Natural Killer Cell Activity Test Helps to Suspect Aggressive Natural Killer Cell Leukemia - Diagnostic Challenge.

Aggressive natural killer cell leukemia (ANKL) is a rare disease with an aggressive clinical course. We aimed to assess the clinicopathological characteristics of the difficult to diagnose ANKL. During ten years, nine patients with ANKL were diagnosed. All the patients exhibited aggressive clinical course and underwent the BM study to rule out lymphoma and hemophagocytic lymphohistiocytosis (HLH). BM examination showed varying degrees of infiltration of neoplastic cells, which were mainly positive for CD2, CD56, cytoplasmic CD3 and EBV in situ hybridization. Five BM aspirates showed histiocytic proliferation with active heomphagocytosis. Normal or increased NK cell activity test results were obtained from 3 patients who were available for testing. Four had multiple BM studies until diagnosis. An aggressive clinical course and positive EBV in situ hybridization, often with associated secondary HLH, should raise the suspicion of an ANKL. Conducting additional supplementary tests such as NK cell activity and NK cell proportion would be helpful for the diagnosis of ANKL.

Differences in PD-1 expression on CD8+ T-cells in chronic myeloid leukemia patients according to disease phase and TKI medication.

Malignant cells can increase in number using immune escape mechanisms such as immune checkpoints. In this study, we evaluated the expression of an immune checkpoint programmed death 1 (PD-1) on T-cell subsets in chronic myeloid leukemia (CML). We obtained bone marrow aspirate samples from CML patients and from individuals without evidence of hematologic malignancies (controls). PD-1 expression on T-cell subsets was measured using flow cytometric analysis. PD-1 expression levels on CD8+ T-cells were significantly lower in complete hematologic response (CHR) than in controls, chronic phase, and blast phase (BP). In CML patients receiving imatinib and dasatinib, PD-1 expression levels on CD8+ T-cells were lower than that at diagnosis. PD-1 expression levels on CD8+ T-cells were positively correlated with quantitative levels of the BCR/ABL fusion gene. PD-1 expression levels on CD4+ T-cells were higher in BP than in CHR. PD-1 expression levels on CD4+ T-cells did not differ significantly according to different medications or quantitative BCR/ABL1 fusion gene levels. Low PD-1 expression on CD8+ T-cells might play a role in maintaining CHR in CML patients. Immune monitoring of PD-1 expression on CD8+ T-cells may predict the disease course. In cases of refractory disease or resistance to imatinib or dasatinib, the use of PD-1 inhibitors would be helpful.

Masked monoclonal gammopathy in capillary electrophoresis.

BACKGROUND: Although the method of choice to detect M-protein is electrophoresis on an agarose gel, such gel electrophoresis (GE) is labor-intensive, time-consuming, and not standardized. In contrast to GE, capillary electrophoresis (CE) has some merits because it is automated, fast, and highly reproducible. However, CE results occasionally make the interpretation difficult and require additional confirmatory tests like GE. METHODS: In order to assist a correct reporting of CE results and compatible interpretations between two different electrophoresis methods, we report here two unusual cases of monoclonal gammopathy by a pattern of polyclonal gammopathy upon CE interpretation in patients with end stage renal disease and multiple myeloma. RESULTS: In these cases, serum CE showed the broad bumpy peak in the gamma region. This bumpy peak does not drop completely flat after the reaction with anti-FLC. CONCLUSIONS: Because the plasma cell is a B-cell lineage and plays an important role in adaptive immunity, MG accompanying with PG is not rarely found in plasma cell dyscrasia. If the broad bumpy peak is observed in CE, careful examinations must be done to rule out the hidden M-peak. In our cases, a parallel use of gel-based methods was very helpful as it revealed monoclonal bands.

Increased PD-1 expression of bone marrow T-cells in acute myeloid leukaemia patients after stem cell transplantation, and its association with overall survival.

