ALBERT

Description: Genetic instability is a hallmark of chronic myeloid leukemia (CML). Recently, several major abnormalities in DNA repair mechanisms have been identified in primitive CML cells that likely explain the additional mutations these cells develop leading to their selective growth under tyrosine kinase inhibitor (TKI) therapies. It seems likely that such mechanisms also underlie disease progression in CML. However, an understanding of the specific somatic mutations involved and investigations of their resulting effects on the biological behavior of primary sources of primitive chronic phase (CP) CML cells is extremely challenging. As an alternative approach, we have now explored the possibility of applying whole genome sequencing (WGS) to induced pluripotent stem cells (iPSCs) derived from primitive CML cells to determine if such iPSCs, genocopy the mutations present in the diagnostic sample from which they were generated and whether primitive hematopoietic cells derived from these iPSCs might be useful for future drug screening experiments. To this end, we chosen a CML patient whose CP clonogenic cells contained both the Ph1 chromosome and the JAK2 V617F mutation and whose disease progressed into an accelerated phase (AP) during TKI therapy. iPSC were generated from leukemic cells obtained at the time of AP using Oct4, Sox2, Klf4 and c-Myc gene transfer. The presence of both BCR-ABL and JAK2 V617F was confirmed in 24/24 iPSC colonies. A control iPSC line negative for both genes was similarly established from the patient’s CD34+CD31+ endothelial progenitors purified from peripheral blood. We then performed WGS on DNA prepared from the leukemic cells obtained at diagnosis of CP (CML 006), the AP cell-derived iPSCs (PB34), and the control non-leukemic iPSCs (PB13), using a HighSeq Illumina platform. WGS revealed 845,175 somatic SNVs and 68,817 somatic short Indels in the CP leukemic cells at diagnosis that were not present in the non-leukemic iPSCs (PB13). 49,225 of these SNVs and 11,665 of the short Indels were novel (absent in the dbSNP database), and 419 were found in the COSMIC database. We identified 274 novel SNVs (3 missense, 161 nonsense, 108 synonymous and 2 splice site mutations) and 46 short Indels (19 insertions and 27 deletions). Most of the novel coding SNVs and Indels were heterozygous and an estimation of the variant allele frequency indicated these were present in virtually all leukemic cells. In addition to the JAK2 V617F mutation that was present at diagnosis, we found a novel frame shift mutation in exon 12 of ASXL1 gene (p.S871YfsX5) leading to protein truncation, a genetic event that has also been associated with myeloproliferative neoplasms (MPNs) and AML. We also identified several novel SNVs predicted by SIFT, Provean and PolyPhen-2 algorithms to be deleterious for protein structure. These novel mutations were found in genes relevant for the pathophysiology of MPNs, including the catenin (CTNNA1 R204C, and AIDA K235T), RAS (RREB1 P789T), autophagy (ULK1 R553C) cellular antioxidant defense (GSR S293C), RNA nuclear transport (NUP160 start loss) pathways. Individual sequencing confirmed the presence of these mutations in PB34 and their absence in PB13 (non-leukemic iPSC). We next compared the sequence data from the AP leukemic cell-derived iPSCs (PB34) with the diagnostic data (CML006). This analysis showed only 799 additional somatic SNVs and 96 new short Indels compared with those already evident in the cells present at diagnosis. Only 4 (3 non synonymous and 1 synonymous) SNVs and no Indels were found in exons. These mutations could have appeared during the application of the reprogramming process to the AP leukemic cell-derived iPSCs; none was an obvious contributor to MPN pathophysiology. Finally, we showed that day16 embryoid bodies derived from the PB34 iPSCs contained expected numbers of CD34+ cells (18±11%, n=6) and BCR-ABL-expressing hematopoietic colony-forming cells (CFCs, 143±64 / 105 cells, n= 6). These CFCs showed a slight inhibitory response to imatinib (54±15% colonies obtained in 1 µM IM, n=4) whereas a combination of IM and Pimozide (a STAT5 phosphorylation inhibitor), reduced survival another ∼10-fold. In conclusion, we have provided proof-of-principle results illustrating the potential of iPSC technology in combination with WGS to dissect the clonal evolution of disease progression in CML and develop patient specific drug screens that could build on this data. Disclosures: Turhan: BMS, Novartis: Honoraria, Research Funding.

