ALBERT

Description: Prior studies have established that ALL can be present at birth. However, such inborn hematological malignancies are rare and contrast to the far more frequent hematological (pre-) malignancies, which are typically found in patients in their sixties or later. Thus, the point mutation V617F in the JAK2 gene in myeloid cells has been found to be a significant player for many chronic myeloproliferations, e.g. polycythemia vera (PCV). In fact, its prevalence in the elderly population is as high as 0.1%. In addition, studies in B-cell derived chronic lymphocytic leukemia (B-CLL) has found that this malignancy is preceded by a clinically silent phase of monoclonal B-cell lymphocytosis (MBL) in the majority of patients. In contrast to leukemic childhood twins, where a high (10-15%) concordance is thought to reflect the inborn initiating mutations, much less is known about the balance between genetics and environment for the pre-leukemic conditions mentioned above. This report relates to a pair of homozygous twins, who came to our attention when they were 73 years old in 2009. Twin A presented at a hematological department for the first time in 2005 at the age of 70 years. He had experienced a 1 h episode of amaurosis fugax and during a neurological workup mild thrombocytopenia (126x109/L), increased hematocrit (0.52) and lymphocytosis (7.95x109/L) without abnormalities in other leukocyte subsets. Immunophenotyping at this time revealed CD5/CD19/CD23+ monoclonal B-cells at the 7-8 109/L levels, but no lymphadenopathy or organomegaly. While no therapy for the latter cells has been instituted, venesectio 3-4 times /year has been deemed necessary to keep the hematocrit below 0.45. Twin B went for a checkup at his GP subsequent to the brother experiencing the above episode. He, too, was diagnosed with lymphocytosis, but his hematocrit was only slightly increased levels. No intervention has been instituted in this twin. While the extent of lymphocytosis in twin A is consistently between 5 and 10 bill/L, that of twin B remains below 5 bill/L. Remarkably, the B-cell light chain expression differed between these identical twins, with twin A being kappa- and twin B lambda positive. High-resolution arrayCGH on blood sample revealed that twin A had microdeletions on chromosome 2p11.2 involving the Ig kappa gene cluster, on chromosome 7p11.2 involving the processed pseudogene ENST00000448242, chromosome 13 involving the RB gene, and on chromosome 17 involving the azacytidine induced gene. In contrast, twin B exhibited no clear aberrations by arrayCGH. For both twins, we have performed whole exome sequencing using FACS purified T-lymphocytes as surrogate germline cells and compared to whole the BM MNC exome in order to identify mutated genes. This analysis revealed (Figure) that twin A predominantly displayed mutations, which have been related to myeloid cells in the literature, while twin B mainly exhibited alterations in genes linked to lymphoid cells. In fact, only the three genes, NTSR2, ATP2C2 and ACOX3, were altered in the BM cells and present on both twins, neither of these readily explaining the shared monoclonal B-cell disorders. Interestingly, mutations in the genes TET2 and RUNX1, which have been closely related to myeloid cells, were solely found in twin A. In conclusion, we have described a pair of homozygous twins who in their seventies were diagnosed with benign monoclonal B-cell lymphocytosis (MBL) with different B-cell light chain expression. Additionally, one twin, but not the other, was found to be JAK2+ with clear polycythemia vera (PCV) features requiring frequent phlebotomies. Extensive molecular analyses including whole exome sequencing have revealed that while both twins harbored point mutation in NTSR2, ATP2C2 and ACOX3, the mutations displayed in twin A with PCV were predominantly genetic abnormalities that have been associated with the myeloid cells underlining that extensive alterations in myelopoiesis have taken place postnatally in this twin. Thus, for these two pre-leukemic conditions, while predisposition to MBL seems to be prenatal, that related to JAK2 is postnatal. Ongoing studies are aimed at discovering a link between the 3 shared genes and B-cell development in an effort to link them to BML development. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction The World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia was revised in 2008 and is based on genetic characteristics and morphology. The classification incorporates newly recognized entities and emphasizes, more than previously, the pivotal role of cytogenetic abnormalities aiming at identifying biological and clinical entities as basis for a stratified treatment. The classification of acute myeloid leukemia (AML) is primarily based on adult studies. However, the frequency of cytogenetic changes varies among children and adults and the relevance of the 2008 revised WHO classification in pediatric AML has not yet been elucidated. The aim of this study was to evaluate the usability of the WHO classification when applied to a large population-based pediatric AML cohort. Methods We included children 0-18 years of age diagnosed with de novo AML in the Nordic countries (Sweden, Norway, Finland, Iceland and Denmark) and Hong Kong treated according to the NOPHO-AML-1993 and 2004 protocols in the period January 1993 to December 2012. Clinical, morphologic, and genetic data were