BACKGROUND: Immune checkpoints are involved in mechanisms by which tumours escape from the host immune system. Our aim was to evaluate acute myeloid leukaemia (AML) patients to determine expression levels of checkpoint molecules according to diagnosis and treatments, and to identify optimal candidates for checkpoint blockade. METHODS: Bone marrow (BM) samples were obtained from 279 AML patients at different disease status and from 23 controls. Flow cytometric analyses of PD-1 and PD-L1/PD-L2 expression were performed. RESULTS: Programmed death-1 (PD-1) expression levels on CD8+ T-cells at AML diagnosis were increased compared to controls. PD-L1 and PD-L2 expression levels on leukaemic cells at diagnosis were significantly higher in secondary AML than in de novo AML. PD-1 levels on CD8+ and CD4+ T-cells after allo-SCT were significantly higher than those at diagnosis and after CTx. PD-1 expression on CD8+ T-cells increased in the acute GVHD group than in the non-GVHD group. The overall survival of patients with high PD-1 expression on CD8+ T-cells was significantly shorter than that of patients with low PD-1 expression. CONCLUSIONS: In conclusion, patients who underwent allo-SCT exhibited high PD-1 expression, suggesting that allo-SCT increases PD-1 expression on T-cells, and the patients with high PD-1 expression on CD8+ T-cells after allo-SCT showed the poor prognosis. For these patients, PD-1 blockade could be an immunotherapeutic strategy.

Mean platelet volume/platelet count ratio in hepatocellular carcinoma.

Mean platelet volume (MPV) has been actively investigated in liver disease such as steatosis, cirrhosis and hepatitis. Recently, MPV/platelet count (PC) ratio has been proposed as a predictor of long-term mortality after myocardial infarction. As PC is known to be decreased in various liver diseases such as cirrhosis, hepatosplenomegaly and malignancy, we planned to evaluate MPV/PC ratio in patients with hepatocellular carcinoma (HCC) in this study. Mean of MPV levels showed significant difference, which were 8.69 fl (range 6.7-12.2 fl) in patients group and 8.02 fl in control group (range 6.7-11.0 fl). In receiver operating characteristic (ROC) curve analysis, the MPV/PC ratio (fl/(10(9)/l)) presented 74.5% of sensitivity and 96.5% of specificity at the criterion > 0.0491 (area under the curve (AUC) = 0.884), while MPV alone showed 57.4% of sensitivity and 81.4% of specificity at the criterion > 8.4 fl. Further studies should evaluate underlying pathogenic mechanisms of MPV/PC ratio difference and various possibilities of this ratio as an indicator of presence of a tumor in HCC.

Laboratory tools for oligo-secretory myeloma diagnosis: capillary electrophoresis versus free light chain assay.

BACKGROUND: To investigate how capillary electrophoresis (CE) works in oligo-secretory myeloma (OSM), we report a case here of OSM using multiple diagnostic methods including gel electrophoresis (GE), CE, and free light chain assay (sFLC). Also, we provide a brief review of laboratory methods to compare their diagnostic utilities in OSM. METHODS: A 72 year-old Korean male suffering from low back pain during the past 6 months was transferred to the department of neurosurgery in order to evaluate abnormal findings in an imaging study, suggesting plasma cell myeloma (PCM) with multiple bone metastasis. CE showed no suspicious M-component; however, it showed increased Kappa components and skewing Kappa/Lambda ratio (K/L). Bone marrow examination revealed plasma cells observed up to 70%, which were compatible with sFLC results. RESULTS: Based on these results, the diagnosis turned out to be OSM with multiple bone metastases. Thereafter, the patient started the first cycle of chemotherapy accompanied by palliative radiation therapy. CONCLUSIONS: In our case, sFLC showed abnormal Kappa and K/L results from both serum and urine specimen. Therefore, it seems to be more sensitive and appropriate than both GE and CE to diagnose OSM.

Immune Checkpoint Programmed Cell Death Protein-1 (PD-1) Expression on Bone Marrow T Cell Subsets in Patients With Plasma Cell Myeloma.

BACKGROUND: Plasma cell myeloma (PCM) is caused by immune dysregulation. We evaluated the expression of immune checkpoint programmed cell death protein-1 (PD-1) on T cell subsets in PCM patients according to disease course and cytogenetic abnormalities. This study aimed to find a target group suitable for therapeutic use of PD-1 blockade in PCM. METHODS: A total of 188 bone marrow (BM) samples from 166 PCM patients and 32 controls were prospectively collected between May 2016 and May 2017. PD-1 expression on BM T cell subsets was measured using flow cytometry. RESULTS: At diagnosis, the median PD-1 expression on CD4+ T cells was 24.6%, which did not significantly differ from that in controls. After stem cell transplantation, PD-1 expression on CD4+ T cells was higher than that at diagnosis (P<0.001), regardless of residual disease. PD-1 expression on CD4+ T cells in patients with residual disease after chemotherapy was significantly higher than that at diagnosis (P=0.001) and after complete remission following chemotherapy (P=0.044). PD-1 expression on CD8+ T cells was higher in PCM patients with cytogenetic abnormalities, including monosomy 13, 1q gain, complex karyotype, and hypodiploidy. CONCLUSIONS: PD-1 blockade might have therapeutic potential in refractory PCM patients after chemotherapy, especially in those with high- or intermediate-risk cytogenetic abnormalities.