Description: Abstract 2932 Poster Board II-908 Post-transplant lymphoproliferative disorders (PTLD) are one of the worse prognostic complications after solid organ or bone marrow stem cells transplantation. Most of them are associated to EBV known to activate NF-kB pathway especially by constitutive p65 expression. BAFF cytokine, a B cell-activating factor belonging to the tumour necrosis factor (TNF) family, have been described to modulate cell growth and survival in non Hodgkin lymphomas. However, little is known on the association between EBV, BAFF/BAFF-R signalling and canonical and non canonical NF-kB pathways expression in PTLD. Thus, we intend to study the role of EBV, NF-κB and BAFF/BAFF-R expression in PTLD.Our study has been investigated in two different contexts of diffuse large B-cell lymphoma (DLBCL). 20 cases of DLCBL resulting from immunocompetent patients (DLBCL/IC) and 13 from post-transplant recipients (DLBCL/PTLD) were compared. Indeed, all cases were characterized by histology and immunohistochemistry (IHC) was used to detect B-cell markers and to identify their germinal center (GC) or non GC (NGC) origin. EBV was detected by in situ Hybridisation (ISH) using EBER probe. Latent proteins LMP1 and EBNA2 as well as replicative protein ZEBRA were also detected by IHC. In addition, p50 and p52 proteins expression was carried out to study NF-kB pathway activation. We showed in DLBCL/IC, regardless of the ontogenic profile GC/NGC, that BAFF-R is expressed in 40% of cases, while in DLBCL/PTLD NGC pattern, BAFF-R is expressed in only one case out of 13 (7,7%) (p

Description: Abstract 3728 The occurrence of JAK2-V617F mutation has been reported during the evolution of several patients with Ph1-positive CML. In all cases where a clonal analysis has been performed, the involvement of two different hematopoietic stem cells (HSC) has been demonstrated, with the presence of two different myeloproliferative disorders (MPDs). The occurrence of the V617F mutation in a leukemic stem cell (LSC) harboring the BCR-ABL rearrangement has not been reported so far. In a 63- year old patient with a diagnosis of high Sokal score Ph1+ CML, the initial therapy with Imatinib led to efficient cytoreduction and complete cytogenetic response but with persistent splenomegaly. Upon disease progression, he was treated successively with IFN-alpha, ARA-C, Dasatinib and Nilotinib with transient responses and persistent splenomegaly. The progressive increase of platelet counts led to the discovery of JAK2-V617F mutation, which appeared to be present from the diagnosis of CML with a progressive increase (40 % at diagnosis up to 70% at the time of the discovery of JAK2 mutation). To determine if BCR-ABL and JAK2 mutation were present in the same cells, a clonogenic assay was performed and single BFU-E's (n=11) were analyzed individually by q-RT-PCR. All 11 BFU-E expressed BCR-ABL at high levels with a mean BCR-ABL/ABL ratio of 157% (Range 91–207 %). The same individual BFU-E also revealed the presence of a heterozygous JAK2-V617F mutation. To generate a HSC model harboring simultaneously these two major molecular events, we have generated induced pluripotent stem cells (iPSC) from leukemic cells using Sendai-virus mediated Oct4, Sox2, Klf4 and c-Myc gene transfer. We have also generated iPSC from non-leukemic (BCR-ABL-negative, JAK2-V617F-negative) endothelial progenitor cells (EPC) obtained from peripheral blood. iPSC generated from both sources expressed pluripotency markers (Tra-1–60, SSEA-3, SSEA-4) and generated teratoma in NOD/SCID mice. In 24 individual leukemic iPSC clones which were amplified, Ph1-chromosome was found to be present in all mitoses and BCR-ABL was highly expressed (Mean ratio 140%). Each iPSC exhibited the same V617F mutation which co-existed in the primary leukemic BFU-E. None of the non-leukemic iPSC expressed BCR-ABL neither V617F. To determine the hematopoietic