Jungwon Huh

Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms

Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number-neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders.

Mutations of E3 Ubiquitin Ligase Cbl Family Members Constitute a Novel Common Pathogenic Lesion in Myeloid Malignancies

PURPOSE Acquired somatic uniparental disomy (UPD) is commonly observed in myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), or secondary acute myelogenous leukemia (sAML) and may point toward genes harboring mutations. Recurrent UPD11q led to identification of homozygous mutations in c-Cbl, an E3 ubiquitin ligase involved in attenuation of proliferative signals transduced by activated receptor tyrosine kinases. We examined the role and frequency of Cbl gene family mutations in MPN and related conditions. METHODS We applied high-density SNP-A karyotyping to identify loss of heterozygosity of 11q in 442 patients with MDS, MDS/MPN, MPN, sAML evolved from these conditions, and primary AML. We sequenced c-Cbl, Cbl-b, and Cbl-c in patients with or without corresponding UPD or deletions and correlated mutational status with clinical features and outcomes. RESULTS We identified c-Cbl mutations in 5% and 9% of patients with chronic myelomonocytic leukemia (CMML) and sAML, and also in CML blast crisis and juvenile myelomonocytic leukemia (JMML). Most mutations were homozygous and affected c-Cbl; mutations in Cbl-b were also found in patients with similar clinical features. Patients with Cbl family mutations showed poor prognosis, with a median survival of 5 months. Pathomorphologic features included monocytosis, monocytoid blasts, aberrant expression of phosphoSTAT5, and c-kit overexpression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution. CONCLUSION Mutations in the Cbl family RING finger domain or linker sequence constitute important pathogenic lesions associated with not only preleukemic CMML, JMML, and other MPN, but also progression to AML, suggesting that impairment of degradation of activated tyrosine kinases constitutes an important cancer mechanism.

Prognostic impact of SNP array karyotyping in myelodysplastic syndromes and related myeloid malignancies

Single nucleotide polymorphism arrays (SNP-As) have emerged as an important tool in the identification of chromosomal defects undetected by metaphase cytogenetics (MC) in hematologic cancers, offering superior resolution of unbalanced chromosomal defects and acquired copy-neutral loss of heterozygosity. Myelodysplastic syndromes (MDSs) and related cancers share recurrent chromosomal defects and molecular lesions that predict outcomes. We hypothesized that combining SNP-A and MC could improve diagnosis/prognosis and further the molecular characterization of myeloid malignancies. We analyzed MC/SNP-A results from 430 patients (MDS = 250, MDS/myeloproliferative overlap neoplasm = 95, acute myeloid leukemia from MDS = 85). The frequency and clinical significance of genomic aberrations was compared between MC and MC plus SNP-A. Combined MC/SNP-A karyotyping lead to higher diagnostic yield of chromosomal defects (74% vs 44%, P < .0001), compared with MC alone, often through detection of novel lesions in patients with normal/noninformative (54%) and abnormal (62%) MC results. Newly detected SNP-A defects contributed to poorer prognosis for patients stratified by current morphologic and clinical risk schemes. The presence and number of new SNP-A detected lesions are independent predictors of overall and event-free survival. The significant diagnostic and prognostic contributions of SNP-A-detected defects in MDS and related diseases underscore the utility of SNP-A when combined with MC in hematologic malignancies.

New Lesions Detected by Single Nucleotide Polymorphism Array–Based Chromosomal Analysis Have Important Clinical Impact in Acute Myeloid Leukemia

PURPOSE Cytogenetics is the primary outcome predictor in acute myeloid leukemia (AML). Metaphase cytogenetics (MC) detects an abnormal karyotype in only half of patients with AML, however. Single nucleotide polymorphism arrays (SNP-A) can detect acquired somatic uniparental disomy (UPD) and other cryptic defects, even in samples deemed normal by MC. We hypothesized that SNP-A will improve detection of chromosomal defects in AML and that this would enhance the prognostic value of MC. PATIENTS AND METHODS We performed 250K and 6.0 SNP-A analyses on 140 patients with primary (p) and secondary (s) AML and correlated the results with clinical outcomes and Flt-3/nucleophosmin (NPM-1) status. RESULTS SNP-A is more sensitive than MC in detecting unbalanced lesions (pAML, 65% v 39%, P = .002; and sAML, 78% v 51%, P = .003). Acquired somatic UPD, not detectable by MC, was common in our AML cohort (29% in pAML and 35% in sAML). Patients with SNP-A lesions including acquired somatic UPD exhibited worse overall survival (OS) and event-free survival (EFS) in pAML with normal MC and in pAML/sAML with abnormal MC. SNP-A improved the predictive value of Flt-3 internal tandem duplication/NPM-1 status, with inferior survival seen in patients with additional SNP-A defects. Multivariate analyses confirmed the independent predictive value of SNP-A defects for OS (hazard ratio [HR] = 2.52; 95% CI, 1.29 to 5.22; P = .006) and EFS (HR = 1.72; 95% CI, 1.12 to 3.48; P = .04). CONCLUSION SNP-A analysis allows enhanced detection of chromosomal abnormalities and provides important prognostic impact in AML.