Increased expression of immune checkpoint programmed cell death protein-1 (PD-1) on T cell subsets of bone marrow aspirates in patients with B-Lymphoblastic leukemia, especially in relapse and at diagnosis.

BACKGROUND: We analyzed expression profiles of immune checkpoint receptors on T cell subsets and ligands on leukemic blasts in patients with B-lymphoblastic leukemia (B-ALL). METHODS: Total 149 bone marrow (BM) samples obtained from 65 B-ALL patients with four different clinical status (41 at diagnosis, 54 in complete remission [CR], 34 in persistence, and 20 in relapse), and 32 BM control samples were prospectively enrolled. Expression of immune checkpoint receptor (programmed cell death protein-1 [PD-1]) on T cell subsets and ligands (PD-L1, PD-L2) on leukemic blasts was evaluated by flow cytometry, and was compared between patient subgroups. RESULTS: Relapsed patients demonstrated highest PD-1 expression proportion and intensity on CD3+ CD4+ T cells with statistical significance when compared to patients in persistence/CR/BM controls (p = .027/<.001/<.001 and .012/.001/<.001, respectively). Newly diagnosed patients showed significantly lower PD-1 expression proportion on CD3+ CD4+ T cells than relapsed patients (p < .001), but their intensity was not significantly different. Relapsed patients showed significantly higher PD-1 expression proportion and intensity on CD3+ CD8+ T cells than patients in CR/BM controls (p = .022/.045 and .049/.005, respectively), but PD-1 expression status on them were not significantly different between relapsed and newly diagnosed patients. PD-L1/L2 expression on leukemic blasts was not significantly different between patient subgroups. CONCLUSIONS: In BM aspirates from B-ALL patients, PD-1 expression on T-cell subsets is increased at diagnosis, and to a greater extent, at relapse. These data suggest the potential usefulness of PD-1 blockade in the treatment of B-ALL, particularly at relapse.

The Incidence and Immunophenotypic and Genetic Features of JL1 Expressing Cells and the Therapeutic Potential of an Anti-JL1 Antibody in De Novo Pediatric Acute Leukemias.

BACKGROUND: JL1 is a newly identified CD43 epitope that specifically recognizes leukemic cells. We analyzed the incidence of JL1 expression and compared the clinical, immunophenotypic, and genetic characteristics of de novo pediatric acute leukemia patients with respect to JL1 expression status to determine the therapeutic potential of an anti-JL1 antibody. METHODS: Seventy-eight patients with pediatric acute leukemia (52 with ALL, 26 with AML) diagnosed between December 2014 and January 2016 were enrolled prospectively. Flow cytometry for JL1 expression was performed at diagnosis. Clinical, immunophenotypic, and genetic characteristics were compared with respect to JL1 expression status by the Student t-test/Mann-Whitney U test and chi-square test/Fisher's exact test. RESULTS: The incidence of JL1 expression was 76.9% and 84.6% in ALL and AML patients, respectively. ALL patients with JL1 expression showed higher CD10 and cytoplasmic IgM expressions than those without JL1 expression (P=0.022 and 0.003, respectively) and were associated with TCF3-PBX1 and KMT2A-MLLT1 translocations. AML patients with JL1 expression showed higher CD13 and lower CD65 and CD15 expressions than those without JL1 expression (P=0.013, 0.007, and 0.024, respectively) and were associated with RUNX1-RUNX1T1, PML-RARA, and CBFB-MYH11 translocations. The JL1 expression incidence did not differ between ALL and AML, and the JL1 expression status did not affect prognosis. CONCLUSIONS: Our findings support the potential therapeutic role of anti-JL1 monoclonal antibodies; JL1 expression was associated with specific immunophenotypes and genetic abnormalities. Future studies should examine the prognostic impact of JL1 expression in pediatric acute leukemias.

Frequency and Clinicopathologic Features of RUNX1 Mutations in Patients With Acute Myeloid Leukemia Not Otherwise Specified.

