Telomere integrated scoring system of myelodysplastic syndrome.

INTRODUCTION: Recently, multigene target sequencing is widely performed for the purpose of prognostic prediction and application of targeted therapy. Here, we proposed a new scoring system that encompasses gene variations, telomere length, and Revised International Prognostic Scoring System (IPSS-R) together in Asian myelodysplastic syndrome. METHODS: We developed a new scoring model of these variables: age ≥ 65 years + IPSS-R score + ASXL1 mutation + TP53 mutation + Telomere length (<5.37). According to this new scoring system, patients were divided into four groups: very good score cutoff (≤3.0), good (3.0-4.5), poor (4.5-7.0), and very poor (>7.0). RESULTS: The median OS was 170.1, 100.4, 46.0, and 12.0 months for very good, good, poor, and very poor, retrospectively (p < 0.001). Meanwhile, according to the conventional IPSS-R scoring system, the median OS was 141.3, 50.2, 93.0, 36.0, and 16.2 months for very low, low, intermediate, high, and very high, retrospectively (p < 0.001). CONCLUSIONS: The newly developed model incorporating molecular variations and TL yielded more clear separations of the survival curves. By adding the presence of gene mutation and telomere length to the existing IPSS-R, its predictive ability can be further improved in myelodysplastic syndrome.

Telomere length and somatic mutations in correlation with response to immunosuppressive treatment in aplastic anaemia.

We investigated the frequencies of cytogenetic aberrations and somatic mutations of prognostic relevance in 393 patients with aplastic anaemia (AA). Clonality was determined by G-banding/fluorescence in situ hybridization (FISH) (n = 245), and targeted capture sequencing was performed for 88 haematopoiesis-related genes (n = 70). The telomere length (TL) of bone marrow nucleated cells was measured at the single cell level by FISH (n = 135). Eighteen (4·6%) patients showed disease progression, and monosomy 7 (50·0%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (32·9%) presented at least 1 mutation; the most frequently mutated genes were NOTCH1, NF1, SCRIB, BCOR and DNMT3A. The patient group with clonal changes (30·7%) showed an adverse response to immunosuppressive treatment (IST), compared to the non-clonal group, but this finding did not show statistical significance. The TL of AA patients was significantly shorter than normal control and patients with clonal changes showed significantly shorter TLs. Patients with TL>5·9 showed a higher response rate to IST (P = 0·048). In conclusion, the patients with clonal changes or TL attrition showed a poor response to IST. Shorter TL can be used not only as a biomarker, but also as a predictive marker for treatment response to IST.

Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones.

OBJECTIVES: We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. METHODS: We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. RESULTS: A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. CONCLUSIONS: Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

The Implication of Cytogenetic Alterations in Pancreatic Ductal Adenocarcinoma and Intraductal Papillary Mucinous Neoplasm Identified by Fluorescence In Situ Hybridization and Their Potential Diagnostic Utility.

Background/Aims: We investigated chromosomal aberrations in patients with pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) by fluorescence in situ hybridization (FISH) to identify cytogenetic changes and molecular markers that may be useful for preoperative diagnosis. Methods: Tissue samples from 48 PDAC and 17 IPMN patients were investigated by FISH analysis using probes targeting chromosomes 7q, 17p, 18q, 20q, and 21q and the pericentromeric region of chromosome 18 (CEP18). Results: The PDAC samples harbored 17p deletion (95.8%), 18q deletion (83.3%), CEP18 deletion (81.2%), 20q gain (81.2%), 21q deletion (77.1%), and 7q gain (70.8%). The IPMN samples had 17p deletion (94.1%), CEP18 deletion (94.1%), 21q deletion (70.6%), 18q deletion (58.8%), 20q gain (58.8%), and 7q gain (58.8%). A significant difference in CEP18 gain was identified between the PDAC and IPMN groups (p=0.029). Detection of 17p or 18q deletion had the highest diagnostic accuracy (80.0%) for PDAC. Conclusions: Chromosomal alterations were frequently identified in both PDAC and IPMN with similar patterns. CEP18 gain and 17p and 18q deletions might be involved in the later stages of PDAC tumorigenesis. Chromosome 17p and 18q deletions might be excellent diagnostic markers.

