Leukemic Transformation In Patients Research Articles

Risk of thrombosis, hemorrhage and leukemic transformation in patients with myeloproliferative neoplasms: A nationwide longitudinal cohort study

IntroductionThere are few large-scale, population-based studies detailing the risks of thrombosis, hemorrhage, leukemic transformation in patients with myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). MethodsWe performed a nationwide longitudinal cohort study using the Korean National Health Insurance System (NHIS) database. MPN patients (n = 11,991) and their 1:4 age- and sex-matched controls (n = 47,964) were enrolled. The risk of thrombosis, hemorrhage, leukemic transformation was estimated using a Cox proportional hazards regression, and stratified analyses were performed for related factors. ResultsDuring a median of 7.8 years of follow-up, 30.1 % of MPN patients (3614/11,991) and 19.0 % of the matched controls (9141/47,964) developed arterial thrombosis, 11.6 % of MPN patients (1397/11,991) and 6.4 % of the matched controls (3099/47,964) developed venous thrombosis and 18.7 % of MPN patients (2251/11,991) and 12.1 % of the matched controls (5836/47,964) developed hemorrhage. 4.9 % of MPN patients (597/11,991) and 0.1 % of matched controls (50/47,964) developed leukemia. The overall risk of developing thrombosis, hemorrhage, leukemic transformation was higher in MPN patients (adjusted hazard ratio [aHR] 1.695, 95 % confidence interval [CI]: 1.629–1.765 for arterial thrombosis, aHR 1.963, 95 % CI: 1.838–2.096 for venous thrombosis, and aHR 1.714, 95 % CI: 1.630–1.802 for hemorrhage) than in the controls. Patients with MPNs had a 10-year cumulative incidence of leukemic transformation of 6.2 %. ConclusionThe patients with MPNs have a higher risk of thrombosis, hemorrhage, and leukemic transformation than matched controls. Strategies are warranted to reduce the risk of thrombosis, hemorrhage, and leukemic transformation in MPN patients.

Polycythemia Vera with Elevated Erythropoietin Level: A Review

According to the 2016 World Health Organization (WHO) recommendations, polycythemia vera (PV) should be suspected at hemoglobin levels of 16.5 gm/dl in males and 16 gm/dl in females. Depending on the underlying driving factor, polycythemia is classified to primary and secondary. Secondary polycythemia is driven by erythropoietin (EPO), which can be released due to hypoxemia, as seen in people living at high altitudes, those with chronic lung diseases, obstructive sleep apnea (OSA) and smoking. It can also be directly released by an underlying EPO secreting tumor, such as renal cell carcinoma. On the other hand, primary polycythemia or polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) in which erythrocytosis is driven by an acquired activating mutation that increases sensitivity to EPO or causes EPO independent erythroid colony formation. Manifestations of PV include fatigue, excessive itching, abdominal pain, weight loss, and fever. However, the most critical complications of PV, which impact survival, are thrombotic complications such as stroke and venous thromboembolism and leukemic transformation. The risk for leukemic transformation in patients with PV is about 2-5% over 15 years and is more common in individuals older than 60. History and physical examination should focus on smoking history and signs and symptoms of cardiopulmonary diseases causing chronic or intermittent hypoxemia, as seen in OSA. On the other hand, findings of abdominal pain, early satiety, weight loss, erythromelalgia, and hepatosplenomegaly would be suggestive of PV. Laboratory workup classically starts with measuring EPO level which further directs workup toward PV or secondary polycythemia. If workup is suggestive of PV, the following steps would be testing for activating Janus Kinase 2 (JAK2) mutation (V617F), which has a sensitivity of 97% for PV, and bone marrow biopsy which shows hypercellularity with trilineage growth (panmyelosis) in cases of PV. EPO measurement constitutes an essential step in polycythemia workup. It is widely accepted that elevated EPO levels are associated with secondary polycythemia; and suppressed EPO level was considered a criterion to diagnose PV, according to the WHO 2016 diagnostic criteria for PV. We conducted a systematic review searching the entity of PV with elevated EPO to study the clinical significance and outcomes. Our search yielded 4 cases summarized in Table 1. The cases were of three males and one female, with a mean age of 50. Two of them were healthy, with two with comorbid diseases, and as seen in the table; only one was a smoker. The most common symptom was headache, followed by erythromelalgia. Thrombotic phenomena happened in a single case in the form of Budd Chiari syndrome. Hepatosplenomegaly was found in a single case, and splenomegaly was found in two cases. The mean Hb level was 20.2 gm/dl, and the mean EPO level was 213. EPO is a glycoprotein hormone that is mainly produced by the kidneys. However, it is also produced in small amounts in the liver, spleen, bone marrow, lungs, and brain. The primary regulator of EPO production is the partial pressure of oxygen (PaO2). Multiple case reports and case series suggested an association between PV with elevated EPO and Budd Chiari Syndrome. Thumures et al. described four patients that were diagnosed with Budd Chiari Syndrome with elevated red cell mass and were found to have elevated serum erythropoietin levels. PV was confirmed with JAK2 mutation assay and bone marrow biopsy examination. It is speculated that liver hypoxia with subsequent hepatocyte injury and necrosis caused by Budd Chiari Syndrome leads to EPO level elevation. Accordingly, we assume that patients with PV and elevated EPO have some degree of organ injury that results in the release of EPO into circulation. This highlights the importance of a systematic approach in medicine, where signs and symptoms remain the cornerstone in diagnosis and where lab tests should be taken in their appropriate clinical context. Thus, we recommend testing for JAK2 mutation in the proper clinical scenario regardless of the EPO levels. It still remains to be clarified whether there are any prognostic importance of the elevated EPO level in these patients and whether it would alter the management.