OBJECTIVES: To evaluate the frequency and clinicopathologic characteristics of RUNX1 mutations, focusing on patients with acute myeloid leukemia not otherwise specified (AML NOS). METHODS: Diagnostic samples from 219 patients with AML NOS were analyzed for RUNX1 mutations using standard polymerase chain reaction and direct sequencing. RESULTS: Thirty-one RUNX1 mutations were detected in 33 (15.1%) patients. Mutations clustered in the Runt homology (61.3%) and transactivation domains (25.8%). Frameshift mutations were most common (51.6%), followed by missense (41.9%) and nonsense (6.5%) mutations. Patients with RUNX1 mutations had a lower platelet count (P = .013) and shorter relapse-free survival (P = .045) than those without. The presence of RUNX1 and NPM1 or CEBPA mutations was mutually exclusive. A literature review, including our study, showed that patients with RUNX1 mutations were associated with intermediate risk; coexisting mutations such as FLT3-ITD, ASXL1, TET2, and DNMT3A; and a relatively cytogenetic heterogeneity. CONCLUSIONS: Our findings strengthen previous data concerning RUNX1 mutations in AML and support the notion that RUNX1 mutational status should be integrated into a diagnostic workup of AML, particularly for AML NOS or an intermediate-risk group.

Evaluation of IL-2, IL-10, IL-17 and IP-10 as potent discriminative markers for active tuberculosis among pulmonary tuberculosis suspects.

INTRODUCTION: Although interferon gamma release assays (IGRAs) are useful for specifically detecting Mycobacterium tuberculosis, they are limited by their inability to differentiate between active tuberculosis (active TB), latent tuberculosis infection (LTBI), and patients with prior TB infection. The purpose of this study was to rapidly and accurately identify active TB patients among patients with suspected respiratory TB by combining interferon-gamma (IFN-γ) with additional cytokines. MATERIALS AND METHODS: The present study was conducted on patients who required TB screening to discern active TB due to respiratory complaints. Among all patients screened, 80 who tested positive in the QunatiFERON TB GOLD in-tube assay (QFT-GIT) were retrospectively selected and divided into the active TB group (n = 40) and the non-active TB group (n = 40). Supernatants from the Nil tube and TB antigen (Ag)-stimulated tube from the QFT-GIT test were used. Interleukin (IL)-2, IL-10, IL-17, and IP-10 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In comparing the differences significant differences in TB Ag tube response of IL-17 and IP-10, and 'TB Ag - Nil' response of IL-10 and IP-10 were evident between the active TB and non-active TB groups. From the calculation of diagnostic performance for differentiating active TB patients from QFT-positive patients, among the measured values, the value of IL-17 TB Ag tube with the cut-off value set to ≤0.82 (AUC 0.742) showed sensitivity, specificity, positive predictive value, and negative predictive value of 69.2%, 97.5%, 96.4%, and 76.5%, respectively. DISCUSSION: Among TB suspects, IL-17 TB Ag response had the highest ability to distinguish active TB followed by IP-10 TBAg-Nil response and the IL-17/IFN-γ-TB Ag response combination improved the detection response. Differentiating active TB is best done using IL-17 as a biomarker, with IP-10 and IL-10 being potential useful.

A case series of autoimmune diseases accompanied by incidentally diagnosed monoclonal gammopathy: is there a link between the two diseases?

AIM: Although the etiology of plasma cell dyscrasia is poorly understood, there is evidence for immune dysregulation or sustained immune stimulation playing a pivotal role in the pathogenesis of these diseases, including chronic infection and autoimmune disorders. In this study, we report four autoimmune disease cases where monoclonal gammopathy (MG) was incidentally found during follow-up. METHODS: We retrospectively reviewed the medical charts and laboratory test results in the following four cases: neuromyelitis optica, Kikuchi disease, Sjögren syndrome and ankylosing spondylosis. RESULTS: The four patients were older than 55 years and the male-to-female ratio was 2 : 2. The autoimmune disease in each case developed differently because two patients had coincidental detection of MG, whereas MG was detected 2 years and 10 years after diagnosis in the other two patients. The amount of M-components in the blood for two cases was ≤ 1 g/dL. For the other two subjects, M-components were ≥ 3 g/dL. CONCLUSION: A high prevalence of MG of undetermined significance (MGUS) has been noted in a series of patients with immune disorders, suggesting a possible association with MG. Further studies should focus on determining how MG relates to various clinical information and laboratory parameters, such as disease duration, disease activity and higher sedimentation rate. In the future, we also need to identify which stimuli, such as cytokine types and levels, can induce lymphocyte clonal transformation and the production of monoclonal antibodies.

