Development Of Overt Leukemia Research Articles

NPM 1 Mutations in AML—The Landscape in 2023

Acute myeloid leukemia (AML) represents 80% of acute leukemia in adults and is characterized by clonal expansion of hematopoietic stem cells secondary to genomic mutations, rendering a selective growth advantage to the mutant clones. NPM1mut is found in around 30% of AML and clinically presents with leukocytosis, high blast percentage and extramedullary involvement. Considered as a "gate-keeper" mutation, NPM1mut appears to be a "first hit" in the process of leukemogenesis and development of overt leukemia. Commonly associated with other mutations (e.g., FLT 3, DNMT3A, TET2, SF3B1), NPM1 mutation in AML has an important role in diagnosis, prognosis, treatment and post-treatment monitoring. Several novel therapies targeting NPM1 are being developed in various clinical phases with demonstration of efficacy. In this review, we summarize the pathophysiology of the NPM1 gene mutation in AML, clinical implications and the novel targeted therapies to date.

ETV6/RUNX1 fusion gene and its active role

Recent investigation successfully identified a pre leukemic ETV6/RUNX1-positive clone in the healthy twin of a patient diagnosed with ETV6/RUNXI-positive acute lymphoblastic leukemia (ALL) and also some studies with ETV6/RUNX1 knock in mice showed that the expression of the fusion gene is not sufficient for the invivo induction of ALL. Taken together, these data indicate that ETV6/RUNX1-positive leukemia is .generated through a multi-step mechanism, and that accumulation of additional genetic changes is necessary for the development of overt leukemia. Hence, to understand fully the genetic evolution of this disorder, identification of the complete spectrum of genetic changes that accompany the ETV6/RUNX1 fusion gene is necessary. Moreover, critical patho genetic insights may be gained from studying the correlation pattern of the different copy number changes.

Acute Myeloid Leukemia (AML) Patients Demonstrate Increased Prevalence of AML-Defining Mutations in Peripheral Blood Years Prior to the Development of Overt Leukemia: A Case-Control Study

Background: It is known that AML is preceded by the serial acquisition of somatic mutations but the pattern is unknown due to the lack of availability of samples prior to disease development. Clonal hematopoiesis of indeterminate potential has been associated with increased risk of hematologic malignancies but the specific contribution of subclonal genetic lesions to AML risk is unknown.We analyzed 725 samples from the Women's Health Initiative (WHI) cohort, which followed participants aged 50-79 from 1993-2010 for an average of 10.8 years. Cases and controls were selected as follows:Cases (n=212) included subjects with centrally adjudicated AML diagnosis. Peripheral blood samples were available from baseline and follow up (year 1 and 3) all prior to diagnosis of overt AML (average time to AML was 9.06 years).Controls (n=212) (healthy non-AML), were matched by age, timing of serial sample collection and history of cancer at WHI baseline and follow-up. Matching was done in a time forward manner to ensure similar follow up times.Methods: Ultra-deep targeted exome sequencing was performed using a 200 gene capture panel of recurrently mutated genes in hematologic malignancies. Variants present at 2.5% VAF minimum were selected and categorized as pathogenic significance vs. variant of uncertain significance (VUS) following rigorous quality control and filtration of SNPs. Analysis was focused on 67 genes recurrently mutated in MDS/AML in addition to categorical analysis of mutations (epigenetic regulators, activated signaling, spliceosome). Chromosomal rearrangements were assessed using information from split and paired end reads mapped to intronic regions of known breakpoints captured by the gene panel.Baseline characteristics of cases and controls were compared by using t-test or Wilcoxon rank-sum test for continuous variables, and the chi-square test or Fisher's exact test for categorical variables. Fisher's exact test was used to calculate the mutation status among cases and controls and to calculate the association of baseline mutation with AML diagnosis. Time to development of AML in cases was assessed by using regression analyses with the mutation status as a covariate. All p-values are two-sided with statistical significance evaluated at the 0.05 alpha levels.Results: The median age of cases and controls were 65.5 and 65 years respectively with average time to AML being 9.06 years. Myeloid specific mutations were detected in 35.4 % of controls and 59.04% of cases. The most commonly found mutations and their frequencies among cases and controls respectively were DNMT3A (22.8% and 10%), TET2 (19.5% and 4.2%) and ASXL1/ASXL2 (8% and 6.8%). TP53 (6.6%) and RUNX1 (2%) mutations were only seen in participants who eventually developed AML and were absent in controls. Subclonal mutations in DNMT3A, TET2, TP53, and SF3B1 revealed a general pattern of mutual exclusivity. Among cases, a >30-fold enrichment in pathogenic mutations was observed relative to controls (14.76 % vs.0.47%). One case demonstrated detectable MLL-ELL rearrangement 6 years prior to diagnosis of AML. There was no association of WBC, platelets or hemoglobin count with the presence of any mutation at the same time.There was an increased prevalence of myeloid specific mutation among cases compared to controls (OR 2.87, C.I. 1.90-4.37, p value <0.001). The presence of a pathogenic myeloid mutation was also significantly different among cases and controls (p value <.0001). In this matched case control analysis, the presence of a myeloid specific mutation at baseline was associated with the development of AML (OR 2.9, p value <0.001). Among cases, time to development of AML in participants with and without a prior myeloid specific mutation was 8.2 years and 9.6 years respectively (p value >0.05). Strikingly, all participants with a RUNX1 and TP53 and majority with the targetable IDH2 R140Q mutation (10/11) eventually developed AML many years later.Conclusions: The likelihood of having a myeloid -specific mutation at baseline, prior to the development of AML, was significantly higher in cases compared to controls from this WHI cohort. IDH2, TP53 and RUNX1 mutations were almost always predictive of development of AML. These findings suggest that subclonal stepwise acquisition of mutations may occur years prior to disease onset and that deep molecular monitoring using NGS may enable early intervention in selected patients. DisclosuresMencia-Trinchant:Cellectis: Research Funding. Lee:Clinipace: Consultancy; Baxalta: Consultancy; Amgen: Consultancy; Alexion Pharmaceuticals: Consultancy. Ritchie:Bristol-Myers Squibb: Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau; Pfizer: Consultancy, Other: Research funding to my institution; Celgene: Consultancy, Other: Travel, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Astellas Pharma: Other: Research funding to my institution. Guzman:Cellectis: Research Funding. Roboz:AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy; Cellectis: Research Funding. Hassane:Cellectis: Research Funding.

