The Role of Granzyme B Containing Cells in the Progression of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Lung inflammation plays a vital role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the characteristics of the inflammatory process remain unclear. There is growing interest in the role of granzyme B (GzmB) because CD8+ T cells can induce apoptosis of target cells by releasing GzmB, which in turn may cause tissue injury and remodeling. However, GzmB is also expressed by regulatory cells, which are able to suppress CD8+ T cell. The role of GzmB+ cells needs to be defined in COPD. METHODS: GzmB+ and CD8+ cells on alveolar wall of surgically resected lungs of microscopically classified 12 nonsmoking control, 12 panlobular emphysema (PLE) and 30 centrilobular emphysema (CLE) subjects were localized by immunohistochemical method. Positively stained cells on alveolar wall were counted and length of corresponding alveolar wall was measured. The results were expressed as mean number of positively stained cells per mm of alveolar wall in each subject. RESULTS: The number of GzmB+ and CD8+ cells on alveolar wall of CLE was greater than that of control or PLE subjects (p<0.05 and p<0.001, respectively). There was a positive relationship between the number of alveolar GzmB+ cells and forced expiratory volume in 1 second (FEV1) (r=0.610, p=0.003) in CLE subjects. The number of alveolar GzmB+ cells progressively decreased with decline of FEV1. CONCLUSION: Our finding that number of alveolar GzmB+ cells was associated with FEV1 suggests that GzmB+ cells might have protective role in the progression of lung destruction and airflow limitation in CLE, which is the predominant emphysema subtype of COPD.

Prognostic impact of germinal center B-cell-like and non-germinal center B-cell-like subtypes of bone marrow involvement in patients with diffuse large B-cell lymphoma treated with R-CHOP.

The prognostic significances of the germinal center B-cell-like (GCB) and non-germinal center B-cell-like (non-GCB) types of diffuse large B-cell lymphoma (DLBCL) have been reported to be different. We analyzed the effect of the cell of origin (COO) of bone marrow (BM) involvement in patients with DLBCL who were treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in a single institute.The subtype of BM involvement was evaluated in 633 patients who were diagnosed with primary DLBCL and had been treated with R-CHOP. BM trephine biopsies were analyzed, and immunohistochemical staining of CD20, CD79a, and CD3 was performed. Additional staining of CD10, Bcl-6, and MUM1 was performed to determine the COO based on a previously reported algorithm.BM involvement was present in 81 patients (12.8%). Among them, 30 patients (37.0%) had GCB-type BM involvement and 51 (63.0%) showed non-GCB-type involvement. Kaplan-Meier survival analysis showed that the non-GCB type had the worst progression-free survival (PFS) and overall survival (OS) (P <.001). In multivariate analysis controlled for the International Prognostic Index (IPI) score, non-GCB type was an independent predictor of PFS (P <.004) and OS (P =.042), whereas GCB type was not a prognostic factor independent of the IPI score.Further prognostication based on the COO of BM involvement is a useful indicator of PFS, independent of IPI score. Accurate staging based on the COO should be included in the examination of BM in DLBCL.

Blast Percentage of Bone Marrow Aspirate on Day 14 of Induction Chemotherapy Predicts Adult Acute Lymphoblastic Leukemia Treatment Outcomes.

The prognosis of adult acute lymphoblastic leukemia is much worse than that of pediatric acute lymphoblastic leukemia, even when patients achieve complete remission. Early response to treatment can be an important alternative indicator of treatment outcomes. The purpose of our current study was to identify the prognostic value of the blast percentage of the induction interim bone marrow, which might predict relapse-free survival and overall survival in patients with adult acute lymphoblastic leukemia. A retrospective analysis was performed on 80 adult patients diagnosed with Philadelphia chromosome-negative acute lymphoblastic leukemia from 1994 to 2011. Complete remission was observed in 75 (93.8%) patients after induction chemotherapy. On multivariate analysis, a reduction of blasts to a level of 5% or less in the induction interim bone marrow and CD20 positivity were significant prognostic predictors of relapse-free survival (hazard ratio, HR = 2.88, p = 0.006, and HR = 2.67, p = 0.010) and overall survival (HR = 2.10, p = 0.033, and HR = 2.39, p = 0.013). The blast percentage of the induction interim bone marrow may be a useful prognostic factor to predict outcome.