Comparison of two molecular methods for detecting toxigenic Clostridium difficile.

BACKGROUND: Clostridium difficile is one of the most common causes of nosocomial diarrhea, and diagnostic methods for detecting C. difficile infection have shifted from conventional to more recent molecular techniques. This study aimed to compare the performance of two molecular assays (Meridian Illumigene™ and AdvanSure CD real-time PCR) in detecting C. difficile using a toxigenic culture as a reference standard. MATERIALS AND METHODS: This study was conducted at Kyung Hee University Hospital, a tertiary university teaching hospital in Seoul, Korea, from July 2010 to February 2011. The study used 203 fresh diarrheal stools. All fecal specimens were immediately tested by culture and the VIDAS C. difficile toxin A & B assay using an automated VIDAS immunoanalyzer. The remainder was stored at -70°C until required for AdvanSure CD real-time polymerase chain reaction and Illumigene™. The alcohol shock procedure was then performed. Aliquots were inoculated directly on C. difficile-selective agar and blood agar and then incubated in an anaerobic jar for 48 h at 35°C. The Rapid ID 32 A test was used for specifying colonies on plates. The AdvanSure CD real-time PCR was used to detect the tcdA and tcdB gene, and PCR Illumigene™ kits were used to detect the tcdA gene of the pathogenicity locus (PaLoc) harboring toxigenic C. difficile. RESULTS: Of 203 clinical samples, 197 showed identical results between the two molecular assays, with a concordance rate of 97.0%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: Illumigene: 92.3, 99.4, 96.0, and 98.9, respectively; AdvanSure CD real-time PCR: 84.6, 98.3, 88.0, and 97.8, respectively. CONCLUSIONS: Both molecular assays demonstrated good sensitivity and specificity. Additionally, both molecular assays showed comparable results to those of a toxigenic culture, albeit with a slight decrease in test sensitivity and specificity.

Interpretation and clinical significance of small monoclonal peaks in capillary electrophoresis.

BACKGROUND: Although the testing mechanism and interpretation criteria for capillary electrophoresis differ from those for gel-based electrophoresis, there are not that many reports on the efficacy of capillary electrophoresis. MATERIALS AND METHODS: We performed a retrospective analysis, using the Laboratory Information System (LIS) to review a total of 163 capillary electrophoresis results from 117 different patients treated in our hospital between March and August 2012. Capillary electrophoresis was performed on capillary2 (Sebia, Lysse, France). RESULTS: Among the patients' group, 4 patients presented very small M-peaks in capillary electrophoresis. By using the zoom function in capillary electrophoresis, two of them were confirmed to have monoclonality, but the remaining two required reconfirmation in gel electrophoresis, leading to confirmation of a discrete monoclonal band. CONCLUSION: In this study, we found that small peaks in capillary electrophoresis accompanying a skewed K/L ratio deserve particular attention as they can grow into larger peaks within a few months. We suggest that any trivial M-peak in capillary electrophoresis should not be overlooked and that a combination of platform tests such as gel electrophoresis or FLC assay be implemented in order to confirm monoclonality.

Factors influencing discordant results of the QuantiFERON-TB Gold In-tube test in patients with active TB.

OBJECTIVES: Indeterminate or negative results from the QuantiFERON-TB Gold In-tube test (QFT-GIT) for TB-confirmed patients indicate the lower sensitivity of this method. The aim of this study was to determine the factors associated with indeterminate and negative QFT-GIT results in active TB patients. METHODS: We analyzed retrospectively the laboratory and clinical data of patients diagnosed with TB between December 2009 and April 2012 at a tertiary university hospital in Seoul, Korea. RESULTS: Among 1301 patients who underwent QFT-GIT, TB-PCR and TB-culture, 168 (12.9%), those with positive TB-PCR or TB-culture were diagnosed with TB. Thirty-nine (23.2%) had indeterminate or negative results by QFT-GIT assay, which did not correlate with positive results of TB-PCR or TB-culture. These patients were older, had lower lymphocyte, total protein and albumin levels, and showed significantly higher CRP levels than the positive group. Multivariate logistic regression analysis showed that the probability of indeterminate and negative QFT-GIT results increased as CRP (odd ratio, 1.069; 95% CI, 1.013-1.127; P = 0.014) or age (1.030, 1.005-1.056, 0.02) increases. CONCLUSIONS: When levels of markers of inflammation, such as CRP, are high or the patient is older, QFT-GIT results should be interpreted carefully and correlated with additional tests for TB.