FISH-negative cryptic PML–RARA rearrangement detected by long-distance polymerase chain reaction and sequencing analyses: a case study and review of the literature

Although a normal karyotype according to conventional cytogenetic analysis in association with cryptic t(15;17) has been infrequently reported in cases of acute promyelocytic leukemia (APL), a fluorescence in situ hybridization (FISH)-negative cryptic PML-RARA rearrangement is even more rare, with only 12 such APL cases of FISH-negative cryptic PML-RARA rearrangements in the literature. Reported here is an additional clinical APL case with a FISH-negative cryptic PML-RARA rearrangement, confirmed by long-distance DNA polymerase chain reaction method. Discussion includes a relevant literature review of similar cases. DNA-PCR can be a useful tool for the analysis of complex and cryptic rearrangements.

Mobilization Kinetics of CD34+ Cells in Association with Modulation of CD44 and CD31 Expression during Continuous Intravenous Administration of G‐CSF in Normal Donors

The aim of the present study is to evaluate the kinetics of CD34+ cells and investigate the potential modulation of CD44 and CD31 expression on CD34+ cells during continuous i.v. administration of G‐CSF, thus to elucidate the possible mechanism of peripheral blood progenitor cell (PBPC) mobilization. Fifteen healthy donors were enrolled in this study. G‐CSF (10 μg/kg/day) was administered for four consecutive days through continuous 24‐h i.v. infusion. For measurement of complete blood counts, CD34+ cell levels and their expression of CD44 and CD31, PB sampling was performed immediately before the administration of G‐CSF (steady‐state) and after 4, 8, 24, 48, 72, 96, and 120 h of G‐CSF administration. The percentage and absolute number of CD34+ cells significantly increased at day 3 (0.55 ± 0.09%, 51.12 ± 24.83 × 103/ml) and day 4 (0.47 ± 0.09%, 46.66 ± 24.93 × 103/ml), compared to the steady‐state level (0.06 ± 0.09%, 2.03 ± 5.69 × 103/ml). At day 3 to day 5 following the onset of G‐CSF administration, a strong decrease of CD44 and CD31 expression was observed on mobilized CD34+ cells compared to controls: the relative fluorescence intensity of CD44 and CD31 was, respectively, 50%‐70% and 40%‐90% lower than that of controls. We conclude that continuous i.v. administration of G‐CSF apparently results in more rapid mobilization of CD34+ cells, and downregulation of CD44 and CD31 on CD34+ cells is likely to be involved in the mobilization of PBPC after treatment with G‐CSF.

Adverse Prognostic Impact of Abnormal Lesions Detected by Genome-Wide Single Nucleotide Polymorphism Array–Based Karyotyping Analysis in Acute Myeloid Leukemia With Normal Karyotype

PURPOSE This study attempted to analyze the prognostic role of single nucleotide polymorphism array (SNP-A) -based karyotying in 133 patients with acute myeloid leukemia with normal karyotype (AML-NK), which presents with diverse clinical outcomes, thus requiring further stratification of patient subgroups according to their prognoses. PATIENTS AND METHODS A total of 133 patients with AML-NK confirmed by metaphase cytogenetics (MC) and fluorescent in situ hybridization analysis were included in this study. Analysis by Genome-Wide Human SNP 6.0 Array was performed by using DNAs derived from marrow samples at diagnosis. RESULTS Forty-three patients (32.3%) had at least one abnormal SNP lesion that was not detected by MC. One hundred thirteen abnormal SNP lesions included 55 losses, 23 gains, and 35 copy-neutral losses of heterozygosity. Multivariate analyses showed that detection of abnormal SNP lesions by SNP-A karyotyping results in an unfavorable prognostic value for overall survival (hazard ratio [HR], 2.69; 95% CI, 1.50 to 4.82; P = .001); other significant prognostic factors included secondary AML (HR, 5.55; 95% CI, 1.80 to 17.14; P = .003), presence of the FLT3 mutation (HR, 3.17; 95% CI, 1.71 to 5.87; P < .001), and age (HR, 1.03; 95% CI, 1.01 to 1.05; P = .020). CONCLUSION Our data demonstrated that abnormal SNP lesions detected by SNP-A karyotyping might indicate an adverse prognosis in patients with AML-NK, thus requiring a more sophisticated treatment strategy for improvement of treatment outcomes.

