ALBERT

Description: Cancer is caused by accumulated genomic and epigenetic abnormalities during the development of an individual, particularly during the neonatal period, when developmental plasticity is actively occurring. Myeloid-specific deletion of pten in embryos or after 3 weeks of age causes acute monocytic or myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) following a transient myeloproliferative neoplasm (MPN) in adult mice, which can mimic the human diseases to varying degrees. However, it is not clear how the timing of genomic and epigenetic abnormalities contributes to the disease phenotype in a mouse that is of an age comparable to human children. We hypothesized that during the development/aging process, the timing of when the genomic abnormality or “hit” occurs, such as loss of Pten or Nf1, is a critical determinant of the disease phenotype. We tested this by investigating the effect of somatic deletion of Pten at an age of 8 days, one of the most vulnerable stages for malignancy development in mice with or without a germline mutant Nf1. Through crossbreeding, we generated mice with Ptenfl/flNf1Fcr/+Mx1-Cre+ on a C57BL6/129 genetic background, and conditionally deleted Pten in a myeloid-specific manner by intraperitoneal injection of Poly(I:C). Mice with a pten deletion and mutant Nf1 (ptenkoNf1mut, hereafter referred as double mutant) showed signs of sickness at the end of the 2nd week of life, and all died by age 3-5 weeks (equivalent to 1-3 years old in humans). The natural survival in double mutant mice (n=10) was significantly shorter than those with wild type pten and Nf1 (ptenwt; Nf1wt, hereafter referred as WT, n=6, median 0.9 vs 〉14 months, 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. 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: Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1V623A, JAK1T478S, DDR1A803V, and NTRK1S677N, once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis.

Description: Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1T478S, and JAK1V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis.

Description: Abstract 764 Anti-apoptotic Bcl2 family genes have been implicated in the pathogenesis of acute myelogenous leukemia (AML), but the functional significance and relative importance of individual proteins (i.e. BCL2, BCL-XL, MCL1) remains poorly understood. We examined the expression of BCL2, BCL-X and MCL1 in primary human hematopoietic subsets and in leukemic blasts from AML patients, and found MCL1 transcripts were consistently expressed at high levels in all samples tested (100%, n=111). Mcl1 protein was also consistently expressed at high levels in myeloid leukemic blasts in a murine Myc-induced AML model, and we used this model to test the hypothesis that Mcl1 facilitates AML development by allowing myeloid progenitor cells to evade oncogene-induced cell death. Activation of Myc for seven days in vivo substantially increased myeloid lineage cells while hematopoietic stem, progenitor and B-lineage cells were depleted. Haploinsufficiency for Mcl1 (Mcl1flox/null) abrogated the development of AML (median survival 56 days vs. not reached), and deletion of a single allele of Mcl1 from fully transformed AML cells significantly prolonged the survival of transplanted mice. In contrast, the rapid lethality of disease (median survival 25 days) was restored by co-expression of Bcl2 with Myc in Mcl1flox/null cells. Together, these data demonstrate a critical and dose-dependent role for Mcl1 in AML pathogenesis and suggest that Mcl1 may be an ideal therapeutic target in patients with de novo AML. Disclosures: DiPersio: Genzyme: Honoraria.

Description: Key Points Early postnatal loss of Pten protein in mice with Nf1 haploinsufficiency causes a fatal juvenile myeloproliferative neoplasm. Akt and MAPK activities are elevated in juvenile mice with Nf1 haploinsufficiency and Pten protein loss.