potential of both iPSC, we have generated day-16 embryoid bodies (EB) followed by induction of hematopoiesis. Day 16 EB's generated variable numbers of CD34+ cells (12–40 %, n=4) and clonogenic potential (25–340 CFC/105 D16-EB) with evidence of growth-factor-independent CFC's. Interestingly, IM and Dasatinib had partial inhibitory effects on CFC activity, but a combination of Pimozide (STAT5 inhibitor) and IM had a major synergistic effect. As neither normal iPSC nor human embryonic stem cells (hESC) are able to generate definitive, long-term hematopoiesis, we have tested the long-term HSC potential of these iPSC in long-term culture initiating cell (LTC-IC) assays as compared to human ESC line H1. In these conditions, leukemic iPSC generated hematopoietic cells with terminal granulocytic and erythroblastic differentiation and gave rise for the first time to an LTC-IC- derived progeny at 6 weeks (200 CFU-C/105 Day-16 EB) whereas in the same time H1-derived cells did not generate any significant LTC-IC potential (3 CFU-C/105 D-16 EB). Interestingly, non-leukemic iPSC generated also hematopoietic cells but exhibited a major genetic instability in culture with appearance after several passages, of a t (5;7) translocation and major deletions/duplications in CGH arrays. To determine a potential upstream molecular event which might be involved at the origin of the occurrence of BCR-ABL and JAK2 mutation in the same HSC, a whole genome sequencing strategy is underway. In conclusion, we report for the first time the occurrence of two major molecular events involved in CML and non-CML MPDs in the same LSC and demonstrate the utility of iPSC modeling to study the earliest HSC involved. These results suggest strongly that the co-existence of BCR-ABL and V617F enables the specification of iPSC towards hematopoieis which could lead to the identification of specific hematopoietic signaling pathways. Finally, clonal analysis of hematopoietic cells in CML patients with demonstration of Jak2V617 mutations is worthwhile to determine the prevalence of the simultaneous occurrence of these two molecular events in the same cell. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Description: Congenital dyserythropoietic anemia type I (CDA I) is an inherited disorder characterized by macrocytic anemia and occasionally also by distal bone malformations. The disease has recently been shown to be caused by mutations in the CDAN1 gene, encoding codanin-1. The aim of the study was to characterize the CDAN1 mutations in 12 French patients with CDA I. The clinical data of the 12 French patients (10 kindreds) with CDA I were reviewed. Each of the 28 CDAN1 exons was amplified and sequenced. Half of the patients had a severe disease with prominent neonatal manifestations, complex bone disease, or both. Nine disease-causing mutations were identified: 6 described previously (P1130L, P671L, F869I, R681X, R713W, S1034F) and 3 novel mutations (R687W, F52L and IVS 8 G to A). Seven were missense mutations located in exons 14 to 28. Twenty seven per cent were identified in exon 14. No patient was homozygous for null type mutations. Only in two monozygotic twin patients did we fail to uncover a mutation. Mutations P671L, F52L and R713W were found on four, two and two alleles, respectively. One of the P671L alleles carried a second mutation (R687W). In conclusion the CDAN1 gene is apparently involved in most cases of CDA I in the French patients explored. Our study supports the previous findings that homozygosity for the null type mutations may be lethal, and that CDAN1 mutations are localized mainly in exons 12–28. In the present small group of patients, no apparent phenotype-genotype correlations were observed, in particular concerning abnormalities of the bones. Analysis of a larger number of patients from different ethnic groups is required to further characterize phenotype-genotype correlation in CDA I.