Mutation of ten-eleven translocation-2 is associated with increased risk of autoimmune disease in patients with myelodysplastic syndrome.

BACKGROUND/AIMS: Myelodysplastic syndrome (MDS) is caused by genetic and epigenetic alteration of hematopoietic precursors and immune dysregulation. Approximately 20% of patients with MDS develop an autoimmune disease (AID). Here, we investigated whether particular genetic mutations are associated with AID in patients with MDS. METHODS: Eighty-eight genetic mutations associated with myeloid malignancy were sequenced in 73 MDS patients. The association between these mutations and AID was then analyzed. RESULTS: The median age of the 73 MDS patients was 70 years (interquartile range, 56 to 75), and 49 (67.1%) were male. AID was observed in 16 of 73 patients (21.9%). Mutations were detected in 57 (78.1%) patients. The percentage (68.8% vs. 80.7%, p = 0.32) and the mean number of mutations (1.8 ± 1.6 vs. 2.2 ± 1.8, p = 0.34) in MDS patients with or without AID were similar. However, the ten-eleven translocation- 2 (TET2) mutation rate was significantly higher in patients with AID than in those without (31.3% vs. 5.3%, respectively; p = 0.001). All TET2 mutations were variants of strong clinical significance. CONCLUSION: Mutation of TET2 in patients with MDS may be associated with increased risk of developing AID.

Telomere length and its correlation with gene mutations in chronic lymphocytic leukemia in a Korean population.

Telomere length (TL) is a prognostic indicator in Caucasian chronic lymphocytic leukemia (CLL), but its significance in Asian CLL remains unknown. To investigate the prognostic significance of TL and its correlation with cytogenetic aberrations and somatic mutations, we analyzed TL measurements at the cellular level by interphase fluorescence in situ hybridization in patients with CLL in Korea. The present study enrolled 110 patients (41 females and 69 males) diagnosed with CLL according to the World Health Organization criteria (2001-2017). TLs of bone marrow nucleated cells at the single-cell level were measured by quantitative fluorescence in situ hybridization (Q-FISH) in 71 patients. The correlations of TL with clinical characteristics, cytogenetic aberrations, genetic mutations, and overall survival were assessed. The median value of mean TL in CLL patients (T/C ratio 7.46 (range 1.19-18.14) was significantly shorter than that in the normal controls (T/C ratio 15.28 (range 8.59-24.93) (p < 0.001). Shorter TLs were associated with complex karyotypes (p = 0.030), del(11q22) (p = 0.023), presence of deletion and/or mutation in ATM and/or TP53 (p = 0.019), and SH2B3 mutation (p = 0.015). A shorter TL was correlated with lower hemoglobin levels and adverse survival (mean TL < 9.35, p = 0.021). When the proportion of cells with extremely short TLs (< 7.61) was greater than 90%, CLL patients showed poor survival (p = 0.002). Complex karyotypes, TP53 mutation, and the number of mutated genes were determined to be significant adverse variables by multivariable Cox analysis (p = 0.011, p = 0.002, and p = 0.002, respectively). TL was attrited in CLL, and attrited telomeres were correlated with adverse survival and other well-known adverse prognostic factors. We infer that TL is an independent adverse prognostic predictor in Korean CLL.

Replicative senescence of hematopoietic cells in patients with idiopathic cytopenia of undetermined significance.