Assessment of bone marrow involvement in patients with lymphoma: report on a consensus meeting of the Korean Society of Hematology Lymphoma Working Party.

In September 2011, the Korean Society of Hematology Lymphoma Working Party held a nationwide conference to establish a consensus for assessing bone marrow (BM) involvement in patients with lymphoma. At this conference, many clinicians, hematopathologists, and diagnostic hematologists discussed various topics for a uniform consensus in the evaluation process to determine whether the BM is involved. Now that the discussion has matured sufficiently to be published, we herein describe the consensus reached and limitations in current methods for assessing BM involvement in patients with lymphoma.

Clinical Application of Revised Laboratory Classification Criteria for Antiphospholipid Antibody Syndrome: Is the Follow-Up Interval of 12 Weeks Instead of 6 Weeks Significantly Useful?

Background. According to revised classification criteria of true antiphospholipid antibody syndrome, at least one of three antiphospholipid antibodies should be present on two or more occasions at least 12 weeks apart. However, it can be inconvenient to perform follow-up tests with interval of 12 weeks. We investigated clinical application of follow-up tests with interval of 12 weeks. Method. Totals of 67, 199, and 332 patients tested positive initially for the lupus anticoagulants confirm, the anti-ß 2 glycoprotein-I antibody, and the anti-cardiolipin antibody test, respectively, from Jan 2007 to Jul 2009. We investigated clinical symptoms of patients, follow-up interval, and results of each test. Results. Among patients with initial test positive, 1.5%-8.5% were subjected to follow-up tests at interval of more than 12 weeks. Among 25 patients with negative conversion in tests, patients with interval of more than 12 weeks showed clinical symptom positivity of 33.3%, which was higher than that of 12.5% with 6-12 weeks. Among 34 patients with persistent test positive, clinical symptoms positivity trended to be more evident in patients at interval of 6-12 weeks (47.4% versus 26.7%, P = 0.191) than more than 12 weeks. Conclusion. Less than 10% of patients with initial test positive had follow-up tests at interval of more than 12 weeks and the patients with persistent test positive at interval of more than 12 weeks showed trends toward having lower clinical symptoms than 6-12 weeks. More research is needed focused on the evidence that follow-up test at interval of more than 12 weeks should be performed instead of 6 weeks.

Prognostic relevance of the Ki-67 proliferation index in patients with mantle cell lymphoma.

BACKGROUND: A high Ki-67 proliferation index (PI) in neoplastic cells is associated with poor survival in mantle cell lymphoma (MCL). We aimed to determine the cut-off values for the Ki-67 PI as a prognostic factor in MCL according to bone marrow findings. METHODS: Immunohistochemical (IHC) staining for Ki-67 was performed on formalin-fixed paraffin-embedded biopsy tissues from 56 patients with MCL. Patients were grouped based on their Ki-67 PI values. Survival analyses were carried out and the cut-off value for the Ki-67 PI was determined. RESULTS: Of the 56 patients, 39 (69.6%) showed bone marrow involvement of MCL; 21 of these patients had leukemic manifestations at the time of diagnosis. The results of the Ki-67 IHC staining were as follows: ≤10% in 22 patients, 11-20% in 14 patients, 21-30% in 3 patients, 31-40% in 4 patients, 41-50% in 4 patients, and >50% in 9 patients. A cut-off value of 20% revealed significantly different survival rates with mean survival times of 69.8 months (Ki-67 PI≤20%) and 47.9 months (Ki-67 PI>20%), irrespective of bone marrow findings (P=0.034). Clinical outcomes did not differ, regardless of bone marrow findings. However, in cases with bone marrow involvement, the Ki-67 cut-off value of 30% for overall survival was required to yield statistical significance (P=0.033). CONCLUSION: The 20% cut-off value for the Ki-67 PI was clinically meaningful, regardless of bone marrow involvement of MCL. For patients with bone marrow involvement, the statistically significant cut-off value increased to 30%.