Description: BAFF, a member of the TNF family, has been shown to be implicated in B cell maturation and survival through an interaction with its three receptors, BCMA, TACI and BAFF-R. Recent data revealed that it also plays a role in B cell malignancies. For exemple, an autocrine activation of the BAFF receptors could participate to oncogenesis during multiple myeloma or chronic lymphocytic leukemia. BAFF-R, the sole BAFF specific receptor, is expressed in a subset of Diffuse Large B Cell Lymphomas (DLBCL). Since BAFF-R activation in B cells leads to cell proliferation and survival, expression of this receptor in DLBCL could correlate with more aggressive lymphomas. DLBCL can be divided into prognostically important subgroups with germinal center B cell-like (GCB), activated B cell-like (ABC) and type 3 gene expression profiles using a cDNA microarray. The germinal center origin of malignant B cells in DLBCL can also be characterized by immunohistochemistry (IHC) according to CD10, Bcl6 CD138 and MUM1 expression. In this work, we investigated whether expression of BAFF-R in DLBCL correlate or not with the GCB vs non GCB phenotype of lymphomatous cells. Lymph nodes biopsies from 23 DLBCL (among whom 3 post-transplantation lymphomas and 1 AIDS-related DLBCL) were analyzed. In a first step, we characterized by IHC the precise phenotype of each DLBCL: CD10+/Bcl6+ DLBCL were classified into the germinal center (GCB) group (n=9), whereas CD10-/BCL6-/MUM1+ lymphomas defined the non germinal center (NGCB) one (n=14). Each DLBCL was then analyzed for BAFF-R expression by IHC. In agreement with a recent report, most of the DLBCL were found to be negative for BAFF-R expression (16 out of 23). Interestingly, 4 of the 9 GCB DLBCL were found to be BAFF-R positive (44%), whereas only 3 out of 14 (21%) in the NGCB group. Although the difference did not reach statistical significance, we concluded from this observation that a consequent percentage of GCB DLBCL express the specific receptor BAFF-R. Thus, we actually analyze the clinical and biological characteristics of BAFF-R positive DLBCL and investigate whether expression of BAFF-R could represent a bad prognosis factor in term of clinical outcome and response to treatment.

Description: Abstract 3726 Recent data showed that BCR-ABL expressing leukemic stem cells (LSCs) persist in vivo in patients in complete molecular remission (CMR) according to European Leukemia Net criteria (Chomel et al, Blood 2011, Chu et al, Blood 2011). It has also been established that, in this category of patients (CMR 4.5– 5), Imatinib Mesylate (IM) discontinuation leads to relapse in the majority of cases (60%), especially during the first 6 months. Although very likely, a formal link has not been established between these two phenomena and, in particular, the prevalence of LSC persistence in a cohort of patients treated with TKI as a first line therapy has not been established. To determine the prevalence of LSC persistence in CMR induced by first line TKI therapies, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL expressing LSCs in marrow samples of 26 patients in CMR for 〉 2 years. CMR was induced by IM (n=22), Dasatinib (n=3) or Nilotinib (n=1) as a first line treatment. CD34+ cells were isolated from bone marrow aspirates (2–4 ml) using immunomagnetic columns and a clonogenic assay was performed. At week+2, clonogenic progenitors were counted and 20 to 40 individual hematopoietic colonies were plucked from methylcellulose for RNA extraction. In some cases, pooled CFU-Cs were also analyzed (20 pools of 10 hematopoietic colonies). The same CD34+ marrow sample was used to start LTC-IC assays, which were performed in murine MS5 cell feeders. At week+6 the cultures were sacrificed and the number of LTC-IC-derived clonogenic cells was determined. Hematopoietic colonies were plucked as decribed above for CFU-Cs for RNA extraction and BCR-ABL expression was quantified by q-RT-PCR. For 4 patients, the yield of CD34+ cells was not sufficient for stem cell assays (3 pts treated with IM; 1 with Dasatinib). qRT-PCR analyses of CFU-Cs revealed the presence of BCR-ABL expressing cells in 3 patients out of 16 tested so far. In all three patients in CMR induced by IM, this involvement can be estimated to approximately 100–360 leukemic CFU-GM/ml of bone marrow. Concerning LTC-IC assays, 2 patients out of 6 tested so far had evidence of BCR-ABL expressing LTC-IC-derived progenitors, one of these patients having also involvement of the clonogenic compartment. In both patients, there was a significant involvement of LTC-IC-derived progeny estimated to 1000–4000 LTC-IC derived progenitor/ml of bone marrow. In the 4 patients in whom LTC-IC compartment was found to be