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: Background: Bing-Neel syndrome (BNS) or central nervous system (CNS) involvement in Waldenströms macroglobulinemia (WM) is rare and typically results from the infiltration of CNS by neoplastic lympho-plasmacytoid cells. The aim of this study is to present a review of the clinical characteristics, practices for treatment and overall prognosis of BNS. We present the largest systematic review to date of BNS cases reported in the literature. Material and Methods: A systematic review of PUBMED (http://www.pubmed.gov) was conducted to search for primary articles and case reports under keywords "Bing-Neel syndrome" and "Waldenströms macroglobulinemia", "leptomeningeal", "central nervous system", and "lympho-plasmacytoid cells". The search was extensive, ranging from 1955 to 2014. All adult cases written in English language were included. The search yielded 35 results and was individually examined by two authors. All studies that described hyperviscosity due to WM aside from BNS were excluded. The effect of regimens with high CSF penetration or intrathecal chemotherapy (IT) on overall survival (OS) was analyzed using Kaplan-Meier curves and Wilcoxon test. Results: This review summarizes the clinical characteristics and treatment modalities for 40 patients with BNS reported in the literature. The mean age at diagnosis was 62 years (range 36-82 years); 37.5% were females and 62.5% were males. Mean time from diagnosis of WM to BNS was 4.8 years, however about 10% (n=4) had neurologic symptoms due to BNS at the time of diagnosis of WM. The longest time from diagnosis of WM to BNS was 17 years. Patients presented with a wide spectrum of neurological symptoms and signs. Most common clinical manifestation was a progressive cognitive decline, seen in 43% (n=17) of patients. Other common presenting symptoms were acute or insidious neurologic deficits, memory impairment, persistent headaches and ataxic gait. Ninety percent of patients (n=36) had an elevated serum IgM (average 7.8 g/L), of which 97% (n=35) had IgM kappa, compared to 3% (n=1) with IgM lambda paraproteinemia. CSF lymphoplasmacytic pleocytosis was present in 89% (n=25/28) of patients, with a mean CSF WBC count of 76/µL at diagnosis. Most commonly these clonal B cells had an immune phenotype profile characterized by CD5+/-, CD10-, CD19+, CD20+, CD79b+, sIgM with light chain restriction (κ/λ). Tissue histology was performed in 55% (n=22/40) of cases and revealed either a diffuse perivascular or tumor like infiltration by small mature lymphocytes or lymphoplasmacytoid cells, immunohistochemically positive for CD20, surface IgM and light chain restriction. Radiographically, BNS presents either as a pseudo-tumoral form (mass lesions) or diffuse form (infiltration into cerebral parenchyma, cranial/spinal nerves or leptomeninges). Two-thirds (n= 30/40) of all cases presented with a diffuse pattern of CNS involvement, 15% (n=6/40) with pseudo-tumoral form, and 5% (n=2/40) had a combined diffuse and tumoral pattern. Magnetic resonance imaging (MRI) is the most sensitive imaging modality, used for diagnosis in 90% of patients (n=35/40). Characteristic findings on MRI are hyperintensities on T2 weighted imaging (diffuse or localized), increased signal intensity on FLAIR and diffusion weighted imaging with low ADC values. Mean time from diagnosis to death, reported in 15 publications, was 7 months (range: death during work up-2 years). Twenty patients were alive at last follow-up, with a mean time from diagnosis to last follow-up of 33.2 months (range 3 months - 14 years). Patients treated with multi-agent chemotherapy (R-DHAC, R-DT-PACE, FR,BR) along with IT chemotherapy (MTX or AraC) or CNS penetrating systemic therapy (HD MTX, RHyperCVAD), with or without RT or ASCT (3 patients only) had improved outcomes, with 71% (n=15/21) of such patients reported alive at last follow up (mean follow of 25 months). Median OS in this group was 19.6 months compared to 3.3 months (p=0.045) in patients receiving older systemic chemotherapy alone (cyclophosphamide, 2Cda, chlorambucil etc.), suggesting that CNS penetrating regimens may improve outcomes. Conclusion: BNS is a rare and aggressive complication of WM. Newer multi-agent chemotherapy combined with CNS penetrating systemic therapy or intra-thecal chemotherapy may improve outcomes. This study highlights an unmet need in this subset of patients with WM. Disclosures Atrash: University of Arkansas for Medical Sciences: Employment. Alapat:University of Arkansas for Medical Sciences: Employment.

Description: Activating mutations in receptor tyrosine kinase (RTK) genes (including FLT3 and KIT) occur in more than 30% of newly diagnosed patients with acute myeloid leukemia (AML); we and others have speculated that mutations in other TK genes may be present in the remaining 70%. We therefore examined the expression of all annotated RTK and cytoplasmic tyrosine kinase (CTK) genes to prioritize these genes for sequencing. We performed high-throughput re-sequencing of the kinase domains of 24 TK genes (9 RTK and 15 CTK) using amplified genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML, and validated positive findings in an additional 94 AML tumor samples (14.4 million base pairs of double-stranded coverage). In addition to previously reported somatic mutations in FLT3, KIT, and JAK2 (which occurred at expected frequencies), we found novel somatic mutations in four patients in JAK1, NTRK1 and DDR1. Unexpectedly, we also identified novel non-synonymous germline sequence changes in 14 genes, including TYK2. We examined frequencies of known polymorphisms in our patients versus controls. We determined that the previously reported JAK3P132T allele is a germline variant that occurs in 19% of normal African Americans. Even when controlling for race, the TYK2G363S allele was found significantly less frequently in AML samples (12/376 alleles, 3.2%) compared to 147 normal controls (27/294 alleles, 9.2%, p=0.0013). Notably, there was loss of heterozygosity (LOH) at TYK2 in 2 patients. Additional population based studies and biologic validation will be required to define the significance of these sequence changes for AML pathogenesis. Lastly, we compared the expression of RTK and CTK genes in AML samples (n=92) to highly enriched normal human CD34+, promyelocyte, or polymorphonuclear neutrophil populations (n=5 each). We found several RTKs (FLT3, KIT, LTK) and CTKs (FYN, LCK, ITK, HCK and FGR) were tightly regulated in normal hematopoietic development but were dysregulated in many AML samples. Taken together, our data suggest that RTK or CTK mutations are not required for AML development but may be disease modifying events. Our data also suggest that germline variants and dysregulated expression of RTK and CTK genes may play significant roles AML pathogenesis.