Correlation of NPM1 Type A Mutation Burden With Clinical Status and Outcomes in Acute Myeloid Leukemia Patients With Mutated NPM1 Type A.

BACKGROUND: Nucleophosmin gene (NPM1) mutation may be a good molecular marker for assessing the clinical status and predicting the outcomes in AML patients. We evaluated the applicability of NPM1 type A mutation (NPM1-mutA) quantitation for this purpose. METHODS: Twenty-seven AML patients with normal karyotype but bearing the mutated NPM1 were enrolled in the study, and real-time quantitative PCR of NPM1-mutA was performed on 93 bone marrow (BM) samples (27 samples at diagnosis and 56 at follow-up). The NPM1-mutA allele burdens (represented as the NPM1-mutA/Abelson gene (ABL) ratio) at diagnosis and at follow-up were compared. RESULTS: The median NPM1-mutA/ABL ratio was 1.3287 at diagnosis and 0.092 at 28 days after chemotherapy, corresponding to a median log10 reduction of 1.7061. Significant correlations were observed between BM blast counts and NPM1-mutA quantitation results measured at diagnosis (γ=0.5885, P=0.0012) and after chemotherapy (γ=0.5106, P=0.0065). Total 16 patients achieved morphologic complete remission at 28 days after chemotherapy, and 14 (87.5%) patients showed a >3 log10 reduction of the NPM1-mutA/ABL ratio. The NPM1-mutA allele was detected in each of five patients who had relapsed, giving a median increase of 0.91-fold of the NPM1-mutA/ABL ratio at relapse over that at diagnosis. CONCLUSIONS: The NPM1-mutA quantitation results corresponded to BM assessment results with high stability at relapse, and could predict patient outcomes. Quantitation of the NPM1-mutA burden at follow-up would be useful in the management of AML patients harboring this gene mutation.

Genetic Characteristics of Polycythemia Vera and Essential Thrombocythemia in Korean Patients.

BACKGROUND: Despite recent advances in the investigation of myeloproliferative neoplasms (MPN), the impact of genetic heterogeneity on its molecular pathogenesis has not been fully elucidated. Thus, in this study, we aim to characterize the genetic complexity in Korean patients with polycythemia vera (PV) and essential thrombocythemia (ET). METHODS: We conducted association studies using 84 single-nucleotide polymorphisms (SNPs) in 229 patients (96 with PV and 133 with ET) and 170 controls. Further, whole-genome sequencing was performed in six patients (two with JAK2 V617F and four with wild-type JAK2), and putative somatic mutations were validated in a further 69 ET patients. Clinical and laboratory characteristics were also analyzed. RESULTS: Several germline SNPs and the 46 haplotype were significantly associated with PV and ET. Three somatic mutations in MPDZ, IQCH, and CALR genes were selected and validated. The frequency of the CALR mutation was 58.0% (40/69) in ET patients, who did not carry JAK2/MPL mutations. Moreover, compared with JAK2 V617F-positive patients, those with CALR mutations showed lower hemoglobin and hematocrit levels (P = 0.004 and P = 0.002, respectively), higher platelet counts (P =0.008), and a lower frequency of cytoreductive therapy (P = 0.014). CONCLUSION: This study was the first comprehensive investigation of the genetic characteristics of Korean patients with PV and ET. We found that somatic mutations and the 46 haplotype contribute to PV and ET pathogenesis in Korean patients.

Establishing a Population-Based HLA-Antibody Panel for Flow Cytometric Monitoring of Chimerism in HLA-Haploidentical Stem Cell Transplantation.