negative for LSCs, CMR was induced by IM in 3 Pts (IM 400 mg n= 2, IM 800 mg for accelerated phase n=1) and by Dasatinib (100 mg) in 1 Pt. Interestingly, in all q-RT-PCR assays, the amount of BCR-ABL mRNA (evaluated by the BCR-ABL/ABL ratio) in individual CFU-Cs appeared to be low, confirming our previous results (Chomel et al Blood 2012) and suggesting that these cells are not addict to BCR-ABL-TK activity for their survival. In 12 patients, the evaluation of LTC-IC compartment is underway. Overall, These data reveal that in patients in CMR induced by IM as a first line therapy, our strategy could identify two types of stem cell response, with some patients presenting a complete eradication of the leukemic stem cell compartment (within the limits of the sensitivity of the test) and others showing high numbers of BCR-ABL expressing stem cells despite the continuous CMR status on TKI therapy. It remains to be determined if the involvement of LTC-IC-compartment (in 2/6 analyzed so far) by LSC expressing low levels of BCR-ABL can be overcome by long-term TKI therapy in some patients. In addition, it will be of interest to determine if a correlation exists between LSC-positive patients the potential occurence of a molecular relapse upon discontinuation of TKI therapy. From the mechanistic point of view, the persistence of residual marrow LSCs in some CML patients in CMR strongly suggests the presence of active mechanisms of either LSC retention and/or LSC quiescence-promoting interactions within in the hematopoietic niche. Finally, the comparison of the LSC persistence phenomenon in CMR induced by TKI2 versus IM as first line treatment will be of major interest to determine the LSC eradication potential of these drugs. Disclosures: Rea: Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Turhan:Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Description: Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.

Description: Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA). Two of the RPS19 mutations identified in DBA patients are located in the sequence upstream of the translational start site: a missense mutation g-〉t at −460 and a 4 bp insertion, gcca, 3 nt upstream of the 5′ end of the RPS19 promoter. All of the RPS19 mutations identified to date are expressed in the heterozygous state. In the present study, in a DBA patient from Ivory Coast we identified the gcca insertional mutation on both alleles. Diagnosis of DBA was made at the age of 3 months. No malformations were noted. The patient responded neither to steroid nor to IL-3 therapies. The patient was regularly transfused but without an effective iron chelation therapy. As a consequence DBA was complicated by a severe hemochromatosis with thyroid, parathyroid, and liver damage. The patient was enrolled into a metoclopramide protocol in our hospital when he was 12 year old. He had a partial response, with increased time intervals between transfusions. While analysis of the RPS19 gene did not identify a mutation in the coding sequence, the 4 bp insertion, gcca at nt − 631 was noted on both alleles. Mother was heterozygous for this mutation and strikingly the father was homozygous. Both parents are apparently healthy. In screening of 200 Caucasian control chromosomes and 100 chromosomes from Ivory Coast we did not identify the 4bp insertion, ruling out the possibility that it is a common polymorphism. A parental disomy was eliminated by a genescan microsatellite analysis using the microsatellites: D19S200, D19S197, and LIPE. To evaluate the effect of this mutation on erythroid differentiation, we isolated 30,000 CD34+ cells from peripheral blood of the patient and normal individuals. CD34+ cells were cultured for 7 days in methylcellulose with EPO, SCF, and IL-3. At day 7, erythroid colonies were isolated and cultured for additional 3 and 5 days in liquid medium. At D7, D10 and D12, aliquots of cells were collected and cloning efficiency and apoptosis was quantitated. RPS19 mRNA was assayed by quantitative RT-PCR and level of protein expression determined by Western blot analysis. The cloning efficiency of DBA erythroid progenitors was decreased by 2.6 fold compared to normal between D7 and D10 and by 3.5 fold between D10 and D12. No difference in apoptosis was noted between DBA and normal erythroid progenitors. Strikingly, during terminal erythroid differentiation, RPS19 mRNA and protein expression levels was similar in the DBA and normal erythroid cells. The presence of the homozygous mutation in the healthy father in conjunction with normal expression of RPS19 in the DBA erythroid cells imply that the gcca insertion at nt −631 is not deleterious and cannot by itself account for the DBA phenotype of our patient.