Incorporation of somatic panels for the detection of haematopoietic transformation in children and young adults with leukaemia predisposition syndromes and with acquired cytopenias.

Detection of somatic mutations may help verify the diagnosis of myelodysplastic syndrome (MDS) in patients with persistent cytopenias or with MDS-predisposition syndromes, prior to the development of overt leukemia. However, the spectrum and consequences of acquired changes in paediatric patients have not been fully evaluated, and especially not in the context of an underlying syndrome. We incorporated a targeted next-generation-sequencing panel of 54 genes for the detection of somatic mutations in paediatric and young adult patients with inherited or acquired cytopenias. Sixty-five patients were included in this study, of whom 17 (26%) had somatic mutations. We detected somatic mutations in 20% of individuals with inherited MDS-predisposition syndromes, including in patients with severe congenital neutropenia and Fanconi anaemia, and with germline mutations in SAMD9L. Thirty-eight per cent of children with acquired cytopenias and suspected MDS had somatic changes, most commonly in genes related to signal transduction and transcription. Molecularly abnormal clones often preceded cytogenetic changes. Thus, routine performance of somatic panels can establish the diagnosis of MDS and determine the optimal timing of haematopoietic stem cell transplantation, prior to the development of leukaemia. In addition, performing somatic panels in patients with inherited MDS-predisposition syndromes may reveal their unique spectrum of acquired mutations.

Development of a Data Portal for Aggregation and Analysis of Genomics Data in Familial Platelet Disorder with Predisposition to Myeloid Malignancy - the RUNX1.DB

Development of a Data Portal for Aggregation and Analysis of Genomics Data in Familial Platelet Disorder with Predisposition to Myeloid Malignancy - the RUNX1.DB

2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns.The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected.Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened.Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented.Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of a novel acquired der(1)del(1)(p13p31)t(1;15)(q42;q15) in a high risk t(12;21)-positive acute lymphoblastic leukemia

The t(12;21)(p13;q22) with ETV6-RUNX1 fusion occurs in 25% of cases of B-cell precursor acute lymphoblastic leukemia (BCP-ALL); and is generally associated with favorable prognosis. However, 15–20% of the t(12;21)-positive cases are associated with high-risk disease due to for example slow early responses to therapy. It is well-known that development of overt leukemia in t(12;21)-positive ALL requires secondary chromosomal aberrations although the full spectrum of these cytogenetic alterations is yet unsettled, and also, how they may be associated with disease outcome. This report describes the case of an adolescent male with t(12;21)-positive ALL who displayed a G-banded karyotype initially interpreted as del(1)(p22p13) and del(15)(q15). The patient was treated according to NOPHO standard risk protocol at diagnosis, but had minimal residual disease (MRD) at 6,4% on day 29 as determined by flow cytometric immunophenotyping. Because of MRD level>0.1% he was then assigned as a high risk patient and received intensified chemotherapy accordingly. Further molecular cytogenetic studies and oligo-based aCGH (oaCGH) analysis characterized the acquired complex structural rearrangements on chromosomes 1 and 15, which can be described as der(1)del(1)(p13.1p31.1)t(1;15)(q42;q15) with concurrent deletions at 1q31.2-q31.3, 1q42.12-q43, and 15q15.1-q15.3. The unbalanced complex rearrangements have not been described previously. Extended locus-specific FISH analyses showed that the three deletions were on the same chromosome 1 homologue that was involved in the t(1;15), and that the deletion on chromosome 15 also was on the same chromosome 15 homologue as involved in the t(1;15). Together these findings show the great importance of the combined usage of molecular cytogenetic analyses and oaCGH analysis to enhance characterization of apparently simple G-banded karyotypes, and to provide a more complete spectrum of secondary chromosomal aberrations in high risk t(12;21)-positive BCP-ALLs.