MPN-398: U2AF1 and SRSF2 Drive Poor Prognosis in Myelofibrosis Through Different Mechanisms

Context: Splicing factor mutations (SRSF2, U2AF1, SF3B1, and ZRSR2) are present in ∼50% of myelofibrosis (MF) patients. They are associated with cytopenias, the management of which is an area of unmet clinical need in MF. SRSF2 and U2AF1 Q157 are considered high-risk mutations, while the prognostic significance of ZRSR2 and SF3B1 has not been well-defined. Objective: To describe the clinical characteristics, treatment approaches, and outcomes in MF patients with splicing mutations. Design: Single-institution, retrospective analysis of 133 MF patients with splicing mutations. Baseline variables were compared between patients harboring different splicing factor mutations. Median overall survival (OS) measured diagnosis to date of death or censored at last follow up/transplant. Results: Among 133 MF patients with a splicing mutation, SRSF2 mutations were most common (n = 48), followed by U2AF1 (n = 36), SF3B1 (n = 27) and ZRSR2 mutations (n = 24). Most SRSF2 mutations occurred at P95. Twenty-nine (81%) U2AF1 occurred at Q157, with 7 (19%) at S34. Eleven (41%) SF3B1 mutations occurred K666, with 9 (33%) at K700. Twelve (50%) ZRSR2 mutations were in-frame insertions/deletions, 2 (8%) frameshift mutations, 6 (25%) nonsense mutations and 4 (17%) missense. Spliceosome mutations were mutually exclusive but for 2 cases. Baseline characteristics were similar between splicing mutations. U2AF1 mutations showed increased prevalence of anemia and thrombocytopenia at presentation, with higher DIPSS/DIPSS+. IMiDs (lenalidomide/thalidomide) were frequently used and associated with encouraging response rates (43–57%), except in patients with SRSF2 mutations (25% response rate). Median OS was decreased in patients with U2AF1 (44 months) and SRSF2 mutations (33 months). Median OS in SF3B1 and ZRSR2 were 181 and 106 months, respectively. SRSF2 mutations were associated with an increased risk of transformation to AML (24% vs 3%, p = 0.002). Conclusions: In MF patients with splicing mutations, U2AF1 mutations are uniquely associated with anemia and thrombocytopenia. IMiD treatment is beneficial, though less so in patients with SRSF2 mutations. The presence of either U2AF1 or SRSF2 mutation is associated with poor prognosis; however, this appears to be driven by cytopenias in U2AF1-mutated patients and leukemic transformation in patients with SRSF2 mutations.

Patterns of leukemic transformation in patients with TP53-mutant myelodysplastic syndromes.

7054 Background: TP53 is the most frequently mutated gene in human cancers, including 7-13% of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. Although it is associated with transformation to AML in patients with MDS, the additional genomic events leading to transformation are poorly understood. Methods: We retrospectively evaluated 312 patients with TP53-mutated AML or MDS diagnosed between 2013-2016. Patient characteristics and bone marrow data, including cytogenetic and next generation sequencing information, were assessed at the time of diagnosis and progression to AML. Results: There were 151 TP53-mutated MDS patients and 161 TP53-mutated de novo AML patients with a median follow-up time of 34.1 months. Forty-one patients with TP53-mutated MDS transformed to AML. Sequencing data at transformation was available in 17 patients (41%). At diagnosis, median age was 67 with 2 patients with intermediate-risk, 7 patients with high-risk, and 32 patients with very high-risk MDS by IPSS-R. Complex karyotype was seen in 40 patients, and 12 patients had 1, 25 patients had 2, and 4 patients had 3 TP53 abnormalities. Predictors of transformation to AML include TP53 loss of heterozygosity (p = 0.008), 3 TP53 abnormalities (p = 0.049), complex cytogenetics (p = 0.023), and female gender (p = 0.002). Median time to transformation was 10.4 months. At transformation, an increase in TP53 variant allelic frequency was observed in 7 patients (41%), and new mutations, particularly NRAS, IDH1, TP53, MLL, KDM6A, TET2, and NOTCH1, were acquired by 10 patients (59%). Cytogenetic evaluation revealed identical clones in 5 patients, linear acquisition of cytogenetic abnormalities in 12 patients, and new clones in 8 patients. Patients with TP53-mutated AML from MDS also had worse median overall survival than patients with TP53-mutated de novo AML at 2.5 months vs. 5.7 months respectively (p < 0.001). Conclusions: TP53 mutations confer a worse prognosis, especially in the setting of AML transformed from MDS. This may be due to the acquisition of new mutations and cytogenetic abnormalities. Further exploration of the biological mechanisms leading to transformation in TP53-mutant MDS is warranted.

Diagnosis of GATA2 haplo-insufficiency in a young woman prompted by pancytopenia with deficiencies of B-cell and dendritic cell development