Determining the chimerism in stem cell transplantation (SCT) is important in the monitoring of engraftment. Conventional monitoring methods such as short tandem repeat polymerase chain reaction (STR-PCR) are labor intensive and difficult in showing the dynamics of cell subpopulations. In HLA-haploidentical SCT, flow cytometric analysis using anti-HLA antibody for the mismatched HLA can be useful in observing changes of cell subpopulations and determining chimerism. We designed a specific panel of HLA antibody reagents for the Korean population, and verified its clinical application in flow cytometric monitoring of chimerism after haploidentical stem cell transplantation. A total of 12 anti-HLA-A, -B-antibodies were selected, which could cover 82.5% of HLA-A and 16.5% of HLA-B in Korean population. This HLA panel distinguished donor and recipient cells in 22 of 23 HLA-haploidentical SCT cases. In one case, the patient had HLA-A*02/A*24, B*48/B*61 while the donor had HLA-A*02/A*33, B*44/B*48. The donor type HLA-B*44(+) and CD3(+) T cells, and HLA-B*44(+) and CD56(+) NK cells were seen at day 14 and day 8, respectively. Increased HLA-B*44(+) cells throughout the study period indicated the engraftment of donor stem cells. We were able to design a population specific panel of HLA-antibodies, and verified that flow cytometric analysis using HLA antibody for the detection of chimerism in HLA-haploidentical SCT was a simple and sensitive monitoring technique. This method allowed us to observe the dynamic changes in cell subpopulations after HLA-haploidentical SCT. Flow cytometric analysis can be considered as a strong tool for the monitoring of engraftment in HLA-haploidentical SCT.

Reconstitution of lymphocyte subpopulations after hematopoietic stem cell transplantation: comparison of hematologic malignancies and donor types in event-free patients.

The reconstitution of different immunocyte subsets after hematopoietic stem cell transplantation (HSCT), follows different timelines. We prospectively investigated changes in lymphocyte subsets after HSCT and their associations with primary diagnosis, conditioning regimen, and HSCT type in event-free patients. A total of 95 patients (48 with acute myeloid leukemia, 22 with acute lymphoid leukemia, and 25 with myelodysplastic syndrome) who underwent allogeneic HSCT (34 sibling matched, 37 unrelated matched, and 24 haploidentical HSCT) but did not experience any events such as relapse or death were enrolled in this study. Lymphocyte subpopulations (T cells, helper/inducer T cells, cytotoxic/suppressor T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK-T cells, and B cells) were quantified by flow cytometry of peripheral blood from recipients 7 days before and 1, 2, 3, 6, and 12 months after HSCT. Leukocyte counts recovered within 1 month after HSCT. However, the number of T and B lymphocytes recovered at 2 months after HSCT. NK cell counts recovered shortly after haploidentical HSCT. However, T lymphocytes and their subpopulations showed delayed recovery after haploidentical HSCT. Lymphocyte subsets showed different sequential patterns according to HSCT type but no differences were seen according to primary diagnosis or conditioning regimen.

Long-term follow-up of imatinib plus combination chemotherapy in patients with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia.

The effects of imatinib plus chemotherapy were assessed in 87 patients with newly diagnosed Philadelphia chromosome-positive (Ph(+) ) acute lymphoblastic leukemia (ALL). Imatinib was administered continuously, starting from the eighth day of remission induction chemotherapy, then through five courses of consolidation or until allogeneic hematopoietic cell transplantation (HCT). Patients who were not transplanted were maintained on imatinib for 2 years. Eighty-two patients (94.3%) achieved complete remission (CR). Among these 82 CR patients, 40 experienced recurrence of leukemia. The 5-year relapse free survival (RFS) rate and overall survival (OS) rates were 39.0% and 33.4%, respectively. In total, 56 patients underwent allogeneic HCT in first CR. The 5-year cumulative incidence of relapse and OS rate of them were 59.1% and 52.6%, respectively. Six of seven patients who were maintained on imatinib after completion of consolidation relapsed and the median time of RFS was 40.7 months. In total patient, cumulative molecular CR rate was 88.5% and median time of molecular CR duration was 13 months. Initial imatinib dose intensity was significantly associated with median CR duration (P < 0.0001), and overall survival (P = 0.002). During the initial phase of treatment of patients with Ph(+) ALL, it is important to maintain imatinib dose intensity.