Oligo-based aCGH analysis reveals cryptic unbalanced der(6)t(X;6) in pediatric t(12;21)-positive acute lymphoblastic leukemia

Secondary chromosomal aberrations are necessary for development of overt leukemia in t(12;21)/ETV6-RUNX1-positive acute lymphoblastic leukemia (ALL). Conventional cytogenetic analysis supplemented with locus-specific FISH analyses is gold standard to detect important clonal aberrations in this disease group. However, adequate chromosome banding analysis may often be hampered by poor chromosome morphology and banding patterns in pediatric ALL cases, which may hinder identification of possible clinical important additional chromosomal aberrations.We used oligo-based high-resolution aCGH (oaCGH) analysis as an adjunct tool to enhance conventional cytogenetic analysis in pediatric acute B-cell lymphoblastic leukemia in a prospective single center study during a 4-year period (2012–2015).In a consecutive series of 45 pediatric B-ALLs, we identified eight patients with t(12;21)/ETV6-RUNX1 fusion by FISH analysis. In three of the patients, oaCGH analysis revealed concurrent Xq duplication and 6q deletion, which was cryptic by G-banded analysis. FISH analyses with whole chromosome painting probes confirmed the imbalances and showed an unbalanced translocation der(6)t(X;6) in all three patients. A search in the literature revealed two additional pediatric patients with cryptic der(6)t(X;6) in t(12;21)-positive ALLs. No common break points on Xq or 6q could be determined between the five patients.This study highlights the importance of oaCGH analysis as an adjunct cytogenetic tool to detect cryptic chromosomal aberrations. Further, the study adds to understanding the full spectrum of secondary chromosomal aberrations in the very common t(12;21)-positive pediatric ALL disease group. We suggest that the unbalanced der(6)t(X;6), which is cryptic to conventional cytogenetics, is a non-random secondary event in this disease group. It might be that the specific combination of concurrent Xq duplication and 6q-deletion results in gain of possible oncogenes on Xq and loss of possible tumor suppressor genes on 6q that are important for the leukemic propagation of t(12;21)-positive hematopoietic cells in a subset of ALLs.

Gipfel – a Novel Method for Unbiased Molecular ETV6-RUNX1 Screening of Healthy Newborns

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns.The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product.In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented.In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. DisclosuresNo relevant conflicts of interest to declare.

Hhex regulates Kit to promote radioresistance of self-renewing thymocytes in Lmo2-transgenic mice.

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute leukemias, in particular poor prognosis early T-cell precursor-like (ETP-) acute lymphoblastic leukemia (ALL). The primary effect of Lmo2 is to cause self-renewal of developing CD4(-)CD8(-) (double negative, DN) T cells in the thymus, leading to serially transplantable thymocytes that eventually give rise to leukemia. These self-renewing thymocytes are intrinsically radioresistant implying that they may be a source of leukemia relapse after therapy. The homeobox transcription factor, Hhex, is highly upregulated in Lmo2-transgenic thymocytes and can phenocopy Lmo2 in inducing thymocyte self-renewal, implying that Hhex may be a key component of the Lmo2-induced self-renewal program. To test this, we conditionally deleted Hhex in the thymi of Lmo2-transgenic mice. Surprisingly, this did not prevent accumulation of DN thymocytes, nor alter the rate of overt leukemia development. However, deletion of Hhex abolished the transplantation capacity of Lmo2-transgenic thymocytes and overcame their radioresistance. We found that Hhex regulates Kit expression in Lmo2-transgenic thymocytes and that abrogation of Kit signaling phenocopied loss of Hhex in abolishing the transplantation capacity and radioresistance of these cells. Thus, targeting the Kit signaling pathway may facilitate the eradication of leukemia-initiating cells in immature T-cell leukemias in which it is expressed.

Novel Genomic Structural Variations in Relapsed Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias Identified by Next Generation Sequencing

