Eunhee Han

Characteristics of hematologic malignancies with coexisting t(9;22) and inv(16) chromosomal abnormalities

Background The coexistence of t(9;22)(q34;q11.2) and inv(16)(p13q22) chromosomal abnormalities is extremely uncommon, and only a small number of such cases have been reported. Here, we characterized 7 cases of hematologic malignancy exhibiting t(9;22) and inv(16) coexistence. Methods We reviewed the cytogenetic data for hematologic malignancies treated at the Catholic Blood and Marrow Transplantation Center between January 2004 and June 2013. We identified 7 cases exhibiting t(9;22) and inv(16) coexistence. In addition, we analyzed mutations in the IKZF1, NPM1, FLT3, N-RAS, K-RAS, c-KIT, and TP53 genes. Results Four cases of chronic myelogenous leukemia (CML; 1 chronic phase, 2 accelerated phase, and 1 blast phase) and 3 cases of acute myeloid leukemia (AML; 1 de novo and 2 therapy-related) were identified. The percentages of circulating blasts and bone marrow eosinophils were higher in AML cases than in CML cases (53% vs. 5% and 30% vs. 5.5%, respectively). The proportions of each chromosomal abnormality were used along with follow-up karyotyping results to identify secondary changes. In BCR/ABL, a p210 fusion transcript was associated with CML, whereas a p190 fusion transcript was associated with AML. One patient with AML harbored 2 mutations: c-KIT D816V and TP53 E11Q. All patients except 1 with CML blast phase sustained clinical remission after treatment, which included an imatinib mesylate regimen. Conclusion This study shows that observations of bone marrow morphology, initial and follow-up cytogenetic studies, and karyotyping of BCR/ABL1 and CBFB/MYH11 provide valuable information for characterizing hematologic malignancies exhibiting t(9;22) and inv(16) coexistence.

Hereditary dehydrated stomatocytosis with splicing site mutation of PIEZO1 mimicking myelodysplastic syndrome diagnosed by targeted next-generation sequencing

To the Editor: Hereditary stomatocytosis is a rare autosomal dominant disorder that presents with various degrees of hemolytic anemia and abnormal red blood cell (RBC) morphologies.1 Among hereditary stomatocytoses, hereditary dehydrated stomatocytosis (DHSt) is osmotically resistant with elevated mean corpuscular hemoglobin concentration.2 DHSt can be misdiagnosed as myelodysplastic syndrome (MDS) because of hypercellular bone marrow (BM) with erythroid hyperplasia and dysmegakaryopoiesis.3 A 23-year-old man had been initially diagnosed with idiopathic thrombocytopenic purpura and showed no response to steroid treatment or intravenous immunoglobulin. The laboratory findings at the time of admission to our hospital were summarized in Supplementary Table S1. A minor proportion of RBCs appeared as stomatocytes on peripheral blood (PB) smear (Figure 1A). BM showed hypercellularity with less than 1% blasts. Erythroid precursors revealed profound megaloblastoid maturation pattern and multiple Howell–Jolly bodies (Figure 1B). Mitotic cells were frequently found (Figures 1C and 1D). The presumptive BM morphologic diagnosis was MDS with multilineage dysplasia. We performed targeted next-generation sequencing (NGS) to exclude the underlying genetic cause of the conspicuous “stomatocytosis”, even though various poikilocytoses of RBCsmay be seen in case of MDS.3 Molecular analysis using a custom capture/NGS gene panel of 199 known BM failure syndromes/hemolytic anemias including the PIEZO1 gene was performed in the proband. The study was approved by the institutional review board of The Catholic University of Korea. Massively parallel sequencingwas performedonHiSeq 2000 Sequencing System (Illumina, Inc., San Diego, CA). Single nucleotide variants and small insertions/deletionswere identified by theGATKHaplotypeCaller and annotated with SnpEff v4.2. An intronic mutation c.465+1G > T of PIEZO1 was identified (RefSeq ID: NM_001142864.2). Its variant frequency was reported as 0.000804 in the Korean Reference Genome Database (http://152.99.75.168/KRGDB/menuPages/intro.jsp), but not in any public sequence databases. Sanger sequencing was performed in the proband and his family members and the presence of splice site mutation was confirmed only in the proband (Figure 1E). Cloning of reverse transcription-polymerase chain reaction products revealed two clones: a wild-type allele (gt of c.465+1_c.465+2) and a pathogenic allele with alternative weak donor site gc of intron 5 (gc of c.465+7_c.465+8) (Figure 1F). Based on these results, the patient was diagnosed as DHSt with PIEZO1mutation. The two major findings of DHSt are unexplained nonimmune hemolytic anemia and abnormal stomatocytes ranging from 40% to 60%on the PB smear.4 Identification ofmutations in PIEZO1 as the primary cause of DHSt has led to both an increased awareness of the disease and an increased importance of genetic diagnosis in unexplained anemia.5 In this study, it was possible to diagnose the patient with DHSt through genetic testing because he did not present the findings relevant to MDS. The potential role of PIEZO1 in RBC physiology is most clearly demonstrated by numerous gain-of-function mutations in PIEZO1 that have been identified in DHSt.6,7 The mutations in PIEZO1 lead to increased activity of the channel due to delayed channel inactivation and increased cation transport.6 Several PIEZO1mutations may exhibit an altered response to osmotic stress and impaired membrane trafficking, with some possibly resulting in increased erythrocyte dehydration.8 This report describes DHSt caused by a novel intronic PIEZO1mutation.