BackgroundGATA2 deficiency presents with a spectrum of phenotypes including increased susceptibility to viral and bacterial infections, multi-lineage cytopenias, aplastic anemia, leukemic transformation and lymphedema. Allogeneic transplantation is only curative therapy for GATA2 deficiency, but is associated with significant treatment related morbidity and mortality. Given the spectrum of clinical presentation, accurate diagnosis of GATA2 deficiency is necessary to identify patients early in their disease course when allogeneic bone marrow transplantation may be of clinical benefit.Case presentationIn this report, we present a GATA2 mutation diagnosed in 23-year-old woman presenting with pancytopenia, recurring oral blisters, fatigue and chronic pain. We describe markedly low levels of mature B-cells in the blood and bone marrow and the absence of detectable blood dendritic cells with normal serum immunoglobulin levels and normal numbers of marrow plasma cells. She was ultimately diagnosed with GATA2 haplo-insufficiency due to a GATA2 germ-line mutation and underwent a successful allogeneic bone marrow transplant from a 10/10 HLA matched unrelated donor.ConclusionsThe case illustrates the diagnostic difficulties in identifying GATA2 deficiencies and the importance of family history and genetic testing. GATA2 plays an important role in B-cell and dendritic cell development, and decreased numbers of those cells is a characteristic feature that should prompt consideration of GATA2 deficiency in a patient with pancytopenia. Maturation of B-cells to long-lived plasma cells is relatively unaffected in GATA2 deficiency. Allogeneic stem cell transplantation can correct immune-deficiencies and prevent leukemic transformation in patients with GATA2 deficiency.

Leukemic transformation in patients with myeloproliferative neoplasms: a population-based retrospective study.

This study determined the epidemiology of developing leukemic transformation in patients with myeloproliferative neoplasms (MPN). We utilized the Surveillance, Epidemiology and End Results 13 database to identify 83 cases of leukemic transformation in MPN (n = 9335). The 5-year cumulative incidence of leukemic transformation was higher in male versus female (2.17 vs 1.09%; p < 0.001), and in myelofibrosis (2.19%; 95% CI: 1.36-3.34%), compared with essential thrombocythemia (0.37%; 95% CI: 0.19-0.65%) and polycythemia vera (0.72%; 95% CI: 0.46-1.07%; p < 0.001). Patients had a median survival of 2 months after leukemic transformation, worse in older patients and without any impact of prior MPN subtypes. Myelofibrosis has a higher risk of leukemic transformation. Overall survival is dismal regardless of MPN subtypes.

A Novel Somatic Mutation in IKZF1 Is Common in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Myelofibrosis

Background:Myeloproliferative neoplasms (MPN) including polycythemia vera, essential thrombocytosis, and primary and secondary myelofibrosis (MF) are characterized by driver mutations in JAK2, CALR, and MPL. Additional somatic variants can be seen. Currently allogeneic hematopoietic stem cell transplant (HCT) is the only treatment with curative potential, but it is associated with a high risk of mortality, exceeding 50% in some patient subsets. The risk of disease progression usually outweighs the risk of transplant in patients with advanced MF. Here, we have evaluated the mutational profile using a targeted next-generation sequencing panel of 54 genes involved in myeloid neoplasms in patients with MF treated with HCT.Patients and Methods:48 patients with a diagnosis of primary (n=18) or secondary (n=30) MF who received a HCT at our center and from whom genomic DNA was available from pre-HCT marrow aspirates were evaluated. DNA was isolated from whole marrow aspirates obtained prior to HCT. Paired-end indexed libraries were sequenced at an average read depth of 1000x on Illumina MiSeq platform. Sequences were aligned to hg19 using the Burrows-Wheeler Aligner, and nucleotide variants were identified using the Genome Analysis Toolkit and Picard tools. Variants were annotated with ANNOVAR. Variants present at >5% variant allele frequency (VAF) with at a minimum of 25 variant reads were selected for further analysis. Variants were called "mutations" if they resulted in (1) non-synonymous changes in amino acid sequence, including nonsense and missense mutations, (2) the variant was known to be associated with hematologic malignancies in COSMIC database, or (3) the variant was present at < 0.5% in 1000 genomes and ExAC databases. Comparisons between clinical variables and mutations were analyzed with chi-square tests, adjusted for multiple testing.Results:The average age of the 48 patients was 55 years (range 23-72) and 52% of patients were female. Disease risk was estimated with DIPSS-Plus (High 33%, INT-2 53%, INT-1 7%, Low 7%). Unfavorable cytogenetics were present in 27% of patients. Donors included a matched sibling donor in 30% (n=14), a matched unrelated donor in 60% (n=29), and an alternative donor in 10% (n=5). At 3 years, overall survival was 52%, 17% of patients had relapsed disease, and 31% had non-relapse mortality.In our cohort, 38% had a mutation in CALR (n=15 type-I and n=3 type-II), 29% in JAK2 (n=14), 0% in MPL, and 33% were triple-negative (n=16; no evidence of CALR, JAK2, or MPL mutation). The average VAF of CALR mutations was 50% (range 20-82%) and JAK2 mutations was 58% (range 25-81%). In addition to driver mutations, 94% of patients had at least one other mutation in 26 of the 54 genes sequenced. On average, patients had 2.1 other mutations (range 0-5 mutations in addition to driver mutation). The most common other mutations were in ASXL1 (31%), IKZF1 (23%), ETV6 (17%), and U2AF1 (17%). The VAF of these other mutations spanned 9-76%.Patients with triple-negative MF were more likely to have primary MF (p=0.019) and platelets <100 (p<0.001), than patients with JAK2 and CALR mutations. Patients with ≥3 other mutations were more likely to have hemoglobin <10 (p=0.02) and a higher rate of relapse (p<0.01) than patients with <3 other mutations. Patients with ASXL1 mutations were more likely to have peripheral blood blasts (p<0.001).Mutations in ASXL1, ETV6, and U2AF1 have been previously reported in myeloid malignancies. Like other groups, we found ASXL1 mutations associated with worse prognosis. Here we report a novel, recurrent mutation in the gene IKZF1. All patients with IKZF1 mutation carried the same insertion of 11 nucleotides leading to an early frameshift and ultimate nonsense mutation (L92fs), which would lead to deletion of the zinc-finger domain. Such a mutation likely leads to loss of IKZF1 tumor suppressor function.Conclusions:Somatic mutations, in addition to driver mutations, are common in patients with MF treated with HCT. Mutation profiling in this patient subset may improve relapse risk prediction. Loss of heterozygosity on chromosome 7 mapping to the IKZF1 locus has been found by others to be associated with leukemic transformation in patients with MPN (Jaeger et al. 2010. Leukemia. 24: 1290). Given their frequency in this high risk MF population, further study of the role of IKZF1 mutations in MPN is warranted. DisclosuresRadich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; TwinStrand: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy.