JAK2 V617F, MPL, and CALR Mutations in Korean Patients with Essential Thrombocythemia and Primary Myelofibrosis.

Mutations in the calreticulin gene, CALR, have recently been discovered in subsets of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). We investigated Korean patients with ET and PMF to determine the prevalence, and clinical and laboratory correlations of CALR/JAK2/MPL mutations. Among 84 ET patients, CALR mutations were detected in 23 (27.4%) and were associated with higher platelet counts (P=0.006) and lower leukocyte counts (P=0.035) than the JAK2 V617F mutation. Among 50 PMF patients, CALR mutations were detected in 11 (22.0%) and were also associated with higher platelet counts (P=0.035) and trended to a lower rate of cytogenetic abnormalities (P=0.059) than the JAK2 V617F mutation. By multivariate analysis, triple-negative status was associated with shorter overall survival (HR, 7.0; 95% CI, 1.6-31.1, P=0.01) and leukemia-free survival (HR, 6.3; 95% CI, 1.8-22.0, P=0.004) in patients with PMF. The type 1 mutation was the most common (61.1%) type among all patients with CALR mutations, and tended toward statistical predominance in PMF patients. All 3 mutations were mutually exclusive and were never detected in patients with other myeloid neoplasms showing thrombocytosis. CALR mutations characterize a distinct group of Korean ET and PMF patients. Triple-negative PMF patients in particular have an unfavorable prognosis, which supports the idea that triple-negative PMF is a molecularly high-risk disease.

Nilotinib combined with multiagent chemotherapy for newly diagnosed Philadelphia-positive acute lymphoblastic leukemia.

We investigated the effects of nilotinib plus multiagent chemotherapy, followed by consolidation/maintenance or allogeneic hematopoietic cell transplantation (allo-HCT) for adult patients with newly diagnosed Philadelphia-positive (Ph-pos) acute lymphoblastic leukemia (ALL). Study subjects received induction treatment that comprised concurrent vincristine, daunorubicin, prednisolone, and nilotinib. After achieving complete hematologic remission (HCR), subjects received either 5 courses of consolidation, followed by 2-year maintenance with nilotinib, or allo-HCT. Minimal residual disease (MRD) was assessed at HCR, and every 3 months thereafter. The molecular responses (MRs) were defined as MR3 for BCR-ABL1/G6PDH ratios ≤10(-3) and MR5 for ratios <10(-5). Ninety evaluable subjects, ages 17 to 71 years, were enrolled in 17 centers. The HCR rate was 91%; 57 subjects received allo-HCT. The cumulative MR5 rate was 94%; the 2-year hematologic relapse-free survival (HRFS) rate was 72% for 82 subjects that achieved HCR, and the 2-year overall survival rate was 72%. Subjects that failed to achieve MR3 or MR5 were 9.1 times (P = .004) or 6.3 times (P = .001) more prone to hematologic relapse, respectively, than those that achieved MR3 or MR5. MRD statuses just before allo-HCT and at 3 months after allo-HCT were predictive of 2-year HRFS. Adverse events occurred mainly during induction, and most were reversible with dose reduction or transient interruption of nilotinib. The combination of nilotinib with high-dose cytotoxic drugs was feasible, and it effectively achieved high cumulative complete molecular remission and HRFS rates. The MRD status at early postremission time was predictive of the HRFS. This trial was registered at www.clinicaltrials.gov as #NCT00844298.

Preferential occurrence of spliceosome mutations in acute myeloid leukemia with preceding myelodysplastic syndrome and/or myelodysplasia morphology.

