Coexistence of JAK2 and CALR mutations and their clinical implications in patients with essential thrombocythemia.

Janus kinase 2 (JAK2) and calreticulin (CALR) constitute the two most frequent mutations in essential thrombocythemia (ET), and both are reported to be mutually exclusive. Hence, we examined a cohort of 123 myeloproliferative neoplasm (MPN) patients without BCR-ABL1 rearrangement and additional ET patients (n=96) for coexistence of JAK2 and CALR mutations. The frequency of CALR mutations was 20.3% in 123 MPN patients; 31.1% in ET (n=74), 25% in primary myelofibrosis (n=4) and 2.2% in polycythemia vera (n=45). JAK2 and CALR mutations coexisted in 7 (4.2%) of 167 ET patients. Clinical characteristics, progression-free survival (PFS), and elapsed time to achieve partial remission across 4 groups (JAK2+/CALR+, JAK2+/CALR-, JAK2-/CALR+, JAK2-/CALR-) were reviewed. The JAK2+/CALR- group had higher leukocyte counts and hemoglobin levels and more frequent thrombotic events than JAK2-/CALR- group. JAK2 mutations have a greater effect on the disease phenotype and the clinical features of MPN patients rather than do CALR mutation. JAK2+ groups showed a tendency of poor PFS than JAK2- groups regardless of CALR mutation. CALR+ was a predictor of late response to the treatment. Our study also showed that thrombosis was more frequent in ET patients with type 2 CALR mutations than in those with type 1 CALR mutations.

Germline TP53 Mutation and Clinical Characteristics of Korean Patients With Li-Fraumeni Syndrome.

BACKGROUND: Little is known of the mutation and tumor spectrum of Korean patients with Li-Fraumeni syndrome (LFS). Owing to the rarity of LFS, few cases have been reported in Korea thus far. This study aimed to retrospectively review the mutations and clinical characteristics of Korean patients with LFS. METHODS: TP53 mutation was screened in 89 unrelated individuals at the Samsung Medical Center in Korea, from 2004 to 2015. Six additional mutation carriers were obtained from the literature. RESULTS: We identified nine different mutations in 14 Korean patients (male to female ratio=0.3:1). Two such frameshift mutations (p.Pro98Leufs*25, p.Pro27Leufs*17) were novel. The recurrent mutations were located at codons 31 (n=2; p.Val31Ile), 175 (n=3; p.Arg175His), and 273 (n=4; p.Arg273His and p.Arg273Cys). The median age at the first tumor onset was 25 yr. Ten patients (71%) developed multiple primary tumors. A diverse spectrum of tumors was observed, including breast (n=6), osteosarcoma (n=4), brain (n=4), leukemia (n=2), stomach (n=2), thyroid (n=2), lung (n=2), skin (n=2), bladder (n=1), nasal cavity cancer (n=1), and adrenocortical carcinoma (n=1). CONCLUSIONS: There was considerable heterogeneity in the TP53 mutations and tumor spectrum in Korean patients with LFS. Our results suggest shared and different LFS characteristics between Caucasian and Korean patients. This is the first report on the mutation spectrum and clinical characteristics from the largest series of Korean LFS patients.

The prognostic impact of mutations in spliceosomal genes for myelodysplastic syndrome patients without ring sideroblasts.

BACKGROUND: Mutations in genes that are part of the splicing machinery for myelodysplastic syndromes (MDS), including MDS without ring sideroblasts (RS), have been widely investigated. The effects of these mutations on clinical outcomes have been diverse and contrasting. METHODS: We examined a cohort of 129 de novo MDS patients, who did not harbor RS, for mutations affecting three spliceosomal genes (SF3B1, U2AF1, and SRSF2). RESULTS: The mutation rates of SF3B1, U2AF1, and SRSF2 were 7.0 %, 7.8 %, and 10.1 %, respectively. Compared with previously reported results, these rates were relatively infrequent. The SRSF2 mutation strongly correlated with old age (P < 0.001), while the mutation status of SF3B1 did not affect overall survival (OS), progression-free survival (PFS), or acute myeloid leukemia (AML) transformation. In contrast, MDS patients with mutations in U2AF1 or SRSF2 exhibited inferior PFS. The U2AF1 mutation was associated with inferior OS in low-risk MDS patients (P = 0.035). The SRSF2 mutation was somewhat associated with AML transformation (P = 0.083). CONCLUSION: Our findings suggest that the frequencies of the SF3B1, U2AF1, and SRSF2 splicing gene mutations in MDS without RS were relatively low. We also demonstrated that the U2AF1 and SRSF2 mutations were associated with an unfavorable prognostic impact in MDS patients without RS.