PCM1-JAK2 Fusion in a Patient With Acute Myeloid Leukemia

Jong-Mi Lee, M.D., Jaewoong Lee, M.D., Eunhee Han, M.D., Myungshin Kim , M.D., Yonggoo Kim, M.D., Kyungja Han, M.D., and Hee-Je Kim, M.D. Department of Laboratory Medicine and Catholic Genetic Laboratory Center, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul; Department of Hematology, Catholic Blood and Marrow Transplantation Center, Seoul St. Mary’s Hospital, Leukemia Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Korea

Association of FLG single nucleotide variations with clinical phenotypes of atopic dermatitis

Background FLG encodes a large protein called profilaggrin, which plays a key role in maintaining an effective skin barrier against the environment. In this study, we identified FLG single nucleotide variations (FLG-SNVs) and evaluated the association of FLG-SNVs with clinical phenotypes including atopic dermatitis (AD)-associated minor clinical features, presence of specific allergic sensitization, and serum parameters. Methods Eighty-one Korean patients with AD were enrolled. AD-associated minor clinical features as well as allergic rhinitis and asthma were diagnosed by specialists. FLG-SNVs were identified by Sanger sequencing of entire exons through long-range PCR. Allergic sensitization to a specific allergen was evaluated by multiple allergen simultaneous test. Serologic parameters such as serum eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) were measured. Results A total of seventy-three SNVs and 4 LOF mutations were successfully genotyped. rs71626704 and rs76413899 were significantly associated with a history of asthma and cheilitis (P = 0.002 and P = 0.033, respectively), however, the associations were not found statistically significant after adjustment by multiple comparisons. In addition, we detected haplotype blocks which were correlated with non-specific hand or foot dermatitis and scalp scale. We identified FLG-SNVs which were associated with sensitization to environmental allergens; rs62623409 and rs71625199 (P = 0.038 and P = 0.008, respectively). Patients with FLG P478S TT and history of allergic rhinitis showed a higher EDN level, and among those patients, the ones with asthma showed a higher ECP level. Conclusion This study revealed the association of FLG-SNVs with AD-associated minor clinical features. We firstly identified rs71625199 which was associated with higher environmental allergic sensitization. We also suggest that FLG P478S is a kind of disease modifier which affects serologic parameters such as EDN and ECP.