We hypothesized that a subset of idiopathic cytopenia of undetermined significance (ICUS) is associated with an increased autonomous proliferation with exhaustion of hematopoiesis. The aim of this study was to investigate the cell turnover rate and replicative history of the bone marrow cells of ICUS patients. To this end, we examined telomere length (TL), proliferation, and apoptosis of the bone marrow cells of ICUS patients and healthy controls (HCs) using telomere quantitative fluorescence in situ hybridization and immunohistochemical staining for Ki-67 and cleaved caspase-3. We also performed targeted sequencing of 88 myeloid-associated genes. A total of 37 patients with ICUS were enrolled in this study, with a median age of 66 years (range: 31-83). TLs were significantly shorter in patients with ICUS than in the HCs (8.8, interquartile range [IQR] 6.8-12.1 vs 18.4, IQR 14.4-22.0, p < 0.0001). Proliferation (Ki-67-positive) and apoptosis (cleaved caspase-3-positive) were significantly increased in patients with ICUS compared to HCs (median = 20.0% vs 5.0%, p = 0.0003; 45.0% vs 22.5%, p = 0.0005, respectively). The shortening of TL and the increased proliferation and apoptotic activity was also prominent in patients with ICUS without mutation and dysplasia than in HCs (p < 0.0001, p < 0.0001, and p = 0.0093, respectively). TL was not associated with mutational profile and clinical characteristics as well in patients with ICUS. To our knowledge, this is the first study to show that ICUS is associated with premature replicative senescence with increased proliferation and apoptosis of bone marrow cells. Further study is needed to address the cause of replicative exhaustion in ICUS patients.

ASXL1 is a molecular predictor in idiopathic cytopenia of undetermined significance.

We analyzed the mutational profile of idiopathic cytopenia of undetermined significance (ICUS) compared with that of myelodysplastic syndrome (MDS). Targeted sequencing of 88 genes associated with myeloid malignancies was performed using samples of bone marrow mononuclear cells from ICUS and MDS patients. Forty patients with ICUS and 128 patients with MDS were included in this study. The median mutational burden was 0.7 mutation/person in the ICUS group and 2.2 mutation/person in the MDS group. ASXL1 (seven patients) was the most frequently mutated gene. ASXL1 was an independent significant prognostic factor for event-free survival (EFS) and overall survival (OS) (hazard ratio (HR) = 10.07 and 30.63, p = .004 and .003, respectively). The ASXL1 mutation which is frequently detected in elderly patients is a molecular predictor for pancytopenia and survival in patients with ICUS. A larger prospective study is needed to validate the role of this genetic mutation in an ICUS prognosis.

Monozygotic twins with shared de novo GATA2 mutation but dissimilar phenotypes due to differential promoter methylation.

A revised WHO classification of hematopoietic neoplasm introduced the new category 'Myeloid Neoplasms with Germline Predisposition', reflecting the growing importance of genetic testing for myeloid neoplasms. Here, we investigated monozygotic twins with the same de novo mutation in GATA2 but different phenotypes. The patient suffering a bleeding tendency was diagnosed with myelodysplastic syndrome (MDS), and her monozygotic twin showed dysmegakaryopoietic features in the bone marrow. Targeted sequencing revealed the same germline mutation in GATA2, c.1192C > T, in both sisters and different somatic mutations in 14 genes between the sisters. The GATA2 mutation was absent in both parents, and their hemograms were normal. The methylation profile of the GATA2 promoter region was different between the twins, showing denser promoter methylation in the patient, correlated with MDS. Thus, we concluded that the twins had acquired a de novo GATA2 mutation but showed different phenotypes, possibly due to the critical role of epigenetic changes.

Characteristics of Waldenström Macroglobulinemia in Korean Patients According to Mutational Status of MYD88 and CXCR4: Analysis Using Ultra-Deep Sequencing.

