, Fisher's Exact test). Conclusion These results indicate that the range of aberrant immunophenotypes seen in adult and pediatric B-cell acute lymphoblastic leukemia minimal residual disease are similar in nature and suggest that the COG-validated assay designed for pediatric B-ALL can be successfully applied to the broader adult population. Because this assay allows for the detection of frank (as well as minimal residual) disease burden, we propose that this flow cytometry approach can be harmonized to accommodate monitoring of B-ALL by flow cytometry regardless of the extent of disease burden. Disclosures No relevant conflicts of interest to declare.

Description: Therapy-related acute myelogenous leukemia (t-AML) is an important late adverse effect of alkylator chemotherapy. Susceptibility to t-AML has a genetic component, yet the specific genes and genetic variations that influence susceptibility are poorly understood. Our lab previously identified mouse strains that are susceptible (SWR/J) or resistant (C57BL/6J and C3H/HeJ) to t-AML induced by the alkylator, ethyl-N-nitrosourea (ENU). To study the genetic basis of these differences, we performed an F2 intercross between susceptible and resistant strains. A single copy of the hCG-PML-RARa (PR) transgene was bred into each mouse. PR is an initiating factor for acute promyelocytic leukemia, but requires cooperating mutations for full leukemic transformation. This provides a platform to detect gene X transgene (PR) and gene X environment (ENU) interactions that promote leukemogenesis. F2 mice were treated (n=141) or untreated (n=141) with ENU and sacrificed and analyzed when moribund. We also analyzed untreated PR+ (n=24) mice from the resistant C57BL/6J X C3H/HeJ (B6C3F1) background. Untreated B6C3F1 PR+ mice developed lethal myeloid leukemia (characterized by splenomegaly 〉 0.25g, WBC 〉 30 K/uL, and increased immature myeloid precursors in PB, BM, and spleen) with an incidence of 12.5% and a latency of 234 days. By contrast, 79.4% of untreated PR+ F2 mice developed myeloid leukemia with a latency of 108 days and median survival of 238 days. The earlier onset and increased incidence of leukemia in F2 mice confirm that SWR/J alleles confer increased susceptibility to AML. ENU treatment further increased the leukemia incidence (90.4% vs. 79.4%, p

Description: Expression of the constitutively activated TEL/PDGFβR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia (CMML). TEL/PDGFβR activates multiple signal transduction pathways in cell culture systems and induces myeloproliferative disease (MPD) in a murine bone marrow transduction/transplantation model of disease. Two TEL-PDGFβR juxtamembrane tyrosines, corresponding to tyrosines activating Stat5 and Src signaling molecules in native PDGFβR, are required for TEL-PDGFRB mediated MPD in mice. We used gene-targeted mice as donors in bone marrow transduction/transplantation experiments to characterize the contribution of Stat and Src genes in the development of TEL-PDGFRB disease. Mice transplanted with cells harboring targeted deletions of both Stat5a and Stat5b genes (TPiGFP→Stat5ab−/−) were protected from rapidly fatal MPD (median survival 〉125 vs. 25 days in TPiGFP→Stat5ab+/+mice, P

Description: Dysregulation of c-Myc is one of the critical oncogenic events required for tumorigenesis in many tissue types. Although c-Myc has been shown to block differentiation of leukemic and normal myeloid cells and is activated by oncogenes commonly found in leukemia, its exact role in myeloid leukmogenesis remains elusive. When c-Myc is over-expressed in the lymphoid compartment, Eμ-Myc transgenic mice develop B-cell lymphomas only after a prolonged latency and require additional mutations to inhibit apoptosis, most commonly by disrupting the Arf-Mdm2-p53 pathway. Although this model has been well used to dissect pathways of Myc-induced lymphomagenesis, there is currently no efficient model with which to study the role of c-Myc in AML. In this study, we utilized a retroviral gene transfer system (MSCV-Myc) to introduce c-Myc into unfractionated murine bone marrow that was subsequently transplanted into syngeneic mice. Surprisingly, 100% of transplanted mice (N=110) from three different strains, developed acute myeloblastic leukemia (AML) characterized by rapid onset, hemiparesis, splenomegaly and infiltration of the marrow and spleen with myeloblasts. c-Myc protein level was comparable to the Eμ-Myc tumors. Disease latency was short (median survival=38 days). MSCV-Myc-induced tumors were oligoclonal, did not display significant cytogenetic abnormalities by SKY analysis, and were exclusively of myeloid origin. These tumors contained germline configuration of the Ink4A locus and wild type coding region of the p53, Arf and Ink4a. Also, the p53 target gene p21 in Myc expressing cells responded normally to gamma irradiation. These data demonstrate that c-Myc induced AML development without disrupting the Arf-p53 pathway and suggest that cooperating mutations were not required for the development of AML. In contrast, using the same retroviral system, lymphoid leukemia developed only if c-Myc and Bcl-2 were co-expressed or if Ink4a was mutated. Tumors from Bcl-2/c-Myc transplanted mice were comprised of a mixture of myeloid and lymphoid leukemic cells. Similarly, mice transplanted with c-Myc transduced Ink4a−/− bone marrow developed leukemia that displayed a mixed phenotype of myeloid and lympoid lineages. Lastly, factor independent methylcellulose colonies induced by Myc were exclusively of myeloid lineage suggesting as intrinsic difference between myeloid and lymphoid cells in response to Myc. Our data imply that dysregulation of c-Myc is sufficient to induce AML and provide direct evidence that Myc is an important downstream target in myeloid leukemogenesis.