Spliceosome mutations are associated with myelodysplasia. Here, we aimed to evaluate the frequency and clinical associations of these mutations in 204 patients with acute myeloid leukemia with myelodysplasia-related changes (AML with MRC) and 37 with therapy-related AML (t-AML). The frequency of mutation-positive patients was 17.0%, including U2AF1 (8.3%), SRSF2 (5.8%) and SF3B1 (2.9%). Mutations were detected almost exclusively in patients with AML with MRC, especially in cases with a preceding myelodysplastic syndrome (MDS) history or myelodysplastic morphology. By contrast, mutations were rare in patients with only MDS-related cytogenetics or t-AML. The presence of a mutation had no impact on survival. In a paired analysis, 16.7% of mutation-negative patients in the MDS phase acquired mutations during leukemogenesis. Our observations highlight the preponderance of spliceosome mutations within a specific AML subgroup with myelodysplasia, and suggest that these mutations might contribute pathologically to leukemogenesis in such patients.

Immunophenotypic markers in adult acute lymphoblastic leukemia: the prognostic significance of CD20 and TdT expression.

BACKGROUND: Efforts to overcome poor outcomes in patients with adult acute lymphoblastic leukemia (ALL) have focused on combining new therapeutic agents targeting immunophenotypic markers (IPMs) with classical cytotoxic agents; therefore, it is important to evaluate the clinical significance of IPMs. METHODS: Baseline characteristics and clinical outcomes of patients with adult ALL were retrospectively analyzed. The percentage of blasts expressing IPMs at diagnosis was measured by multicolor flow cytometry analysis. Samples in which ≥20% of blasts expressed an IPM were considered positive. RESULTS: Among the total patient population (N=230), almost all (92%) were in first or second hematological complete remission (HCR) and 54% received allogeneic hematopoietic cell transplant (allo-HCT). Five-year hematologic relapse-free survival (HRFS) and overall survival (OS) rates were 36% and 39%, respectively, and 45.6% and 80.5% of patients were positive for the IPMs CD20 and terminal deoxynucleotidyl transferase (TdT), respectively. Expression of CD20, CD13, CD34, and TdT was associated with HRFS rate, and expression of CD20 and CD13 was associated with OS rate, as was the performance of allo-HCT. In multivariate analysis, positivity for CD20 (HRFS: hazard ratio [HR], 2.21, P<0.001; OS: HR, 1.63, P=0.015) and negativity for TdT (HRFS: HR, 2.30, P=0.001) were both significantly associated with outcomes. When patients were categorized into three subgroups according to positivity for CD20 and TdT, there were significant differences in HRFS and OS among the subgroups. CONCLUSION: Positivity for CD20 and TdT expression and clinical risk group were prognostic factors in adult ALL.

CD34 and p53 immunohistochemical stains differentiate hypocellular myelodysplastic syndrome (hMDS) from aplastic anemia and a CD34 immunohistochemical stain provides useful survival information for hMDS.

BACKGROUND: The presence of significant dysplasia in bone marrow (BM) aspirates helps to distinguish between hypocellular myelodysplastic syndrome (hMDS) and aplastic anemia (AA). Occasionally, diluted BM aspirates make it difficult to recognize dysplastic changes and can also negatively affect the detection of cytogenetic abnormalities in hMDS. We evaluated the usefulness of CD34 and p53 immunoreactivity for discriminating between hMDS and AA and for estimating survival outcomes in hMDS patients. METHODS: BM clot section (BMC) or BM biopsy (BMB) specimens were obtained from 64 hMDS/AA patients (33 with hMDS and 31 with AA) and seven controls. Immunohistochemical (IHC) staining for CD34 and p53 was performed by using the EnVision detection system (Dako, Denmark). We compared the results of IHC staining, BM findings, and chromosomal analyses, and determined overall survival outcomes. RESULTS: The number of CD34- and p53-positive BM cells was higher among the patients with hMDS than among the patients with AA (P<0.001 and P=0.001, respectively). hMDS patients with increased CD34-positive cells had significantly poorer survival outcomes compared with those with normal number of CD34-positive cells (P=0.013). CONCLUSIONS: CD34 and p53 IHC stains of BMC or BMB provide useful information for differentiating between hMDS and AA. CD34 IHC staining of BMC or BMB also provides useful information for estimating survival outcomes in hMDS patients.