BACKGROUND: Little is known about the mutational frequency of myeloid differentiation factor 88 (MYD88) and C-X-C chemokine receptor type 4 (CXCR4) and the corresponding characteristics in Asian individuals afflicted with Waldenström macroglobulinemia (WM). We investigated the characteristics of WM according to mutational status of MYD88/CXCR4, and attempted to determine the lineage commitment among hematopoietic cells by MYD88L265P single-cell sequencing on bone marrow (BM) smear slides. MATERIALS AND METHODS: CXCR4 mutations (muts) were detected using ultra-deep sequencing using target capture. Mutational burden of MYD88 was assessed using real-time polymerase chain reaction. Single-cell sequencing for MYD88 was performed on lymphocytes, plasmacytoid lymphocytes, plasma cells, and neutrophils using laser microdissection. RESULTS: Among 31 patients, the frequencies of MYD88/CXCR4 muts were as follows: MYD88 wild type (WT) CXCR4WT (6 patients, 19.4%), MYD88L265PCXCR4WT (19 patients, 61.4%), MYD88L265PCXCR4mut (6 patients, 19.4%; 1 frameshift and 5 nonsense muts). Immunoglobulin M levels of MYD88L265CXCR4WT patients were significantly higher than those of MYD88WTCXCR4WT patients (P = .024). Tumor burden in BM was highest in patients with MYD88L265PCXCR4mut (82.0%), followed by MYD88L265PCXCR4WT (52.8%) and MYD88WTCXCR4WT (14.2%) (P < .001). The quantity of MYD88-mutated DNA tended to correlate with tumor burden in BM (correlation coefficient 0.647; P = .009). MYD88L265P was detected in plasma cells, plasmacytoid lymphocytes, and lymphocytes but not neutrophils. CONCLUSION: The frequency of MYD88/CXCR4 muts in Korean and Caucasian patients with WM was similar, however 5 of the 6 CXCR4 muts were nonsense-a proportion higher than reported frequencies in Caucasian individuals. Ultra-deep sequencing was capable of detecting CXCR4 muts not detectable using Sanger sequencing, suggesting a possible replacement of the B-cell sorting.

Shifting of erythroleukemia to myelodysplastic syndrome according to the revised WHO classification: Biologic and cytogenetic features of shifted erythroleukemia.

The 2016 revision of the World Health Organization (WHO) classification of tumours of haematopoietic and lymphoid tissues was published. According to 2016 WHO criteria, diagnostic criteria of acute erythroid leukemia was revised. We reassessed 34 de novo acute erythroid leukemia (AEL) diagnosed by 2008 WHO criteria, according to 2016 WHO criteria. A total of 623 patients (excluding M3) with acute myeloid leukemia including 34 patients with AEL were enrolled. Among 34 patients diagnosed with AEL, diagnosis was shifted to MDS-EB in 28 patients (28/34, 82.3%) and MDS-U in 2 patients (2/34, 5.9%), while remained as AEL in 4 patients (4/34, 11.8%) according to 2016 WHO criteria. Interphase FISH for cytogenetic changes of MDS (-5/del(5q), -7/del(7q), del(20q), +8) revealed cytogenetic aberrations in 50.0% (17/34) of AEL 2008 group. AEL 2008 group showed higher frequency of complex cytogenetic abnormalities and higher MDS related cytogenetic abnormalities than AML excluding AEL group. Transformation to another AML subtype was noted in 10% in AEL shifted to MDS. Majority (88.2%) of AEL by 2008 WHO criteria was reclassified to MDS by 2016 WHO criteria. Clinical characteristics of shifted AEL were similar to those of MDS rather than de novo AML.

Dysregulation of Telomere Lengths and Telomerase Activity in Myelodysplastic Syndrome.