AbstractAbstract 518 IntroductionThe chimeric fusion gene ETV6/RUNX1 generated by the interchromosomal translocation t(12;21) presents the most frequent chromosomal aberration in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of all patients. This ALL subtype is associated with an overall favorable prognosis, nevertheless 10–20% of children will suffer from relapse. ETV6/RUNX1-positive preleukemic clones arise already in utero, but require additional cooperating oncogenic lesions for the development of overt leukemia. The nature of the assisting genetic alterations and the mechanisms driving the development of leukemia and recurrent disease are still not well understood. MethodsWe applied state-of-the-art whole genome and whole exome next-generation-sequencing to comprehensively analyze the assisting oncogenic alterations in pediatric patients with initial and/or recurrent ETV6/RUNX1+ ALL (primary disease n=11, recurrent disease n=7). Matched sample sets taken at initial diagnosis, remission and relapse were compared. Mate pair and/or paired end sequencing was carried out for whole genome analysis with inserts spanning 2 kb or 500 kb, respectively. Constructed libraries were sequenced from both ends with 36- or 50-bp reads on a Genome Analyzer IIx or a HiSeq 2000 (Illumina/Solexa), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Duplicate reads were removed. Unique reads with high mapping quality (q>35) served as input for GASV which detected translocations and inversions based on the mapping coordinates, insert sizes and read orientation. Variations covered by at least three reads in the tumor sample and not detected in the remission sample or the Database of Genomic Variants were reported. For detection of copy number variations, the program FREEC carried out coverage normalization, computation of copy number ratios between paired leukemia and remission samples (with up to 10 kb resolution) and subsequent segmentation. A subset of six selected patients was further investigated by targeted enrichment of whole exomic regions employing SeqCap EZ libraries (Roche) and 100 bp single read next-generation-sequencing on a HighSeq 2000. Mutations were called using GATK and further processed by an in-house bioinformatic pipeline. Putative somatically acquired mutations were validated by PCR, Sanger sequencing and FISH analysis. ResultsGenomes were sequenced to at least 13× physical coverage (mate pair) and 6.7× sequence coverage (paired-end). Exome sequencing achieved a minimum of 25× sequence coverage. In silico we detected 155 tumor-specific intragenic translocations. On average each tumor harbored 9 acquired translocations. With the exception of one case (13 translocations at diagnosis, 9 at relapse), the number of translocations was higher in relapse than in the matched diagnosis sample (additional 3 translocations on average). Ongoing validation studies confirmed the defining ETV6/RUNX1 translocation t(12;21) and identified 13 novel translocations. The genes affected are involved in essential signaling pathways, such as cytokine signaling (LIFR), calcium signaling (RCAN2), insulin and anti-apoptotic signaling (PHIP). Interestingly, also a factor essential for pre-mRNA splicing (IBP160) and genes encoding regulatory RNAs (miRNAs, lincRNAs and RNAs involved in splicing) were rearranged. A validated intragenic deletion of 836 bp leading to a frameshift and premature stop affected a calcium ion sensor of the ferlin protein family. Recurrent deletions in 9 of 11 cases (82%) ranging from 5 to 200 kb were detected in the immunoglobulin lambda variable gene cluster (IGLV) on chromosome 22q11. Some of the deletions were extending into the pre-B lymphocyte 1 gene (VPREB1) locus. In silico the probabilty of illegitimate RAG-mediated recombination at the breakpoint sites was determined by evaluation of RIC scores. RIC scores indicated that aberrant V(D)J rearrangements involving cryptic recombination sequence signals had caused the deletions on chromosome 22q11. ConclusionWe present somatic mutations that are promising novel candicate genes (e.g. LIFR, RCAN2, PHIP, IBP160) for cooperating secondary mutations in ETV6/RUNX1+ ALL and discuss their impact on the molecular pathology of primary and recurrent disease. Disclosures:No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia Shares a Common Cellular Origin with Regulatory B10 Cells