Feasible quantitative detection of cytomegalovirus from urine sediment in stem cell transplant patients.

BACKGROUND: Urine is an important source for the detection of infections caused by CMV in stem cell transplant patients. Currently, there is no agreement about the type of urine specimen. In order to investigate which is the better specimen type for quantitative detection of CMV, we compared the results from urine supernatant and sediment from the same patients. METHODS: Seventy urine specimens were collected from patients with hematological disorders or solid tumors. After performing shell vial culture, residual urine specimens were centrifuged. Then, 10 mL of each urine supernatant and sediment were taken and immediately frozen at -70 degrees C. Afterwards, archived urine specimens were thawed at room temperature and CMV-quantitative PCR was performed on both the supernatant and sediment fraction of urine. The results from each patient were reviewed for CMV antigenemia, blood shell vial culture, CMV-IgM or IgG, and clinical symptoms. RESULTS: CMV-qPCR results for the urine sediment fraction revealed a significant difference (p = 0.012) between the active CMV infection group and the latent CMV infection group. In addition, receiver operating characteristic curves for active CMV infection revealed that CMV-qPCR using urine sediment produced more accurate results than urine supernatant. CONCLUSIONS: These findings suggest that the sediment fraction of urine is a more suitable specimen in CMV-qPCR testing.

Direct confirmation of quiescence of CD34+CD38- leukemia stem cell populations using single cell culture, their molecular signature and clinicopathological implications.

BACKGROUND: The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications. METHODS: Single cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients. RESULTS: Single cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFß super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn't support the prognostic value of the ASCs in AML. CONCLUSIONS: ASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways.

Alteration of the SETBP1 gene and splicing pathway genes SF3B1, U2AF1, and SRSF2 in childhood acute myeloid leukemia.

BACKGROUND: Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. METHODS: Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. RESULTS: Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. CONCLUSIONS: Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.

Presymptomatic identification of CDH1 germline mutation in a healthy korean individual with family history of gastric cancer.

Gastric cancer (GC) is one of the most common cancers with high morbidity and mortality. Familial GC is seen in 10% of cases, and approximately 3% of familial GC cases arise owing to hereditary diffuse gastric cancer (HDGC). CDH1, which encodes the protein E-cadherin, is the only gene whose mutations are associated with HDGC. Screening for the familial GC-predisposing gene has been neglected in high-risk countries such as Korea, China, and Japan, where all the cases have been attributed to Helicobacter pylori or other carcinogens. Screening for the GC-causing CDH1 mutation may provide valuable information for genetic counseling, testing, and risk-reduction management for the as-yet unaffected family members. An asymptomatic 44-yr-old Korean male visited our genetic clinic for consultation owing to his family history of GC. Eventually, c.1018A>G in CDH1, a known disease-causing mutation, was found. As of the publication time, the individual is alive without the evidence of GC, and is on surveillance. To our knowledge, this is the first Korean case of presymptomatic detection of CDH1 mutation, and it highlights the importance of genetic screening for individuals with a family history of GC, especially in high-risk geographical areas.

Profiling of biomarkers for the exposure of polycyclic aromatic hydrocarbons: lamin-A/C isoform 3, poly[ADP-ribose] polymerase 1, and mitochondria copy number are identified as universal biomarkers.

This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs.

Serum parathyroid hormone is a new potential risk factor in multiple myeloma.

We hypothesized that serum PTH might be associated with various clinicopathological parameters in multiple myeloma (MM). So we investigated the implications of serum PTH in MM patients and the relationship with other risk factors of MM. A total of 115 patients who were newly diagnosed with MM were enrolled. Serum PTH level was 24.7 ± 34.9 (ranged 0.0-284.1) pg/mL. Serum levels of IgG, IgM, FLC-lambda, albumin, and LDH were in positive correlation with serum PTH. Compared to non-high PTH (<68.3 pg/mL) group, the hazard ratio (HR) for overall survival was higher for group with high PTH level (≥ 68.3 pg/mL) (HR, 1.710). Furthermore, the patient group with high PTH level showed inferior progression-free survival than non-high PTH group (P = 0.056). Interestingly, subgroup analysis showed that serum PTH level at diagnosis was associated with risk factors and clinical outcome in MM patients, especially in complete remission group, transplantation cases, ISS stage II cases, and cases without chromosome abnormality. In conclusion, this study showed that blood PTH level in MM at diagnosis was associated with risk factors and clinical outcome in MM patients.

Circulating mucosal-associated invariant T cell levels and their cytokine levels in healthy adults.