Impact of Genomic Alterations on Outcomes in Myelofibrosis Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: The impact of genomic alterations, such as mutations in ASXL1, on the risk of disease progression and leukemic transformation in patients with myelofibrosis (MF) is well established. Further, emerging data suggests that the number and type of mutations may impact response to therapies such as ruxolitinib or imetelstat. Allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the only potentially curative treatment for MF patients. However, the impact of somatic mutations on overall survival (OS) and relapse-free survival (RFS) is poorly understood. Using next-generation sequencing of pre-transplant blood and bone marrow samples from a well clinically-annotated cohort of MF patients who underwent allo-HSCT, we sought to determine the impact of mutational burden on outcomes.Methods: A multicenter retrospective analysis of a cohort of 84 patients was carried out. This included 52 patients treated on the MPD-RC 101 prospective study (NCT00572897), 18 patients treated at Prince Margaret Hospital, and 14 patients treated at Memorial Sloan Kettering Cancer Center. Patient and transplant characteristics are displayed in Table 1. DNA was extracted from pre-transplant bone marrow aspirate samples or peripheral blood samples. High-throughput sequencing of a panel of genes was performed. Average coverage of 829x (standard deviation of ±130) was obtained. Mutect was utilized to call single point variants (comparing our samples to a pool of normal samples) and PINDEL was used to call short insertions and deletions. We excluded all mutations present in at least one database of known non-somatic variants (DBSNP and 1000 genomes) and absent from COSMIC. Univariate Cox regression and Kaplan-Meier graphics were used to investigate the association of patient, transplant, and disease characteristics with OS and RFS.Results: JAK2V617F was the most frequent mutation detected in 41(48.8%) patients (Table 2). Eighteen patients (21.4%) had triple negative disease (negative for JAK2, MPL, and CALR mutations). Univariate analysis included the following: patient characteristics (age, gender), transplant characteristics (related vs. unrelated donor, matched vs. mismatched donor and myeloablative vs. reduced intensity conditioning) and disease characteristics (DIPSS and presence of mutations). Decreased OS was associated with unrelated donor status (HR 2.09, 95% CI: 1.03-4.23, p=0.04), reduced intensity conditioning (HR 4.21, 95% CI: 1.01-17.59, p=0.049), triple negative disease (HR 2.09, 95% CI: 1.02-4.30, p=0.04), and presence of U2AF1 (HR 2.53, 95% CI: 1.10-5.81, p=0.03) or SUZ12 mutations (HR 3.92, 95% CI: 1.19-12.21, p=0.02). Decreased RFS was associated with unrelated donor status (HR 2.27, 95% CI: 1.16-4.45, p=0.02), and the presence of SUZ12 mutation (HR 6.97, 95% CI: 2.37-20.49, p<0.001). A descriptive decrease in RFS in patients with U2AF1 (HR 2.15, 95% CI: 0.94-4.88, p=0.07) was observed but did not reach statistical significance. Importantly, mutations previously reported to be associated with reduced OS and RFS in the non-transplant setting, such as ASXL1, EZH2, IDH1/2, and SRSF2, were not associated with poorer outcomes in this analysis in transplanted patients. In an exploratory multivariate analysis including donor type (related vs. unrelated) and presence of U2AF1 and SUZ12 mutations, there was a significantly reduced OS and RFS in patients who harbor these mutations regardless of donor type (OS: HR 5.30, 95% CI: 2.08-13.47, p<0.001; RFS: HR 5.49, 95% CI: 2.27-13.30, p<0.001). In patients without the above mutations, having an unrelated donor was associated with worse OS (HR 2.55, 95% CI: 1.09-5.96, p=0.03) and RFS (HR 2.61, 95% CI: 1.17-5.83, p=0.02, Figure 1).Conclusions: Our analysis demonstrates that mutations previously associated with poor prognosis in MF, such as ASXL1, do not appear to confer a worsened prognosis in patients undergoing allo-HSCT, suggesting transplant may be able to overcome the impact of these mutations. However, mutations in SUZ12 and U2AF1 are associated with reduced OS in univariate and multivariate analysis (together with donor type). Further studies with larger cohorts of patients are indicated to validate these findings, and to elucidate the impact of these mutations on disease biology. [Display omitted] [Display omitted] [Display omitted] DisclosuresRampal:Incye and CTI: Consultancy. Mascarenhas:Janssen: Research Funding; CTi Biopharma: Research Funding; Promedior: Research Funding; Merk: Research Funding; Incyte: Research Funding. Mesa:Galena: Consultancy; Gilead: Research Funding; Promedior: Research Funding; Incyte: Research Funding; CTI Biopharma: Research Funding; Celgene: Research Funding; Ariad: Consultancy; Novartis: Consultancy. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.