retrieved from the database. Patients with myeloid leukemia of Down syndrome, acute promyelocytic leukemia, and therapy-related AML were excluded. The karyotypes from the Nordic countries were centrally reviewed with annual evaluation of the karyograms by the NOPHO cytogenetic working group. Morphological and molecular analyses and immunophenotyping were carried out at regional centers. The patients were classified according to the revised 2008 WHO classification using genetic parameters and the French-American-British (FAB) classification. Results In the NOPHO database we identified 609 children aged 0-18 years diagnosed with AML. In 13 cases, the karyotype analyses either failed or considered uninformative and were excluded. Among the remaining 596 informative cases (98%), 241 (40%) were categorized as AML with recurrent genetic abnormalities; t(8;21)(q22;q22);RUNX1-RUNX1T1 (n=69), inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 (n=47), t(9;11)(p21;q23);MLLT3-MLL (n=61), t(6;9)(p22;q34);DEK-NUP214 (n=5), inv(3)(q21q26.2) or t(3;3)(q21;q26.2);RPN1-EVI1 (n=1), t(1;22)(p13;q13)/RBM15-MKL1 (n=4), NPM1 mutated (n=13), and CEBPA mutated (n=10). Furthermore, 23 children were classified in the provisional entity AML with FLT3-ITD. Based on the karyotype, 92 children (16%) were classified as AML with myelodysplasia-related cytogenetical changes (AML-MDS). This group was dominated by unbalanced abnormalities (9%) with -7/del(7q) being the most common (4% of the total cohort) and complex karyotypes (6% of the total cohort). AML with balanced myelodysplasia-related changes was found in 0.5%. The largest group was the highly heterogeneous AML not otherwise specified (AML-NOS) (n=263) (44%), and included some of the well-defined 11q23 abnormalities. In the 2001 version of the WHO classification, “AML with abnormalities of 11q23; MLL,” was listed separately, but in the 2008 edition only AML with t(9;11)(p22;q23);MLLT3-MLL was listed as an entity. The AML-NOS cases were sub-classified according to morphology, with FAB M5 being most common, found in 22% of NOS, followed by FAB M1, FAB M2, and FAB M4, all found in 18%. In conclusion, 40% of the children were classified in clinically relevant entities, but the WHO classification allocated 16% of the children to AML-MDS, the relevance is unknown in children, and 44% to the NOS group. This large and unspecified group limits the applicability of the WHO classification in children with AML suggesting that additional considerations are warranted for relevant classification of pediatric AML. Disclosures: No relevant conflicts of interest to declare.

Description: The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein “preBLL”). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.

Description: Background: Despite major improvements in survival, relapse is still a frequent and severe event in pediatric acute myeloid leukemia (AML). Cytogenetic abnormalities represent important predictors of outcome. Improved risk stratification encompassing the identification of novel cytogenetic subsets may allow refinements in treatment strategies and increase survival rates. Hypodiploidy, defined as a modal number (MN) below 46 chromosomes, is associated with an adverse outcome in pediatric acute lymphoblastic leukemia. In childhood AML, the predominant cause of hypodiploidy is loss of a sex chromosome accompanying t(8;21). The most commonly lost autosome is chromosome 7, associated with a poor prognosis. However, other chromosome losses in pediatric AML have not been independently explored. We aimed to investigate the occurrence, genetic characteristics, and prognostic impact of hypodiploidy in childhood AML. Methods: This study was conducted as a retrospective cohort study within the I-BFM-AML framework. Children 0-18 years of age diagnosed with de novo AML between January 2000 to December 2015 and a hypodiploid karyotype were eligible for inclusion. Patients with constitutional hypodiploidy, monosomy 7, composite karyotypes, and t(8;21) with loss of a sex chromosome were excluded. Cytogenetic review was performed according to International System for Human Cytogenetic Nomenclature. Hypodiploidy was considered clonal when occurring in at least three metaphases. The clone with the lowest MN defined the ploidy level. The log-rank test was applied for estimating difference in survival. Considering allografting in first complete remission (CR1) as a time-dependent event, the effect of stem cell transplantation (SCT) on overall survival (OS) was estimated by the Mantel-Byar method. Multivariate analyses included sex, complex karyotype (≥3 non-recurrent aberrations), FLT3-ITD mutation status, and white blood cell count as covariates. As all patients with FLT3-ITD mutations had same MN (MN=45) leading to a skewed imputation model, multiple imputation was not performed, and multivariate analyses included complete cases only (n=54). Results: Hypodiploidy was detected in 81/6,409 patients with full karyotyping from 14 collaborative study groups, yielding a frequency of 1.2% in childhood AML. The cohort displayed a balanced sex distribution (male/female: 42/39) and a median age of 6 (range: 0-17). MNs 45 (n=66), 44 (n=10) and 43 (n=5) were observed. No patient had MN below 43. Eight patients (9.8%) had chromosome loss as the only aberration. Forty-eight patients (59%) had a complex karyotype. Core-binding factor abnormalities were observed in three patients (3.7%). Four patients harbored FLT3-ITD mutations (9.3%, 54 tested). Median follow-time for patients alive was 4.5 years (range: 0.2-15.3). Five-year event-free survival (EFS) and OS were 34% (CI 95%: 23% - 45%) and 52% (CI 95%: 40% - 63%), respectively. Cumulative incidence of relapse was 50% with a median time-to-relapse of 0.8 years. Twelve out of 38 relapsed children (32%) were alive at the end of follow-up. Children with MN≤44 (n=15) had lower EFS (21%, CI 95%: 4% - 46%) and OS (33%, CI 95%: 10% - 59%) than children with MN=45 (n=66, EFS: 37%, CI 95%: 24% - 49%, OS: 56%, CI 95%: 42% - 68%). Crude hazard ratios (HR) for MN≤44 vs MN=45 were 1.8 (p=0.08) for EFS and 1.9 (p=0.1) for OS. After adjustment HRs increased to 4.9 (p=0.001) and 6.1 (p=0.003), respectively. Loss of chromosome Y (n=12) displayed a non-significant superior survival (EFS: 58%, OS: 75%). Poor survival rates were especially observed in monosomy 9 (n=11, EFS: 14%, OS 15%), 10 (n=5, EFS: 20%, OS: 20%), and 16 (n=5, EFS: 25%, OS: 25%), although not reaching statistical significance. Allogenic SCT did not offer any advantage in survival. For children receiving SCT in CR1 (n=18), crude HR for OS was 1.4, p=0.39. Multivariate analysis showed HR for OS at 1.5, p=0.42. Exclusion of children not reaching CR1 (n=7) did not significantly change HRs. This large international study reveals that hypodiploidy is rare in pediatric AML. Children with loss of chromosome 9, 10, or 16 all displayed unfavorable survival rates, whereas loss of chromosome Y did not have influence on prognosis. SCT in CR1 did not improve survival. Our results suggest that modal number has an impact on outcome and may be important for risk stratification. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3542 The prognosis of acute promyelocytic leukemia (APL) has improved markedly over the last two decades. Clinical trials have demonstrated excellent survival of patients receiving anthracycline-based chemotherapy in combination with all-trans-retinoid acid (ATRA). It is not clear whether the excellent survival results demonstrated in clinical trials during recent years, also do apply to unselected patients including elderly and frail patients for whom a clinical trial may not be available. In order to investigate survival and death rates in APL over the course of treatment, we conducted a retrospective analysis of survival in 105 consecutive APL-patients diagnosed in Denmark January 2000 through June 2012. Data were retrieved from the Danish National Acute Leukemia Registry which covers 95 – 100 % of all acute leukemia cases diagnosed in Denmark since January 2000 (not including children). A diagnosis of APL was confirmed in 105 (3.9 %) of 2726 adult patients (age ≥ 15 years) with acute myeloid leukemia (AML) diagnosed in Denmark during the 12½ year period. This corresponds to an incidence rate of 1.53 APL cases per million inhabitants per year. The diagnosis of APL was based upon cytogenetic analysis (performed in 96 (91 %) of cases), and/or interphase FISH (iFISH), and/or RT-PCR in a total of 100 cases. In 5 cases clinical signs and morphological changes in bone marrow were highly suggestive of APL and these cases were, thus, deemed to have APL. Median age at diagnosis of APL was 50 years (range 15 to 83 years) and median leukocyte count was 2.65 (range 0.2 to 99.0 × 109/L). In 104 patients with data available for analysis, very early death (VED, within the first week from the date of diagnostic bone marrow) occurred in 10 cases (9.6 %), and early death (ED, death within 30 day from diagnosis) occurred in 22 (21 %) cases. Death between day 30 and 5 years from diagnosis occurred in 11 cases. No deaths were seen after 5 years from diagnosis. For all patients, estimated overall survival at 10 years was 65 % (Figure 1). Patients surviving beyond day 30 from day of diagnosis (82 cases) had an excellent overall survival of greater than 80 % at 10 years (Figure 2). Survival of patients were strongly correlated with WHO-performance status whereas age over 50 years and presenting leukocyte count greater than 10 × 109/L could not be definitely associated with inferior survival (Table 1 and Figure 3). Table 1. Factors of importance to survival in unselected APL-patients Probability of overall survival (Univariate Cox Regression, nevaluable= 104) Probability of overall survival (multivariate Cox Regression, nevaluable= 101) Variable Hazard ratio 95% CI of HR P value Hazard ratio 95% CI of HR P value Age (≤50 vs. 〉50 years) 2.17 1.07–4.42 0.03 1.44 0.67–3.09 NS WHO performance status (0–1 vs. ≥2) 5.20 2.57–10.5 〈 10−4 4.06 1.88–8.78 〈 10−4 WBC (≤10 × vs.〉10 × 109/L) 1.76 0.86–3.61 NS 1.62 0.79–3.33 NS From this population-based analysis we conclude that early death continues to be the predominant hazard to patients with APL. Two thirds of deaths in APL-patients do occur during the first month from diagnosis. Patients with a poor performance status at disease presentation carry a particularly dismal prognosis. For all APL-patients, every possible effort should be made to facilitate prompt diagnosis and speedy initiation of treatment with ATRA and anthracycline-based chemotherapy. When timely applied, currently available treatments are highly effective and result in cure in a high proportion of patients including elderly patients and patients with high leukocyte count at time of disease presentation. Disclosures: No relevant conflicts of interest to declare.