BACKGROUND: Telomere shortening is thought to be involved in the pathophysiology of myeloid malignancies, but telomere lengths (TL) during interphase and metaphase in hematopoietic malignancies have not been analyzed. We aimed to assess the TLs of interphase and metaphase cells of MDS and telomerase activity (TA) and to find out prognostic significances of TL and TA. METHODS: The prognostic significance of TA by quantitative PCR and TL by quantitative fluorescence in situ hybridization (QFISH) of interphase nuclei and metaphase chromosome arms of bone marrow cells from patients with MDS were evaluated. RESULTS: MDS patients had shorter interphase TL than normal healthy donors (P<0.001). Average interphase and metaphase TL were inversely correlated (P=0.013, p arm; P=0.029, q arm), but there was no statistically significant correlation between TA and TL (P=0.258). The progression free survival was significantly shorter in patients with high TA, but the overall survival was not different according to average TA or interphase TL groups. Multivariable Cox analysis showed that old age, higher International Prognostic Scoring System (IPSS) subtypes, transformation to AML, no history of hematopoietic stem cell transplantation and short average interphase TL (<433 TL) as independent prognostic factors for poorer survival (P=0.003, 0.001, 0.005, 0.005, and 0.013, respectively). CONCLUSIONS: The lack of correlation between age and TL, TA, and TL, and the inverse relationship between TL and TA in MDS patients reflect the dysregulation of telomere status and proliferation. As a prognostic marker for leukemia progression, TA may be considered, and since interphase TL has the advantage of automated measurement by QFISH, it may be used as a prognostic marker for survival in MDS.

The high frequency of the U2AF1 S34Y mutation and its association with isolated trisomy 8 in myelodysplastic syndrome in Asians, but not in Caucasians.

Mutational profiles of 153 Korean myelodysplastic syndrome (MDS) patients were investigated. Sequencing of 87 genes presented similar mutational profiles in Korean MDS patients compared with previous reports. The most frequently mutated genes were ASXL1 (22.9%), U2AF1 (16.3%), TP53 (13.7%), RUNX1 (10.5%), TET2 (10.5%), DNMT3A (8.5%), and SRSF2 (8.5%). The U2AF1 mutation frequency was higher, with different frequencies in the mutated sites of U2AF1 (S34Y, 6/25; S34F, 11/25; and Q157P 8/25). The U2AF1 S34Y mutation was strongly associated with isolated trisomy 8 (5/6, 83%) and was characterized by a younger age of MDS onset (median, 39 years). The S34F mutation was associated with trisomy 8 (6/11, 55%) and del(20q) (3/11, 27%). Data from 10 literatures (total 3460 patients) of 229 U2AF1-mutated cases revealed a significant association between the S34Y and trisomy 8 in Asians (P=0.0001), but not in Caucasians (P=0.080). We infer that U2AF1 S34 mutations characterize a distinct subgroup of MDS: younger age of onset and differential associations with particular cytogenetic aberrations depending on specific mutations [S34Y to +8; S34F to +8 and del(20q)]. The impact and causal relationship between U2AF1 S34 and trisomy 8 need to be elucidated, which might contribute to design of tailored treatments.

Idiopathic hypereosinophilia is clonal disorder? Clonality identified by targeted sequencing.

Idiopathic hypereosinophilia (IHE)/idiopathic hypereosinophilic syndrome (IHES) has been defined by a persistent elevation of the blood eosinophil count exceeding 1.5×103/µL, without evidence of reactive or clonal causes. While T-cell clonality assessment has been recommended for unexplained hypereosinophilia, this approach is not often applied to routine practice in the clinic. We hypothesized that the clonality would exist in a subset of IHE/IHES patients. We aimed to investigate the candidate mutations and T-cell clonality in IHE/IHES and to explore the role of mutations in eosinophil proliferation. We performed targeted capture sequencing for 88 genes using next-generation sequencing, T-cell receptor (TCR) gene rearrangement assays, and pathway network analysis in relation to eosinophil proliferation. By targeted sequencing, 140 variants in 59 genes were identified. Sixteen out of 30 patients (53.3%) harbored at least one candidate mutation. The most frequently affected genes were NOTCH1 (26.7%), SCRIB and STAG2 (16.7%), and SH2B3 (13.3%). Network analysis revealed that our 21 candidate genes (BIRC3, BRD4, CSF3R, DNMT3A, EGR2, EZH2, FAT4, FLT3, GATA2, IKZF, JAK2, MAPK1, MPL, NF1, NOTCH1, PTEN, RB1, RUNX1, TET2, TP53 and WT1) are functionally linked to the eosinophilopoietic pathway. Among the 21 candidate genes, five genes (MAPK1, RUNX1, GATA2, NOTCH1 and TP53) with the highest number of linkages were considered major genes. A TCR assay revealed that four patients (13.3%) had a clonal TCR rearrangement. NOTCH1 was the most frequently mutated gene and was shown to be a common node for eosinophilopoiesis in our network analysis, while the possibility of hidden T cell malignancy was indwelling in the presence of NOTCH1 mutation, though not revealed by aberrant T cell study. Collectively, these results provide new evidence that mutations affecting eosinophilopoiesis underlie a subset of IHE/IHES, and the candidate genes are inferred to act their potential roles in the eosinophilopoietic pathway.

Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis.

PURPOSE: KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. MATERIALS AND METHODS: The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. RESULTS: KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. CONCLUSION: KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Burden of cytogenetically abnormal plasma cells in light chain amyloidosis and their prognostic relevance.

We performed cytoplasmic fluorescence in situ hybridization assays of light chain amyloidosis (AL). In total, 234 patients were enrolled: 28 patients with AL, 24 with monoclonal gammopathy of undetermined significance (MGUS), and 182 with multiple myeloma (MM). Chromosomal abnormalities were detected in 13 of 22 (59%) AL patients without MM. All 13 patients demonstrated IGH rearrangement, and t(11;14)/IGH-CCND1 was most frequent (32%). Chromosome gain was not observed in AL patients without MM. These findings were dissimilar to findings in MGUS patients, in whom trisomy 9 was the most frequent abnormality. Of 6 AL patients with MM, 5 (83%) patients had cytogenetic abnormalities: 1q gain (4/6, 67%), gains of chromosome 9 (3/6, 50%), IGH rearrangement and RB1 (13q) deletions (2/6 each, 33%). The percentage of clonal plasma cells among total plasma cells was variable (median, 75%; range, 16-100%) for AL patients without MM, which was lower than the results for MM patients (median 100%). The overall survival of AL patients without MM was not significantly different according to the presence of cytogenetic abnormalities (P=0.510). In summary, among Korean AL patients, IGH rearrangement was the most frequent cytogenetic abnormality and cytogenetic aberration patterns differ compared with MGUS and MM patients.

Short telomere length and its correlation with gene mutations in myelodysplastic syndrome.

BACKGROUND: Telomere erosion can lead to genomic instability and cancer progression. It has been suggested that the shortest telomere, not the average telomere length (TL), is critical for cell viability. Some studies have shown shorter TL in myelodysplastic syndrome (MDS) patients but the critically short telomeres, the variability of TL within individual patient has not been evaluated. Thus, we aimed to investigate the TL of MDS patients and assessed the association of TL with recurrent genetic mutations in MDS. METHODS: We measured the TL of bone marrow nucleated cells for diagnostic samples at a single-cell level by quantitative fluorescence in situ hybridization (Q-FISH) for 58 MDS patients and analyzed the minimum, median, average, standard deviation, average of the 0th to 10th percentile TL within a patient, and the proportion of cells with TL that is shorter than the lowest 10th percentile of the normal control (NC). The correlations of TL to clinical parameters, cytogenetic results, and genetic mutations were assessed. RESULTS: MDS patients showed eroded telomeres and narrow distribution compared to the NC (P < 0.001, P = 0.018, respectively). Patients with mutation showed significantly lesser cells with short TL, below the lowest 10th percentile of the NC (P = 0.017), but no differences in TL were found according to mutations/cytogenetic abnormalities except for CSF3R mutation. However, those patients with a high percentage (≥80 %) of cells with short TL showed poorer overall survival (P = 0.021), and this was an independent prognostic factor, along with TP53, U2AF1 mutation, and high BM blast count (P = 0.044, 0.001, 0.004, 0.012, respectively). CONCLUSIONS: The shortest TL, which determines the fate of the cell, was significantly shorter, and higher burden of cells with short TL were found in MDS, which correlated with poor survival, suggesting the need to measure TL in single cells by Q-FISH.