Second malignancies in Philadelphia-positive and -negative myeloproliferative neoplasms: A single center study

Introduction: Leukemic transformation (LT) of both Philadelphia (Ph) -positive and -negative myeloprolifetarive neoplasms (MPNs) is a well-known subject. However sufficient data are not available in literature from Turkey about the frequency of second malignancies (SMs) except LT in patients with MPNs. In this study, it was aimed to investigate the frequency of SMs in Ph-positive or -negative MPN cases. Materials and Methods: A total of 438 patients diagnosed with classical MPN according to WHO 2008 diagnostic criteria were included in the study.Results: SMs were detected in 15 out of 438 patients (3.4%). In this study, cancer incidence rate was found higher (1149.8/100.000 person-years for males and 540.8/100.000 person-years for females with MPNs) compared with Turkey data. Conclusion: SM frequency is significantly higher than normal population in patients with MPNs. Therefore these patients should be carefully examined for SM symptoms and signs.

SCT for Secondary AML in a Patient with Glycogen Storage Disease Type 1b

AbstractAbstract 4402It is well established that patients with congenital neutropenia due to mutations of ELANE or HAX1 have a strongly increased risk of developing secondary leukemia. In contrast, the leukemia risk is not strongly increased in patients with specific other neutropenia subtypes (e. g. cyclic neutropenia with ELANE mutations). Here we report the first glycogen storage disease type Ib (GSD1b) patient in our registry who developed acute myelogenous leukemia (AML). GSD1b is an autosomal recessive disorder of glycogen metabolism with intracellular accumulation of glycogen caused by mutations in the glucose-6-phosphate translocase gene. Patients present with hepatomegaly, growth retardation, hypoglycemia, lactic acidosis as well as neutropenia and neutrophil dysfunction predisposing to bacterial infections. G-CSF has been shown to be an effective long-term treatment for GSD1b patients leading to an increase in neutrophil counts and enhancement of neutrophil function resulting in fewer infections and improved quality of life. The European Branch of the Severe Chronic Neutropenia International Registry (SCNIR) has collected long-term data from 23 patients with GSD1b. Median age was 17 years, 3 patients expired from infections at an age of 8; 9 and 26 years, respectively. All 23 patients received G-CSF treatment for a median duration of 9.5 years, 22 patients received continuous G-CSF with a median dose of 3.4 mcg/kg/day (range 0.8 to 5 mcg/kg/day), one patient received G-CSF interventionally only.Our patient was diagnosed with AML FAB classification M2 linked to cytogenetic aberrations involving trisomy 21. Leukemia cells also harboured a missense RUNX 1 mutation in exon 3. G-CSF treatment was initiated at age 5 months at a dose of 2.3 mcg/kg/d and was continued until AML diagnosis at age 10 years. The G-CSF dose was increased due to low ANCs and infectious episodes with otitis media and pneumonitis up to 4.05 mcg/kg/day. After initial AML treatment with a hypometylating chemotherapy consisting of azacytidine and cytarabine she developed partially reversible renal insufficiency and oxygen dependent pneumonitis which led to a reduction of chemotherapy despite persistence of AML blasts.Remission was achieved with the use of mylotarg. Subsequently, she received a conditioning regimen with melphalan, fludarabine and treosulfan plus ATG-Fresenius followed by infusion of 10/10 HLA matched peripheral blood stem cells from an unrelated donor. Leukocyte engraftment occurred on day +18 followed by neutrophil engraftment on day +21. Post-SCT she developed grade III skin GvHD on day +8 which was responsible to steroid treatment. Discussion:Up to date 3 GSD1b patients with development of AML have been reported in the literature. Two patients were on long-term G-CSF treatment (Pinsk et al. J Pediatr. 2002; Schroeder et al. J Medical Case Reports 2008), one patient transformed predating the availability of G-CSF (Simmons et al. J Pediatr. 1984). Conclusion:According to the literature, leukemic transformation in patients with GSD1b is a rare event. However, with a total of four patients reported with secondary AML with or without G-CSF treatment, GSD1b patients may have a higher leukemia risk than previously anticipated. Independent from G-CSF treatment, regular bone marrow examinations (morphology, cytogenetics, mutation analysis of candidate genes such as RUNX1) may help to detect malignant transformation early and therefore avoid chemotherapy prior to SCT. SCT is a treatment option for AML in GSD1b, but further data is required. Disclosures:No relevant conflicts of interest to declare.

A patient with a 20-year lag phase between JAK2-V617F+ myeloproliferation and NPM1-mutated AML arguing against a common origin of disease

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.

224 Factors affecting leukemic transformation in patients with del-5q treated with lenalidomide

224 Factors affecting leukemic transformation in patients with del-5q treated with lenalidomide

Short Telomeres Before Lenalidomide Treatment Predict Leukemic Progression In Patients with Myelodysplastic Syndrome and Deletion 5q