Description: The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and cellular dysplasia. Peripheral blood cytopenias and progression to AML are important clinical sequelae of MDS. 10–20% of MDS cases are a consequence of prior treatment with alkylators. The molecular basis of therapy-related MDS (t-MDS) is poorly understood. Point mutations of RAS family members and inactivation of the p53 and p15 tumor suppressor genes by mutation or hypermethylation represent the most frequently reported molecular abnormalities in MDS. Clonal cytogenetic changes, usually involving loss of material from chromosomes 5 and/or 7 are present in 〉90% of t-MDS cases. These recurring deletions suggest that myeloid tumor suppressor genes may be present in these regions, although their identify has not yet been established. Progress in understanding the genetic basis of human MDS has been hampered by a lack of suitable animal models. To develop a mouse model of t-MDS, we screened 32 inbred strains for susceptibility to t-MDS after treatment with the prototypical alkylating agent, N-nitroso-N-ethylurea (ENU). Mice (n=12 per strain) received two doses of ENU (100mg/kg, IP) or no treatment (n=12 per strain) at 9 and 10 weeks of age. Among the strains tested, SWR/J mice were found to be highly susceptible to myeloid malignancies (MDS/AML). We confirmed this in a second cohort. 10 of 33 (30%) ENU-treated SWR/J mice developed key features of MDS, including anemia (mean Hb=10.9 ±1.1 g/dL, compared to mean Hb=14.0 ±0.3 g/dL in 32 untreated age and sex-matched SWR/J mice, p=0.0006) and erythroid dysplasia (megaloblastic maturation, nuclear budding and blebbing in normoblasts) with a latency of approximately 30 weeks after ENU exposure. There was also evidence of dysplasia in the megakaryocytic lineage, manifested by numerous micromegakaryocytes with unilobar nuclei. Mild dysplastic features were detected rarely in untreated controls from this strain. The t-MDS mice developed significant splenomegaly (mean=0.49 ±0.19 g compared to control mean=0.15 ±0.01 g, p=0.004) with histologic evidence of increased extramedullary hematopoiesis. No significant immunophenotypic differences were detected in bone marrow cells from the t-MDS cases compared to controls. Iron stores were normal with no evidence of ringed sideroblasts. In 2 of the 10 affected mice, MDS evolved to AML, manifested by rapid breathing, circulating myeloid blasts, and leukocytosis (21-621,000 cells/μL). An additional 3 ENU-treated SWR/J mice developed AML without evidence of a preceding MDS phase. All AML cases had a Kit+Gr1+CD34- phenotype with no expression of lymphoid markers. The blasts were myeloperoxidase negative. This mouse model recapitulates many key features of human alkylator-associated t-MDS/AML and should be useful for discovery of mutations involved in the pathogenesis of this syndrome. We are employing array-based comparative genomic hybridization and candidate gene resequencing as tools for mutation discovery in this model. Because of their unique sensitivity to t-MDS, the SWR/J strain will also be particularly useful for identification of germline polymorphisms that affect susceptibility to alkylator-associated t-MDS/AML.

Description: Key Points FLs harbor more recurrent mutations in the BCR signaling pathway, SWI/SNF complex, and histone genes than previously known. Novel recurrent mutations affecting BTK, SYK, and HVCN1 may have therapeutic and prognostic implications for FL.

Description: Dendritic cells (DC) play a critical role in the initiation and regulation of immune responses. Based on immunophenotypic and functional charcateristics, murine CD11c+ DC can be classified into CD11c+, CD11b+, and B220− “conventional” DC and CD11c+, CD11b−/dim, B220+, and Gr-1+ interferon-producing cells (IPC) or plasmacytoid dendritic cells (pDC). The developmental origin of IPC remains controversial. In this study, we investigated the capacity of different murine bone marrow subpopulations to acquire immunophenotypic, morphologic and functional characteristics of IPC after in vitro culture with Flt-3L. Total bone marrow (BM) from 129/SvJ mice was sorted into six distinct BM subsets based on surface expression of the myeloid markers CD31 and Ly6C and subsequently cultured with Flt-3L for 8 days. Purified CD31high/Ly6C− BM cells were the only subset that consistently developed characteristics closely resembling that of IPC. Specifically, these cells expressed high levels of CD11c, low levels of CD11b, the lymphoid marker B220, and the IPC-specific marker 440C. Furthermore, these cells displayed the typical plasmacytoid morphology with eccentrically located nucleus showing mature lymphoid chromatin and lack of nucleoli. Functionally, these cells showed a high proliferation rate as demonstrated by the [3H] thymidine release assay and secreted detectable levels of IFN-alpha when stimulated with CPG ODN 2216. In contrast, all other sorted subsets failed to proliferate in the presence of Flt-3L. Similar results were obtained using BM from B6-RAG−/− mice. These findings indicate that, although both the CD31high/Ly6C− and CD31int/Ly6C− BM subsets contain early progenitor cells corresponding to “cobblestone area forming cells”, only the CD31high/Ly6C− is able to give rise to IPC when cultured with Flt-3L. Interestingly, CD31high/Ly6C− BM cells have been previously shown to generate also conventional DC if cultured with GM-CSF. Thus, IPC and DC may derive from common or closely related progenitors. Moreover, our results demonstrate that IPC development, in contrast to that of lymphocytes, does not require RAG proteins.