Systemic Epstein-Barr Virus-positive T-Cell Lymphoproliferative Disease of Childhood With Good Response to Steroid Therapy

Systemic Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease of childhood is a rare disease and has a very fulminant clinical course with high mortality. A 21-month-old female patient was referred to our hospital with a 1 week history of fever and was subsequently diagnosed with systemic Epstein-Barr virus-positive T-cell lymphoproliferative disease of childhood. After starting treatment with dexamethasone, she showed early defervescence and improvement of laboratory parameters, and has remained disease-free after stopping steroid treatment, although longer follow-up is necessary. Our report underscores the possibility that this disease entity may be heterogenous in terms of prognosis.

Practical informativeness of short tandem repeat loci for chimerism analysis in hematopoietic stem cell transplantation

OBJECTIVE Short tandem repeat (STR) loci are most frequently used for chimerism analysis after hematopoietic stem cell transplantation (HSCT). The aim of this study was to evaluate the practical informativeness of STR chimerism by integrating theoretical and analytical points. METHODS Theoretical and practical informativess of 16 STR loci were evaluated from 1249 pairs of recipients and donors who were prepared for HSCT. RESULTS Theoretical informativeness was influenced by genetic diversity including allele frequency and heterozygosity, and was higher in the unrelated HSCT group (90.5±5.3%) compared to the related HSCT group (66.2±4.4%). Practical informativeness was lower than theoretical (6.1±1.7%) because several STR loci were excluded due to stutter peaks and less reliable results, especially in type II-2 donor-recipient match pattern with no recipient-specific allele. We simulated an efficient STR combination for reliable chimerism analysis. Eight informative STR loci were required to analyze chimerism with at least one practically informative locus in the related HSCT group (D18S51, FGA, D2S1338, D13S317, D8S1179, D21S11, D16S539 and D7S820) while only three loci were needed in the unrelated group (D2S1338, FGA and D18S51). A minimum set of 2, 4 or 7 STR loci were required to provide at least 1, 3 or 5 practically informative loci in 95% of the unrelated HSCT group while 3, 8 or 12 loci were required in the related HSCT group. CONCLUSION We deducted the practical informativeness of STR chimerism, identified the major influencing factors on the practical informativeness of each STR locus, and successfully simulated the efficient STR combination for reliable chimerism analysis.

Clinical relevance of combined anti-mitochondrial M2 detection assays for primary biliary cirrhosis

BACKGROUND Antimitochondrial antibody (AMA) is a specific serologic marker in primary biliary cirrhosis (PBC). The aim of this study was to evaluate the clinical relevance of combined AMA assays. METHODS Sera were obtained from 79 patients with PBC and 108 patients with other liver disease. They were tested by indirect immunofluorescence (IIF) using rat kidney/stomach tissue and HEp2 cells as substrate, 4 AMA-M2 assays, anti-sp100, and anti-gp210 assays. RESULTS Using IIF-AMA with cut-off titer of 1:40, the sensitivity and specificity for PBC were 88.6% and 87.0%, respectively. A cut-off titer of 1:80 improved the specificity to 93.5%. The 4 commercial assay kits using AMA-M2 autoantibodies showed sensitivity of 55.7-79.7% and specificity of 91.7-95.4% with moderate to good agreement. AMA-M2 assays using both native and recombinant E2 antigens had higher sensitivity. ANAs on HEp2 cells, anti-sp100, and anti-gp210 were detected in 67.1%, 13.9-15.2%, and 22.8-27.8% of PBC patients, respectively. Additional AMA-M2 specific assays in IIF-AMA negative and low titer positive (1:40) sera increased the sensitivity and specificity to 88.6% and 90.7%, respectively. CONCLUSIONS Serological diagnosis for PBC using IIF with high titer cut-off and additional AMA-M2 specific tests by ELISA or LIA in IIF-negative sera should be used.