AbstractAbstract 30Lenalidomide has been shown to induce transfusion independence and cytogenetic response in a high proportion of patients with MDS and isolated 5q deletion (del5q). However, some of these patients progress into acute myeloid leukemia, particularly those who do not show an erythroid or cytogenetic response. Yet the mechanisms inducing the leukemic transformation in patients with MDS and del(5q) are mainly unclear. Since dysfunctional telomeres play an important role in the development of genetic instability and shorter telomeres are associated with advanced MDS and AML, we reasoned whether telomere shortening may contribute to leukemic progression in patients with MDS and del5q. This study included 14 patients with transfusion-dependent anemia and low- or intermediate-1-risk MDS enrolled in the studies MDS-003 (n=42) or MDS-004 (n=260) who were treated with lenalidomide according to the study protocols. Written informed consent was provided according to the Declaration of Helsinki. Seven patients progressed into RAEB-1 or AML while on study and 7 patients did not. Time span between initial diagnosis and study entry was similar (median 51.3 and 44.4 months respectively in the patients with and without leukemic progression). Combined fluorescence R-banding and T/C-FISH analysis to determine the telomere length of each individual chromosome was performed as described earlier (K Lange et al, Genes Chromosomes Cancer, 2010). Telomere length at study entry was 6.1 kb (range 4.8 to 7.9 kb) in patients with MDS and del5q who later underwent leukemic transformation. Patients without later disease progression had a median telomere length of 9.3 kb (range 8.4 to 10.2 kb). Thus, patients who progressed after a median of 29 months (range 5 to 51 months) had significantly shorter telomeres than patients who had a cytogenetic response or stable disease (p<0.001). Notably, during treatment with lenalidomide, the group of patients who underwent leukemic transformation had a median increase in telomere length of 2.4 kb (range: 0.5 to 3.4 kb, p<0.02). In contrast, no relevant telomere elongation was observed in patients who did not undergo leukemic progression (9.3 kb at study entry and 8.8 kb after a median of 31 months (range 11 to 52 months) following lenalidomide treatment). Furthermore, telomere length was independent of the presence of additional aberrations. Two patients with additional aberrations and subsequent progression had shorter telomeres than another patient with additional aberrations who did not undergo progression (5.7 kb and 7.9 kb versus 10.2 kb). Two patients with disease progression showed a mosaic of normal and aberrant metaphases in the bone marrow. In both cases, telomeres of the normal metaphases were longer than the telomeres of aberrant metaphases (9.1 kb versus 6.9 kb; 9.7 kb versus 5.5 kb, respectively). Three of the patients who underwent leukemic progression acquired additional chromosome aberrations and developed complex karyotypes. They had a median telomere length of 6.4 kb (range: 4.8 to 7.9 kb), which was not significantly different from the median telomere length of 6.0 kb (range: 4.8 to 7.5 kb) in the group of patients who showed an isolated del5q during the leukemic transformation. Thus, telomere shortening not only preferentially occurred in cells with del(5q), it was also independent of clonal evolution. Although the underlying mechanisms leading to excessive telomere shortening in MDS pathogenesis remain unclear, telomere shortening seems to predispose to leukemic transformation in patients with MDS and del5q. Currently, factors predictive for increased risk of leukemic transformation are lacking and only clinical, morphological and cytogenetic follow-up investigations provide meaningful information regarding treatment success and disease stage. Identification of a prognostic marker to identify patients at increased risk for leukemic progression at an early time point before lenalidomide treatment would greatly improve patient care. Telomere length measurement in these patients may therefore provide a diagnostic tool for predicting treatment response and disease outcome following lenalidomide treatment. Clearly, these data need confirmation in larger patient cohorts, before telomere length measurement can be used for risk assessment prior to starting lenalidomide treatment. Disclosures:Göhring:Celgene Corp.: Consultancy. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Nicotinamide Phosphoribosyltransferase (Nampt) Induces NAD+ / SIRT1 Mediated Deacetylation of p53 in Myeloid Cells.

Abstract 3589Poster Board III-526Recently we found that NAMPT, a protein involved in biosynthesis of NAD+ was significantly increased in Severe Congenital Neutropenia (CN) patients treated with G-CSF as compared to healthy individuals (Skokowa et al, Nature Medicine, 2009). Increased NAD+ levels correlated with the elevated levels of SIRT1, a NAD+-dependent deacetylase involved in the deacetylation of histone and non-histone proteins. CN is considered as a pre-leukemic syndrome, since ca. 20% of CN patients develop AML/MDS. SIRT1 mediated deacetylation of p53 at the lysine 382 has been shown to attenuate the transcriptional activity of p53. We asked if inactivation of p53 by NAMPT/ NAD+ / SIRT1 dependent deacetylation plays a role in the leukemic transformation in patients suffering from CN. Here we can demonstrate that presence of NAMPT or NAD+ enhances the SIRT1 mediated deacetylation of acetylated lysine peptide in vitro. Further in 293T cells overexpression of NAMPT induces the SIRT1 mediated deacetylation of p53. Using the promyelocytic cell line NB4 we show that endogenous p53 interacts with SIRT1 and the presence of NAMPT leads to decreased acetylation of p53 at the lysine 382. The compound FK866 which specifically inhibits NAMPT has recently entered clinical trials as a potential chemotherapeutic agent. Acetylation of p53 is considered to be important for its tumor suppressor function and we asked if inhibition of NAMPT using FK866 increases the acetylation of p53. Indeed we demonstrate that the acetylation level of p53 is enhanced when NB4 cells were treated with FK866 and increased acetylation of p53 correlates with the decreased interaction between p53 and SIRT1. Treatment of NB4 with NAMPT further leads to the decreased expression of p53 target gene cyclin-dependent kinase inhibitor 1A (p21, Cip1) protein. p21 is a well known target of p53 and is involved in cell cycle arrest after the cell undergoes stress. Using p21 promoter-luciferase construct we show that SIRT1 inhibits the p53 mediated activation of p21 promoter and this inhibition was further enhanced in the presence of NAMPT or NAD+. Taken together our working hypothesis is that NAMPT/NAD+ activated SIRT1 mediates deacetylation of p53 protein thereby leading to p53 inactivation and downregulation of downstream target genes with tumor suppression functions (e.g. p21) which might possibly resulting in the leukemic transformation in CN patients. Disclosures:No relevant conflicts of interest to declare.

Increased ROS Stress and Alterations in the Bone Marrow Microenvironment Contribute to DNA Damage in Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation.