Genomic Profile of Chronic Lymphocytic Leukemia in Korea Identified by Targeted Sequencing.

Chronic lymphocytic leukemia (CLL) is extremely rare in Asian countries and there has been one report on genetic changes for 5 genes (TP53, SF3B1, NOTCH1, MYD88, and BIRC3) by Sanger sequencing in Chinese CLL. Yet studies of CLL in Asian countries using Next generation sequencing have not been reported. We aimed to characterize the genomic profiles of Korean CLL and to find out ethnic differences in somatic mutations with prognostic implications. We performed targeted sequencing for 87 gene panel using next-generation sequencing along with G-banding and fluorescent in situ hybridization (FISH) for chromosome 12, 13q14.3 deletion, 17p13 deletion, and 11q22 deletion. Overall, 36 out of 48 patients (75%) harbored at least one mutation and mean number of mutation per patient was 1.6 (range 0-6). Aberrant karyotypes were observed in 30.4% by G-banding and 66.7% by FISH. Most recurrent mutation (>10% frequency) was ATM (20.8%) followed by TP53 (14.6%), SF3B1 (10.4%), KLHL6 (8.3%), and BCOR (6.25%). Mutations of MYD88 was associated with moderate adverse prognosis by multiple comparisons (P = 0.055). Mutation frequencies of MYD88, SAMHD1, EGR2, DDX3X, ZMYM3, and MED12 showed similar incidence with Caucasians, while mutation frequencies of ATM, TP53, KLHL6, BCOR and CDKN2A tend to be higher in Koreans than in Caucasians. Especially, ATM mutation showed 1.5 fold higher incidence than Caucasians, while mutation frequencies of SF3B1, NOTCH1, CHD2 and POT1 tend to be lower in Koreans than in Caucasians. However, mutation frequencies between Caucasians and Koreans were not significantly different statistically, probably due to low number of patients. Collectively, mutational profile and adverse prognostic genes in Korean CLL were different from those of Caucasians, suggesting an ethnic difference, while profile of cytogenetic aberrations was similar to those of Caucasians.

CALR, JAK2, and MPL mutation profiles in patients with four different subtypes of myeloproliferative neoplasms: primary myelofibrosis, essential thrombocythemia, polycythemia vera, and myeloproliferative neoplasm, unclassifiable.

OBJECTIVES: We investigated mutation profiles of CALR, JAK2, and MPL in 199 Korean patients with myeloproliferative neoplasms (MPNs). METHODS: In total, 199 patients with MPN (54 primary myelofibrosis [PMF], 79 essential thrombocythemia [ET], 58 polycythemia vera [PV], and eight MPN-unclassifiable [MPN-U]) and 4 patients with acute panmyelosis with myelofibrosis (APMF) were retrospectively subjected to Sanger sequencing for CALR, JAK2, and MPL. RESULTS: The overall frequency of CALR mutations was 12.6% (type 1 mutation, 16 patients; type 2 mutation, nine patients): most frequent in MPN-U (37.5%), followed by ET (17.7%) and PMF (14.8%). CALR mutations were not found in PV or APMF. CALR and JAK2 or MPL mutations were mutually exclusive. In PMF, the CALR mutations were associated with lower levels of leukocytes, lower bone marrow cellularity, and higher number of megakaryocytes. Patients with CALR-mutated ET more frequently progressed to the accelerated or blast phases compared with patients with JAK2 mutations. CALR mutations were frequently observed in the JAK2-negative MPNs, most frequently in MPN-U. CONCLUSIONS: The prognostic significance of CALR mutations likely differs among the MPN subtypes.