Abstract 286 Background:The cell of origin of CLL is unknown. Researchers have proposed various B cell subsets as the normal counterparts based on surface marker similarities or Ig gene utilization comparisons of normal and CLL cells. Regulatory B lymphocytes (“B10” cells), with the capacity to produce IL-10, negatively regulate T cell, B cell, and mononuclear phagocyte function. CLL patients are immunosuppressed with abnormalities in both humoral and cellular immunity. B10 cells have a phenotype similar to CLL cells (CD24hiCD27+CD5+CD19+). B10 cells are increased in autoimmune mice and in humans with autoimmune diseases—situations in which these cells negatively regulate immune-mediated inflammation. Since CLL cells and B10 cells may share common phenotypes and immunosuppressive mechanisms, we sought to determine if mouse and human CLL cells share common cellular origins and regulatory properties. Methods:Mouse spleen, lymph node, and bone marrow cell, and human blood B lymphocyte and CLL cell preparation and culture; IL-10, TNF, IGHV determinations; and flow cytometry were done as we have reported before (Blood 109:1559, 2007; Blood 117:530–541, 2010; Immunity 28:639–650, 2008). After culture for 5 hours with LPS and PMA+ionomycin+brefeldin A (PIB), or CpG+PIB, we assessed for intracellular IL-10 by flow cytometry. We term these IL-10 producing cells “B10” cells. Alternately, cells were cultured 48 hours with CD40 ligand+LPS or CD40L+CpG, and then PIB was added during the last 5 hours, after which cells were assessed for intracellular IL-10. We term these IL-10 positive cells “B10+B10pro” cells. Results:We examined CLL cells from 54 CLL patients. Most had low-risk disease: 90% were either Rai stage 0 or stage 1, 89% were CD38 negative, 46% were Zap70 negative, and 70% had a mutated IGHV. Twenty percent had normal cytogenetics, 48% del13q, 20% trisomy 12, 4% del17p, 4% del11q, and 4% with complex abnormalites. Patients with CLL as compared to healthy controls had higher numbers of B10pro cells compared to those of normal controls (7.7±0.9% and 3.2±0.3%, respectively; p<0.0001). CLL cells had a CD24hiCD27+ memory B cell phenotype similar to normal human B10 cells, and CLL cells secreted IL-10 when treated in vitro with CpG or CD40L/CpG, as do normal human B10 cells. CLL cell TCL-1 protein levels (immunoblot) correlated directly with CLL B10pro percentages (p=0.001) and absolute numbers (p=0.01). CLL patients’ plasma IL-10 levels were 1.5 fold higher than those of age-matched healthy controls (p=0.008), and these levels correlated directly with the absolute numbers of CLL cells that were competent to produce IL-10 after 48 hours stimulation with CD40L/CpG.To validate the precursor/product relationship between B10 cells and CLL, we studied the TCL-1 transgenic mouse model of CLL. TCL-1 transgenic mice had an age-dependent expansion of splenic CD5+B220int cells, and these leukemic cells were IL-10-competent. Likewise, aged TCL-1 mice had increased numbers of B10 cells in the bone marrow, lymph nodes, and peritoneal cavity. The TCL-1 CLL cells were similar in phenotype (IgM+CD11BhiCD23lowCD43hiCD19+) to mouse regulatory B10 cells (CD1dhiIgMhiIgDlowCD19hiCD23lowCD24hiCD43±) that we have previously reported. TCL-1 CLL cells produced IL-10 in vitro and in vivo, and depressed mouse macrophage TNF production. This TCL-1 CLL cell-mediated inhibition of mouse TNF production was blocked by anti-IL-10 antibody. Plasma IL-10 increased with age and with development of overt leukemia in TCL-1 mice. Summary:We demonstrate for the first time that human CLL cells and CLL-like cells from TCL-1 mice share a common origin with regulatory B10 and B10pro cells. Both CLL cells and B10 cells can produce the immuno-inhibitory cytokine IL-10 in vitro and in vivo, and they can suppress mononuclear phagocyte activation in vitro through IL-10-dependent pathways. The immunophenotype of CLL cells matches that of human B10, and B10pro cells. It is likely that IL-10 competent CLL cells derive directly from either regulatory B10pro or B10 cells. B10 cell-derived IL-10 may contribute to the immunosuppression noted in mice and humans with CLL. Future studies may lead to new and better treatments that take advantage of links between B10 cells, IL-10, and CLL. Disclosures:Lanasa:GlaxoSmithKline: Consultancy, Speakers Bureau. Tedder:Angelica: Consultancy, Share holder; Takeda Therapeutics: Consultancy.

Mll-AF9 Initiates Fetal Hematopoietic Stem Cell Transformation but Development of Overt Leukemia Requires Additional Events in Post-Natal Cells

Backtracking studies in twins show that human MLL fusion gene leukemias are often initiated during fetal life. However, important unresolved questions arewhat is the nature of additional genetic or epigenetic events that are necessary for development of overt leukemia in addition to the MLL-fusion gene? anddo these additional events occur directly in fetal hematopoietic cells or, alternatively, in the more differentiated progenitor cells that develop post-natally? In order to address these questions, the murine knock-in mouse model which contains the Mll-AF9 oncogene under endogenous regulatory control in all hematopoietic stem and progenitor cells is useful.In the current experiments, we analyzed the characteristics of day E14.5 fetal liver Lin− CD117+ Sca1+ Thy1.1low stem cells (FL-HSCs) and compared them with bone marrow HSCs (BM-HSCs) from 8-wk old mice for transformability and the ability to produce leukemia when transplanted to secondary recipients. Fetal liver cells were stained with biotin labeled antibodies against lineage markers (B220, CD3e, CD4, CD8, Ter119 and Gr1), and then enriched using MACS Micro Beads/Columns (Miltenyi Biotec). Partially enriched Lineage negative (Lin−) cells were further stained with PE-Cy5-Streptavidin, FITC-Sca1, APC-CD117 and PE-Thy1.1 antibodies, and then sorted with a FACS Aria (BD Bioscience). Sorting profiles showed that Mll-AF9 fetal livers had a higher percentage of CD117+ Sca1+ cells in the Lin− population than the fetal livers from wild type fetuses (1.4% vs. 0.9%, P < 0.05), indicating Mll-AF9 fusion gene induced expansion of FL-HSCs by day E14.5. The selfrenewal capability of FL-HSCs was also studied using myeloid colony forming assays in vitro. Sorted HSCs were cultured in methylcellulose medium containing IL-3, IL-6, SCF and GM-CSF, replated every 7 days for three generations and colony numbers were counted for each generation. The study revealed that Mll-AF9 FL-HSCs form significantly higher numbers of myeloid colonies than wild type FL-HSCs in all three generations. Colony distribution analysis also showed that Mll-AF9 FL-HSCs produce more immature compact colonies than wild type FL-HSCs in methylcellulose medium. 94–96% of cultured cells of the third generation were CD11b positive myeloid cells by FACS. The Mll-AF9 induced increase in FL-HSC numbers and colony growth was similar to the results we recently reported (Chen W et al, Cancer Cell , 13, 432, 2008) for BM-HSCs from 8-wk old Mll-AF9 mice. To compare the ability of Mll-AF9 FL-HSCs and BM-HSCs to produce overt leukemia, cells were injected with rescue bone marrow cells into lethally irradiated wild type recipients. One hundred BM-HSCs from 8-wk old Mll-AF9 mice caused leukemia in 90% of recipients at a median of 165 days. On the other hand, one hundred FL-HSCs recipients developed leukemia later and with lower frequency; in the recipients of one hundred FL-HSCs, 60% of recipient mice died at a median of 307 days (p<0.002). Increasing the injected number of FL-HSCs to 1000 did not increase the number of recipients with fatal leukemia when mice were followed to 355 days. Leukemic mice in all groups had enlarged spleens, high white counts and the CD11b/Mac1 myeloid phenotype characteristic of murine Mll-AF9 leukemia. In summary, our results show that in murine cells the Mll-AF9 fusion gene results in transformation both in fetal liver HSCs and BM HSCs as reflected in an increased number of both HSCs and compact myeloid colonies in vitro. However, MLL fusion gene induced transformation in either fetal liver or early postnatal bone marrow, while necessary, is not sufficient to produce overt leukemia in the mouse. The long delay in leukemia development in recipients of fetal HSCs and the similarities in leukemia organ distribution and phenotype in recipients of fetal and post natal HSCs suggests that leukemogenesis is a multistep process with critical secondary events occurring in cells that are post-natal. Therapeutic elimination of these post-natal leukemia-propagating cells in the mouse and the human should be facilitated by further understanding of the molecular signature of these critical cells.