Abstract 3630Poster Board III-566Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a markedly increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are associated with the development of MDS/AML in SCN. These mutations invariably produce a truncated Csf3r that, though remaining ligand-dependent, transmits a hyperproliferative signal.Knock-in mice carrying a truncation mutation of the Csf3r (termed d715 G-CSFR) have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We recently reported that expression of the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell level that is dependent upon exogenous G-CSF. Though not sufficient by itself, we previously reported that the d715 G-CSFR was able to cooperate with PML-RARa to induce AML in mice. Herein, we explore mechanisms by which CSF3R truncation mutations contribute to leukemic transformation. Specifically, we test the hypothesis that altered signaling by the d715 G-CSFR contributes to genetic instability through induction of reactive oxygen species (ROS) stress. We show that the basal level of ROS in c-KIT+ Sca+ lineage− (KSL) hematopoietic stem/progenitor cells (HSPCs) was similar in cells expressing wildtype and d715 G-CSFR. However, 24 hours after in vivo G-CSF stimulation, whereas the level of ROS was not significantly changed in wildtype KSL cells, it was induced 3.4 ± 0.1 fold in d715 G-CSFR cells (p = 0.009). To determine whether this increased ROS stress contributed to DNA damage, levels of phosphorylated histone gH2AX (pH2AX) were measured by flow cytometry. In wildtype mice, short-term (24 -hour) treatment with G-CSF had no affect on pH2AX levels in KSL cells. However, in d715 G-CSFR mice, G-CSF treatment was associated with a modest but significant increase in pH2AX levels (1.9 ± 0.1 fold; p = 0.0007). We and others previously showed that prolonged (more than 3-4 days), but not short-term (24 hour), treatment with G-CSF is associated with disruption of the osteoblast niche in the bone marrow. Since, most patients with SCN are treated chronically with G-CSF, we measured osteoblast activity in the bone marrow and H2AX phosphorylation in KSL cells after 7 days of G-CSF treatment. Notably, osteoblast activity, as measured by CXCL12 and osteocalcin expression, was reduced to a greater extent in d715 G-CSFR versus wild type mice. Interestingly, compared with short-term G-CSF treatment, pH2AX levels were significantly higher in d715 G-CSFR KSL cells after 7 day G-CSF treatment (2.6 ± 0.3 fold ± SEM; p = 0.006). To establish whether induction of ROS was responsible for the increase in pH2AX, we next co-administered the antioxidant N-acetyl cysteine (NAC) during the 7-day G-CSF treatment. As expected, induction of ROS was markedly suppressed in KSL cells by NAC administration. Moreover, the increase in pH2AX levels in d715 G-CSFR KSL cells by G-CSF was completely blocked by NAC administration.Collectively, these data suggest that both increased ROS stress and altered bone marrow microenvironment may contribute to genomic instability and leukemic transformation in patients with SCN carrying a CSF3R truncation mutation. Moreover, these data raise the possibility that anti-oxidant therapy may be an effective strategy to prevent MDS/AML in SCN. Disclosures:No relevant conflicts of interest to declare.

ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome

Epigenetic gene silencing due to promoter methylation is observed in human cancers like acute myeloid leukemia (AML). Little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a ∼30% risk of transformation into secondary AML. Recent evidence demonstrated that ID4, a negative regulator of transcription, may act as a tumor-suppressor gene. To clarify the role of ID4 in MDS, we employed methylation-specific PCR (MSP) to examine the methylation status of ID4 in 144 adult de novo MDS patients. We found that ID4 methylation was present in 35.4% ( n = 51) of these MDS patients and methylaiton was correlated significantly with World Health Organization (WHO) subtypes and International Prognostic Scoring System (IPSS) risk groups. Patients with advanced stages of WHO subtypes (45.8% vs. 21.3%, P = 0.002) and higher risk IPSS subgroups (45.7% vs. 26.0%, P = 0.014) exhibited a significantly higher frequency of ID4 methylation. The median survival of patients with ID4 methylation was shorter than patients without ID4 methylation (12.2 months vs. 26.9 months, P = 0.005). Multivariate analysis indicated that ID4 methylation status was the independent factor that impacted leukemia-free survival (LFS). Disease in patients with ID4 methylation progressed more rapidly than those without ID4 methylation ( P = 0.047, HR = 2.11). Our results suggest that ID4 may be a therapeutic target in MDS.

Plasma cell leukemia: a highly aggressive monoclonal gammopathy with a very poor prognosis

Plasma cell leukemia (PCL) is an aggressive variant of multiple myeloma and is characterized by the presence of >20% and/or an absolute number of greater 2 x 10(9)/L plasma cells circulating in the peripheral blood. PCL represents approximately 2-4% of all MM diagnosis and exists in two forms: primary PCL (PPCL, 60% of cases) presents de novo, whereas secondary PCL (SPCL, accounts for the remaining 40%) consists of a leukemic transformation in patients with a previously diagnosed MM. Because the mechanisms contributing to the pathogenesis of PCL are not fully understood, immunophenotyping, genetic evaluation (conventional karyotype, FISH, GEP and array-CGH), and immunohistochemistry are really important tools to investigate why plasma cells escape from bone marrow and become highly aggressive. Since treatment with standard agents and steroids is poorly effective, a combination of new drugs as part of the induction regimens and bone marrow transplant (autologous and allogeneic approaches) could nearly overcome the poor prognosis exhibited by PCL patients.