Pseudoeosinophilia associated with malaria infection determined in the Sysmex XE-2100 hematology analyzer

Hemozoin is known to be an end product of hemoglobin digestion by the malaria parasite. Hemozoin is a birefringent crystal, and thus hemozoin-containing white blood cells (WBCs) may show the atypical light scattering pattern. The purpose of this study was to investigate pseudoeosinophilia associated with malaria infection using a Sysmex XE-2100 hematology analyzer (Sysmex Corporation, Kobe, Japan). The study group included 16 patients with malaria infection. Of these, 38% showed erroneously high eosinophil counts and atypical eosinophil distributions in the WBCs scattergram, which was due to the presence of hemozoin-containing neutrophils. In two patients, their erroneously high eosinophil counts declined as the parasitemia decreased with treatment. In conclusion, hematologists should consider the possibility of pseudoeosinophilia as a result of hemozoin-containing WBCs and confirm the WBC differential count by microscopy in cases of malaria infection.

Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte‐colony stimulating factor during ex vivo expansion of human cord blood CD34+ cells

The insufficient number of haemopoietic stem cells (HSCs) in cord blood (CB) is the major potential limitation to widespread use of CB for marrow replacement. Cytokine‐mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine‐mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma‐free liquid culture system with thrombopoietin (TPO), flt3‐ligand (FL), stem cell factor (SCF), and/or granulocyte‐colony stimulating factor (G‐CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7‐amino‐actinomycin D (7‐AAD) along with concurrent immunophenotyping of CD34 and CD44 with three‐colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down‐regulated CD44 appeared from the fourth day up to the second week. G‐CSF also induced apoptosis but in a different manner; the apoptotic fraction without down‐regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down‐regulation of CD44. These findings show that apoptosis is indeed involved in the regulation of CB CD34+ cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G‐CSF in inducing apoptosis, which still remains to be elucidated.

Genome-wide high density single-nucleotide polymorphism array-based karyotyping improves detection of clonal aberrations including der(9) deletion, but does not predict treatment outcomes after imatinib therapy in chronic myeloid leukemia.

The current study investigated molecular cytogenetic characteristics of chronic myeloid leukemia (CML) using genome-wide, single nucleotide polymorphism arrays (SNP-A) capable of detecting cryptic submicroscopic genomic aberrations. Genome-Wide Human SNP 6.0 Array (Affymetrix, CA, USA) was performed in 118 patients having CML, chronic phase. Thirty-nine clonal aberrations (CAs) were identified (35 losses, two gains, two copy neutral loss of heterozygosity) that were not detected by metaphase cytogenetics in 25 patients (21%). The 9q34 deletions were found in 10% of cases, while 22q11.2 deletions were observed in 12% of cases. Seven patients (6%) harbored both 5′-ABL and 3′-BCR deletions adjacent to the t(9;22) breakpoint. Copy number gains were identified at 8p and 9p, and losses at 2q, 7q, 8q, 9q, 11q, 13q, 16p, and 22q. When we compared the treatment outcome of imatinib therapy between patients with and without CAs identified by SNP-A, treatment failure and progression to advanced disease were not significantly different (p > 0.05). In addition, according to the presence of deletions of 9q34 and/or 22q11.2 identified by SNP-A, the treatment outcome did not show any significant differences (p > 0.05). Our data suggests that SNP-A analysis is a useful tool for detection of clonal aberrations including deletions adjacent to the t(9;22) breakpoint in the CML cancer genome. However, clonal aberrations detected by SNP-A could not improve a prognostic stratification in CML patients with chronic phase.