Discordance of MLL-Rearranged (MLL-R) Infant ALL in Monozygotic Twins with Spontaneous Clearance of Preleukemic Clone in Unaffected Twin

The concordance rate for acute leukemia in infant monozygotic twins is estimated to be between 50 and 100%. The leukemic clone is thought to develop in one twin and transfer to the other twin via placental anastamoses. This hypothesis is supported by studies documenting identical, clonal, non-constitutional fusion sequences in concordant leukemias of monozygotic twins. For the ~80% of infant leukemias that are MLL-R+, the twin concordance rate is thought to be 100%, with a very short latency period between the diagnosis of the two twins. This suggests that either the MLL fusion is sufficient to cause leukemia, or it promotes the rapid acquisition of additional mutations required for the development of overt disease.We report a case of discordance in an infant monozygotic twin pair in which twin A presented with MLL-ENL+ ALL and twin B, who was transiently RT-PCR+ for MLL-ENL in bone marrow (BM) and blood, remains healthy 2.5 years later. Twin A was diagnosed at age 9 months (T=0) with CD10(−) pre-B ALL, cytogenetics t(11;19)(q23;p13.3), FISH+ for MLL-R, RT-PCR+ for MLL-ENL fusion transcript. Twin B was healthy with normal CBC and physical exam (PE). At T+7wks, twin B's CBC and PE remained normal, but RT-PCR on peripheral blood mononuclear cells (PBMNC) was MLL-ENL+. Three weeks later (T+10wks), BM aspirate was also RT-PCR+ for MLL-ENL. Morphology, FISH and cytogenetics were normal, as were CBC and PE. Two weeks later (T+12wks), twin B developed neutropenia (WBC= 2,880/ml; ANC=461) and thrombocytopenia (29,000/ml) one week after a viral URI (fever, rhinorrhea, cough, diffuse erythematous rash). He had no lymphadenopathy or hepatosplenomegaly on PE. Review of his PB smear confirmed the cytopenias and revealed atypical lymphocytosis and activated monocytes compatible with a viral process, but no lymphoblasts. Large platelets were noted, suggesting a diagnosis of acute ITP. No treatment was given. Repeat CBC 2 days later showed recovery of counts with WBC of 6,390/ml (ANC=1150) and platelets of 117,000/ml. RT-PCR of PBMNC was negative for MLL-ENL; BM aspirate was not performed. One week later, CBC had normalized. Over the next 4 months, monthly CBC and PE were normal, and monthly RT-PCR of PBMNC were negative. Twin B remains healthy with no clinical or laboratory evidence of leukemia at T+2.5yrs.Twin A was treated according to COG protocol P9407. End-induction BM showed morphologic remission, normal cytogenetics and negative FISH, although RT-PCR remained MLL-ENL+. End-consolidation BM showed continued remission, and was negative for MLL-ENL by cytogenetics, FISH and RT-PCR. At the end of P9407 therapy, an additional 1 year of maintenance therapy was given (total duration of therapy = 2 years). Two months after completion of therapy (26 months from diagnosis), twin A experienced an isolated testicular relapse of CD10(−) pre-B ALL, MLL-ENL+. He received reinduction chemotherapy and testicular radiation and is currently receiving post-induction chemotherapy.To our knowledge, this is the first report of discordant MLL-R leukemia in infant monozygotic twins, and the first report of documented spontaneous clearance of a detectable preleukemic clone in a healthy “twin B”. There are reports of twins discordant for MLL-R leukemia, but placental status was either unknown, dichorionic, or the leukemia developed in the first twin at 5 years of age and was likely postnatally acquired. The temporal association of the disappearance of the preleukemic clone in twin B with an episode of viral-induced, self-limited thrombocytopenia and neutropenia suggests the possibility that the immune process that mediated the cytopenias may also have been responsible for clearance of the preleukemic clone.This report confirms prior reports that clonal MLL-R cells are shared between monozygotic twins. However, this case presents evidence against the hypothesis that MLL rearrangements (specifically the MLL-ENL fusion) are sufficient for the development of overt leukemia. It is possible that a viral-induced autoimmune reaction may have aborted the progression of twin B's MLL-ENL+ preleukemic clone to overt leukemia by eradicating the clone prior to the acquisition of additional mutations. It is also possible that twin B's preleukemic clone persists at a level beneath the sensitivity of our RT-PCR assay. A less speculative conclusion is that short latency concordance of infant MLL-R leukemia in monozygotic twins is not universal.