The Association Between STAT5a Activation, LEF-1 Down Regulation and Leukemic Transformation in Patients with Severe Congenital Neutropenia

Congenital hematological disorders are excellent models for investigating the regulation of hematopoiesis in humans. For instance, Severe Congenital Neutropenia (CN) is a heterogeneous syndrome characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with no mature neutrophils in the peripheral blood. Even though G-CSF treatment results in increased neutrophil numbers in more than 90 % of CN patients, G-CSF-dependent granulocytic differentiation is severely affected in these patients. CN patients are found to be at increased risk of developing acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) (cumulative incidence ~ 20 %). Since AML/MDS are not observed in cyclic or idiopathic neutropenia patients treated with G-CSF, an underlying defect down stream of G-CSF signaling rather than G-CSF therapy per se predisposes to malignant transformation in CN. STAT5 is activated by G-CSF signaling pathway and has also been found to be activated in AML. Recently we found that downregulation of LEF-1 transcription factor and its target gene C/EBPa are a common pathologic mechanism for CN. Therefore, we investigated the effects of G-CSF on the phosphorylation status of STAT5a in CN and whether it leads to the down modulation of LEF-1 expression and functions. Indeed, we detected elevated phosphoSTAT5 expression in CD34 + cells from CN patients before G-CSF stimulation in vitro , as compared to these cells from healthy individuals. Moreover, treatment with G-CSF resulted in a significantly higher phosphorylation of STAT5a in CN. Intriguingly, levels of phosphoSTAT5 in myeloid blast cells from one CN patient who developed AML was even higher and was in line with undetectable LEF-1 protein expression. Transduction of constitutive active STAT5a (STAT5a 1*6) in CD34 + cells from healthy individuals resulted in significant downregulation of LEF1 levels in a dose dependent manner. A screen of 10 kb upstream region of LEF1 gene revealed two putative STAT5 binding sites (−3891bp to −3909bp and −3714bp to −3732bp) and the specificity of this binding was confirmed in the nuclear extracts of CD34 + cells by chromatin immunoprecipitation assay. We found enhanced and prolonged STAT5a binding to the LEF-1 promoter in G-CSF treated CD34 + cells from CN patients, as compared to healthy individuals. Additionally, transfection of CD34 + cells with LEF-1 cDNA resulted in elevation of LEF-1 promoter activity, which suggests a strong LEF-1 autoregulation. Co-transfection with STAT5a 1*6 significantly disrupted LEF-1- dependent activation of LEF-1 promoter. Moreover STAT5a 1*6 severely abrogated the LEF-1 dependent regulation of C/EBPα gene promoter. Taken together phosphorylation of STAT5 is upregulated in hematopoietic progenitors from CN patients which lead to subsequent down regulation of LEF-1. These downstream effects of activated STAT5a may contribute to the malignant transformation of myelopoiesis in CN.

Erythropoiesis‐stimulating agents are effective in myelodysplastic syndromes, but are they safe?

69. 11. Gotlib J, Lavori P, Quesada S, et al. A phase II intra-patient dose-escalation trial of weight-based darbepoetin alfa with or without granulocyte-colony-stimulating factor (G-CSF) in myelodysplastic syndromes. Am J Hematol 10.1002/

The Association Between STAT5a Activation, LEF-1 Down Regulation and Leukemic Transformation in Patients with Severe Congenital Neutropenia

Congenital hematological disorders are excellent models for investigating the regulation of hematopoiesis in humans. For instance, Severe Congenital Neutropenia (CN) is a heterogeneous syndrome characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with no mature neutrophils in the peripheral blood. Even though G-CSF treatment results in increased neutrophil numbers in more than 90 % of CN patients, G-CSF-dependent granulocytic differentiation is severely affected in these patients. CN patients are found to be at increased risk of developing acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) (cumulative incidence ~ 20 %). Since AML/MDS are not observed in cyclic or idiopathic neutropenia patients treated with G-CSF, an underlying defect down stream of G-CSF signaling rather than G-CSF therapy per se predisposes to malignant transformation in CN. STAT5 is activated by G-CSF signaling pathway and has also been found to be activated in AML. Recently we found that downregulation of LEF-1 transcription factor and its target gene C/EBPa are a common pathologic mechanism for CN. Therefore, we investigated the effects of G-CSF on the phosphorylation status of STAT5a in CN and whether it leads to the down modulation of LEF-1 expression and functions. Indeed, we detected elevated phosphoSTAT5 expression in CD34+ cells from CN patients before G-CSF stimulation in vitro, as compared to these cells from healthy individuals. Moreover, treatment with G-CSF resulted in a significantly higher phosphorylation of STAT5a in CN. Intriguingly, levels of phosphoSTAT5 in myeloid blast cells from one CN patient who developed AML was even higher and was in line with undetectable LEF-1 protein expression. Transduction of constitutive active STAT5a (STAT5a 1*6) in CD34+ cells from healthy individuals resulted in significant downregulation of LEF1 levels in a dose dependent manner. A screen of 10 kb upstream region of LEF1 gene revealed two putative STAT5 binding sites (−3891bp to −3909bp and −3714bp to −3732bp) and the specificity of this binding was confirmed in the nuclear extracts of CD34+ cells by chromatin immunoprecipitation assay. We found enhanced and prolonged STAT5a binding to the LEF-1 promoter in G-CSF treated CD34+ cells from CN patients, as compared to healthy individuals. Additionally, transfection of CD34+ cells with LEF-1 cDNA resulted in elevation of LEF-1 promoter activity, which suggests a strong LEF-1 autoregulation. Co-transfection with STAT5a 1*6 significantly disrupted LEF-1- dependent activation of LEF-1 promoter. Moreover STAT5a 1*6 severely abrogated the LEF-1 dependent regulation of C/EBPα gene promoter. Taken together phosphorylation of STAT5 is upregulated in hematopoietic progenitors from CN patients which lead to subsequent down regulation of LEF-1. These downstream effects of activated STAT5a may contribute to the malignant transformation of myelopoiesis in CN.