Mucosal-associated invariant T (MAIT) cells have been reported to play an antimicrobial role in infectious diseases. However, little is known about age- and gender-related changes in circulating MAIT cell level and function in healthy population. The purposes of this study were to examine the level and cytokine production of circulating MAIT cells and their subsets in healthy adults and to investigate potential relationships between clinical parameters and MAIT cell levels or their subset levels. One hundred thirty-three healthy subjects were enrolled in this study. MAIT cells, their subset, and cytokine levels were measured by flow cytometry. Circulating MAIT cell levels were found to vary widely (0.19% to 21.7%) in the study subjects and to be significantly lower in elderly subjects (age, 61-92 years) than in young subjects (age, 21-40 years) (p<0.0005). No significant difference was found in the circulating MAIT cell levels between male and female subjects. A linear regression analysis revealed that circulating MAIT cell levels declined annually by 3.2% among men and 1.8% among women, respectively. Notably, the proportion of CD4+ MAIT cells increased with age, whereas that of CD8+ MAIT cells decreased with age. In addition, the production of interleukin (IL)-4 by MAIT cells was found to be significantly increased in elderly subjects and the ratio of interferon (IFN)-γ/IL-4 was lower as compared with young subjects, showing a Th1 to Th2 shift in cytokine profile in elderly subjects. Our data suggest that aging is associated with a reduction in circulating MAIT cells, accompanied with alterations in subset composition and cytokine profile.

Overnight storage of blood in ACD tubes at 4{degrees}C increases NK cell fraction in peripheral blood mononuclear cells.

A considerable variabilility in the effects of sample handling on NK cytotoxicity has been observed. Using flow cytometry, NK cytotoxicity assays and lymphocyte subset analysis of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMCs) isolated from whole blood stored under various conditions were performed. The NK cytotoxicity of samples in heparin tubes stored overnight at 4 and 22°C, as well as at 22°C in acid citrate dextrose (ACD) tubes, was lower than that of a fresh sample. However, the NK cytotoxicity of samples in an ACD tube stored at 4°C was similar to that of a fresh sample. Based on lymphocyte subset analysis, samples in an ACD tube stored at 4°C showed a lower percentage of CD3+ T cells and a higher percentage of CD16/56+ NK cells compared to samples stored under other conditions. The NK cytotoxicity of fresh samples and samples in ACD tubes stored in a Styrofoam cooler box did not differ significantly; however, the differences were inconsistent. Overnight storage of peripheral blood in ACD tubes at 4°C is optimum for retention of NK cytotoxicity, the level of which is similar to that of fresh blood. This may be associated with an increased NK-cell fraction in Ficoll-Hypaque-separated PBMCs after overnight storage.

Over-expression of Her-2 in colorectal cancer tissue, but not in serum, constitutes an independent worse prognostic factor.

BACKGROUND: Currently, conflicting information exists regarding Her-2 over-expression and its clinicopathological implications in colorectal cancer (CRC). This study was undertaken to determine Her-2 over-expression in both serum and tumor tissue of CRC patients, and to assess its clinicopathological and targeted therapeutic implications. METHODS: Ninety five CRC patients and sixty healthy controls were prospectively enrolled. Her-2 expression status in serum and CRC tissue were examined by chemiluminescent immunoassay and immunohistochemical staining, respectively. The results were confirmed using fluorescent in situ hybridization. Clinicopathological parameters were analyzed according to Her-2 expression status. RESULTS: Serum Her-2 levels were found to be increased in CRC patients as compared to those of healthy controls. However, serum Her-2 levels were not found to be significantly associated with prognostic parameters. Her-2 expression analysis of CRC tissues revealed Her-2 over-expression in 23 patients (25%), i.e., 13 patients (14%) showed moderate over-expression and 10 patients (11%) showed strong over-expression. The overall survival of patients negative for Her-2 expression was significantly better than that of patients positive for Her-2 expression (P=0.018). The disease-free survival of patients with Her-2 over-expression was significantly shorter than that of patients with no Her-2 expression (P=0.021). CONCLUSIONS: Her-2 over-expression in CRC tissue, but not in serum, acts as a significant independent worse prognostic factor. Assessment of Her-2 expression status may be valuable for the targeted therapeutic management of CRC.

Diagnostic and prognostic value of mitochondrial DNA minisatellites after stem cell transplantation.