Myelodysplastic Syndromes: An Historical Perspective

In 1900, Leube described a patient with severe megaloblastic anemia, preceding the development of overt leukemia. This case was followed by similar reports of patients characterized by cytopenia, dysmaturation of marrow precursors, an increase of marrow blasts and a significant risk of evolving to acute myeloid leukemia. The first classification of the myelodysplastic syndromes published in 1982 by the French-American-British (FAB) group, sorted out the differences between subgroups with and without an increase of marrow blasts, defined sideroblastic anemia by its characteristic accumulation of mitochondrial iron in erythroblasts, and included chronic myelomonocytic leukemia. During the 1990s, it became clear that additional clinical variables, and in particular chromosome analysis, largely contributed to clinical outcome and that survival and risk for leukemic transformation was significantly worse in patients with adverse cytogenetics. In 1997, patient cohorts from the United States, Europe and Japan were put together and reviewed thoroughly in order to form the database of the International Prognostic Scoring System (IPSS). Based on the number of significant cytopenias, percentage of marrow blasts, and cytogenetic risk profile the IPSS divided patients into four risk categories with significantly different probabilities for survival and risk for leukemic transformation. The IPSS has been extremely useful, and not until recently have new validated risk factors been introduced, the most important being presence of a transfusion need.

MEIS1 Is a Promising New Target for Leukemia Therapy.

The homeobox transcription factor MEIS1 is expressed in most human acute myeloid leukemias and MLL-rearranged leukemias of both myeloid and lymphoid phenotypes. MEIS1 over-expression in murine hematopoietic progenitor cells accelerates transformation initiated by other oncogenes. All leukemias developing in knock-in MLL-AF9 mice display abundant expression of MEIS1. However, it remains unknown if MEIS1 is essential for the survival of established leukemias. We studied the effect of MEIS1 silencing in both human and murine leukemia cell lines. To knock down the expression of MEIS1, we screened multiple shRNA constructs cloned into a lentivirus expression vector (The RNAi Consortium). Two MEIS1 shRNA lentivirus clones reduced the growth of human leukemia cells RS4-11 and SEMK2 by 50-70% at 5 days post-infection compared to control virus at a multiplicity of infection (MOI) of 10. The murine cell line (4166) was derived from a transgenic MLL-AF9 knock-in leukemic mouse and displays a myelo-monocytic phenotype, similar to human MLL-AF9 leukemias. When transplanted into wild type mice, this cell line induces leukemia with high efficiency. Two murine MEIS1 shRNA clones (M26 and M27) consistently inhibited the growth of 4166 cells at MOI of 10 or greater, detected as early as 24-48h. At 5 days post-transduction with an MOI of 100, growth of M26 and M27 transduced 4166 cells was reduced by 88% and 58% respectively in a representative experiment compared to those transduced with a control virus. In M26 transduced cells, MEIS1 mRNA levels were reduced by > 70% when compared to cells infected with a control virus. Analysis of DNA content by flow cytometry showed that MEIS1 silencing induced apoptosis, as indicated by an increase in the amount sub-diploid DNA, with the sub-diploid fractions in the non-transduced, control virus and M26 transduced cells being 17.4%, 21.7% and 63.9% respectively. When cultured in semi-solid methylcellulose media for 5 days, M26 transduced cells formed significantly fewer colonies than those transduced with a control virus (439±24 and 974±31 colonies per 1000 cells respectively, p<0.0006). The proportion of dense compact colonies, which are composed predominantly of undifferentiated myeloid progenitors was also reduced (37.9±4.8% Vs 90.5±0.8% respectively). To investigate MEIS1 as a molecular target for leukemia therapy, 10,000 cells untreated or infected with either M26 or control virus were injected into lethally irradiated wild type mice (7 mice/group). Animals were sacrificed when moribund. Necropsies with assessment of white blood cell count, spleen weight and FACS analysis were performed to confirm the cause of death. By day 115 post-transplant, 100% of the mice given untreated or control virus-transduced 4166 cells had succumbed to myeloid leukemia, while 43% of the MEIS1 shRNA treated group were still alive. Notably, 28% of mice in this group have survived past 170 days. Log-rank analysis revealed that survival of the three groups was significantly different (p<0.04). Overall, these results show that MEIS1 silencing in leukemia decreases cell growth, induces apoptosis and delays the development of overt leukemia in vivo. These results demonstrate that MEIS1 is an attractive molecular therapeutic target for leukemias that express this transcription factor critical for leukemia cell survival.