Description: Abstract 3965 Poster Board III-901 We have recently established that whole genome sequencing is a valid, unbiased approach that can identify novel candidate mutations that may be important for AML pathogenesis (Ley et al Nature 2008, Mardis et al NEJM 2009). Acute promyelocytic leukemia (APL, FAB M3 AML) is a subtype of AML characterized by the t(15;17)(q22;q11.2) translocation that creates an oncogenic fusion gene, PML-RARA. Our laboratory has previously modeled APL in a mouse in an effort to understand the genetic events that lead to the disease. In our knockin mouse model, a human PML-RARA cDNA was targeted to the 5' untranslated region of the mouse cathepsin G gene on chromosome 14 (mCG-PR). The targeting vector was transfected into the RW-4 embryonic stem cell line, derived from a 129/SvJ mouse. The transfected RW-4 cells were injected into C57Bl/6 blastocysts, and chimeric offspring were bred to C57Bl/6 mice. F1 129/SvJ x C57Bl/6 mice were subsequently backcrossed onto the B6/Taconic background for 10 generations before establishing a tumor watch. About 60% of the mCG-PR mice in the Bl/6 background develop a disease that closely resembles APL only after a latent period of 7-18 months, suggesting that additional progression mutations are required for APL development. Array-based genomic techniques (expression array studies and high resolution CGH) have revealed some recurring genetic alterations that may be relevant for progression (i.e. an interstitial deletion of chromosome 2, trisomy 15, etc.), but gene-specific progression mutations have not yet been identified. To begin to identify these mutations in an unbiased fashion, we sequenced a cytogenetically normal, diploid mouse APL genome using massively parallel DNA sequencing via the Illumina platform. Since the tumor arose in a highly inbred mouse strain, we predicted that 15x coverage of the genome (approximately 40 billion base pairs of sequence) would be necessary to identify 〉90% of the heterozygous somatic mutations. We generated 2 Illumina paired-end libraries (insert sizes of 300-350 bp and 550-600 bp) and generated 59.64 billion base pairs of sequence with 3 full sequencing runs; the reads that successfully mapped generated 15.6x coverage. The sequence data predicted 87,778 heterozygous Single Nucleotide Variants (SNVs) compared to the mouse C57Bl6/J reference sequence, and 23,439 homozygous SNVs. Of the predicted heterozygous SNVs, 695 were non-synonymous (missense or nonsense, or altering a canonical splice site). Thus far, 80 of these putative non-synonymous SNVs have been further analyzed using Sanger sequencing of the original tumor DNA vs. pooled B6/Taconic spleen DNA and pooled129/SvJ spleen DNA as controls. 37/80 were shown to be false positive calls, and 37 were inherited SNPs from residual regions of the129/SvJ genome. 6/80 were present only in the tumor genome, and were candidate somatic mutations. These 6 were screened in 89 additional murine APL tumor samples derived from the same mouse model. Mutations in the Jarid2 (L915I) and Capns2 (N149S) genes occurred only in the proband, and are therefore of uncertain significance. 4/6 mutations were found in additional samples; 3 of these mutations were derived from a common ancestor of the proband and the other affected mice, and were therefore not relevant for pathogenesis. The other recurring mutation was in the pseudokinase domain of JAK1 (V657F), and was identified in one other mouse that was not closely related to the proband. This mutation is orthologous to the known activating mutation V617F in human JAK2, and is identical to a recently described JAK1 pseudokinase domain mutation (V658F) found in human APL and T-ALL samples (EG Jeong et al, Clin Can Res 14: 3716, 2008). We are currently testing the functional significance of this mutation by expressing it in bone marrow cells derived from young WT vs. mCG-PR mice. In summary, unbiased whole genome sequencing of a mouse APL genome has identified a recurring mutation of JAK1 found in both human and mouse APL samples. This approach may allow us to rapidly identify progression mutations that are common to human and murine AML, and provides an important proof-of-concept that this mouse model of AML is functionally related to its human counterpart. Disclosures: No relevant conflicts of interest to declare.