Mitochondrial DNA has been used to investigate phylogenetic relationships and pathophysiologic roles in aging, degenerative diseases, and cancer. We investigated the prognostic usefulness of mitochondrial DNA minisatellite (mtMS) markers compared with nuclear short tandem repeat markers by evaluating the laboratory performance and clinical value of these markers in a large sample of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) with various simulated conditions in vitro and in serial follow-up samples. We examined the value of mtMS markers as a prognostic indicator in 100 patients with various hematologic disorders undergoing allo-HSCT, including 35 patients with longitudinal follow-up for 55 months. The mtMS markers showed high sensitivity and accuracy for the quantitative detection of chimerism compared with nuclear short tandem repeat markers, particularly in unrelated transplantation and under inappropriate sampling conditions. Longitudinal follow-up after allo-HSCT disclosed that chimerism precisely reflected the status of engraftment or relapse during the clinicopathological course. Moreover, changes in mtMS markers in recipients before allo-HSCT were associated with clinical outcomes. Our data indicate that mtMS markers have multiple functions in monitoring mixed chimerism and predicting prognosis after allo-HSCT.

Allelic expression imbalance of JAK2 V617F mutation in BCR-ABL negative myeloproliferative neoplasms.

The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs.

Molecular identification of Schizophyllum commune as a cause of allergic fungal sinusitis.

Schizophyllum commune, a basidiomycetous fungus, rarely causes disease in humans. We report a rare case of allergic fungal sinusitis caused by S. commune in a 14-yr-old girl. The patient presented with nasal obstruction and a purulent nasal discharge. Materials obtained during endoscopic surgery of the frontal recess revealed allergic mucin and a few fungal hyphae. A potato dextrose agar (PDA) culture from the allergic mucin yielded a rapidly growing white woolly mold. Although no distinctive features including hyphae bearing spicules or a clamp connection were present, the case isolate disclosed compatible mycological features including growth at 37℃, susceptibility to cycloheximide, and production of a tart and disagreeable smell. S. commune was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA. We believe this is the first report of allergic fungal sinusitis caused by S. commune in Korea. Moreover, this report highlights the value of gene sequencing as an identification tool for non-sporulating isolates of S. commune.

Age- and gender-related differences in circulating natural killer T cells and their subset levels in healthy Korean adults.

Natural killer T (NKT) cells have been reported to play crucial roles in a variety of diseases, including infectious diseases, autoimmunity, and cancers. Previous studies have reported wide age- and/or sex-related variations in circulating NKT cell levels in healthy subjects, but reported results are discrepant. In this study, the authors examined NKT cell levels in the peripheral blood of healthy Korean subjects and investigated potential relationships between clinical parameters and NKT cells and their subset levels. One hundred and thirty-eight age- and sex-matched healthy subjects were enrolled in this study. NKT cell and NKT subset levels were measured by flow cytometry. Circulating NKT cell levels were found to vary widely (0.01-5.15%) in the study subjects and to be lower in men than in women (P<0.05). Notably, gender-related differences in NKT cell levels were more prominent in elderly subjects (P<0.05). Furthermore, alterations in NKT subset compositions were found in elderly men, in whom the proportion of CD4+ NKT cells was elevated and that of double-negative NKT cells was reduced. Our data suggest that circulating NKT cells and NKT subset levels are affected by age and gender in the Korean population.

Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence of dysplasia.

BACKGROUND: Barrett's esophagus (BE) is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA). The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA) mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities) in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT) and tissue (BT) from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53%) exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS) level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE.

An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells.

Several flow cytometric methods for measuring natural killer cell activity have been developed. Commonly used protocols involve the staining of target cells with various fluorescent dyes. However, these protocols are not applicable to certain experimental settings. Therefore, we used Calcein AM (CAM), which has been reported to be the most suitable dye for use in target cell staining protocols, as a means of developing an improved flow cytometry-based NK cytotoxicity assay involving effector cell staining. Peripheral blood mononuclear cells (PBMCs) isolated by gradient density centrifugation and expanded NK cells were used as effector cells. Cytotoxicity against K562 cells and several hematologic cancer cell lines was measured by a flow cytometry-based method using CAM and propidium iodide. The new assay was compared with a standard (51)Cr release assay (CRA) in terms of its ability to measure the cytotoxicity of NK cells in PBMCs and expanded NK cells against K562 cells. The optimal concentration of CAM for staining effector cells was 0.05 µM, and CAM fluorescence intensity in effector cells was maintained for 4 hours. CAM staining had no significant effect on NK cell activity in human PBMCs or expanded NK cells. Comparison of the CRA and this new assay using K562 cells revealed a good correlation (PBMCs, r = 0.894; expanded NK cells, r = 0.887). Distinct separation between target tumor cells (Daudi, Raji, RPMI8226, U266, U937, and K562 cells) and CAM-stained PBMCs (E:T ratio, 12.5:1 to 50:1) or expanded NK cells (E:T ratio, 0.5 to 4:1) was observed after incubation for 1 or 4 hours. In summary, we successfully developed an effective flow cytometry-based assay for assessing the activity of NK cells in PBMCs and expanded NK cells against K562 cells and various types of hematologic cancer cells.