Trib1 and Evi1 cooperate with Hoxa and Meis1 in myeloid leukemogenesis

AbstractCooperative activation of Meis1 and Hoxa9 perturbs myeloid differentiation and eventually leads myeloid progenitors to leukemia, yet it remains to be clarified what kinds of subsequent molecular processes are required for development of overt leukemia. To understand the molecular pathway in Hoxa9/Meis1-induced leukemogenesis, retroviral insertional mutagenesis was applied using retrovirus-mediated gene transfer. The mice that received Hoxa9/Meis1-transduced bone marrow cells developed acute myeloid leukemia (AML), and Trib1, Evi1, Ahi1, Rarα, Pitpnb, and AK039950 were identified as candidate cooperative genes located near common retroviral integration sites. Trib1 and Evi1 were up-regulated due to retroviral insertions, and coexpression of these genes significantly accelerated the onset of Hoxa9/Meis1-induced AML, suggesting that Trib1 and Evi1 are the key collaborators. Furthermore, Trib1 by itself is a novel myeloid oncogene, enhancing phosphorylation of ERK, resulting in inhibition of apoptosis. These results demonstrate the importance of specific oncogene interaction in myeloid leukemogenesis.

Expression dynamics of drug resistance genes, multidrug resistance 1 (MDR1) and lung resistance protein (LRP) during the evolution of overt leukemia in myelodysplastic syndromes

It is well-known that leukemic cells of overt leukemia (OL) that have transformed from myelodysplastic syndromes (MDS) are more resistant to chemotherapy as compared with de novo AML cells. Thus, to examine the expression levels of drug-resistant genes and their alterations with the development of OL in MDS, the expression of mRNA for MDR1 and LRP was determined in bone marrow samples from control, de novo AML, MDS, MDS at the time of OL transformation (MDS → OL), and after transformation (OL) by quantitative real-time RT-PCR. The expression of MDR1 in MDS bone marrow at the time of initial diagnosis was as low as that for control subjects. However, the expression level was significantly elevated at the time of the development of OL (MDS → OL) compared with the initial MDS subjects ( P < 0.05), while expression was relatively reduced after OL development (OL). The expression of LRP was significantly higher in MDS and MDS → OL samples than control subjects. However, the high expression of LRP in MDS → OL was significantly reduced after OL development (OL). The expression levels of drug-resistant genes in MDS → OL or OL were not significantly higher than those of de novo AML samples, although LRP expression in MDS or MDS → OL was relatively higher than that of de novo AML. Detecting increases in the expression of MDR1 would be useful for predicting OL development in MDS patients.

Nonleukemic Granulocytic Sarcoma in the Bile Duct: A Case Report

Granulocytic sarcoma (GS) is an extramedullary tumor composed of immature myeloid cells, typically occurring during the course of acute myelogenous leukemia. Nonleukemic GS, that is, GS with no evidence of overt leukemia and no previous history of leukemia, is very rare, and even more unusual is nonleukemic GS of the bile duct. We report a case of nonleukemic GS of the bile duct. The patient was initially misdiagnosed as a bile duct carcinoma arising in the hilum of the liver (so-called Klatskin tumor), and received a right lobectomy of the liver. Histological examination of the tumor yielded the diagnosis of GS, and the bone marrow biopsy did not show any evidence of leukemia. Considering the risk of subsequent development of overt leukemia, the patient was treated with two cycles of combination chemotherapy as used in the cases of acute myelogenous leukemia. To date, he has remained free of disease 15 months after treatment.

Expression of IAP family proteins in myelodysplastic syndromes transforming to overt leukemia

Bone marrow cells of patients with myelodysplastic syndromes (MDS) frequently undergo apoptosis, though the apoptotic cell ratio decreases when overt leukemia (OL) develops. Thus, we compared the expression of the inhibitor of apoptosis protein (IAP) gene family proteins in bone marrow samples from control, MDS, OL transformed from MDS (MDS → OL), and de novo acute myelogenous leukemia (AML) subjects by the quantitative real-time RT-PCR method and an immunohistochemical approach. Overexpression of mRNA for survivin, cIAP1, NAIP and XIAP was significant in MDS bone marrow cells compared with control samples. However, the expression of mRNA for survivin, cIAP1 and cIAP2 exhibited a remarkable decrease after the development of OL (MDS → OL). By immunohistochemistry, survivin was found to localize to the nucleus of myeloid cells in the majority of MDS cases. Next, the chronological changes in the expression of IAPs were determined in cases of MDS with evolution of OL. Although the expression of cIAP1 and cIAP2 revealed a sudden or gradual decrease as OL developed, survivin in many cases and XIAP in the majority of cases exhibited a peak of expression before a decline, indicating that these IAPs could be associated with the early events in the development of OL.