Soluble NKG2D Ligands Are Potential Biomarkers and Sentinels of Immune-Mediated Bone Marrow Injury in Bone Marrow Failure Syndromes.

Immune-mediated processes are considered important in the pathogenesis of bone marrow failure syndromes (BFS). We previously reported that natural killer group 2D (NKG2D) ligands were expressed on pathological blood cells of patients with BFS and that NKG2D immunity may be involved in bone marrow failure. In addition to membranous NKG2D ligands on the cell surface, soluble NKG2D ligands can exist in plasma. We therefore examined the relationship between soluble NKG2D ligands and blood cell counts in 86 patients with BFS, including aplastic anemia, myelodysplastic syndrome with single lineage dysplasia, and paroxysmal nocturnal hemoglobinuria. Approximately half of the BFS patients were positive for soluble NKG2D ligands in the plasma by enzyme-linked immunosorbent assay, and soluble NKG2D ligand-positive BFS patients exhibited severe cytopenia regardless of membranous NKG2D ligand expression. In vitroanalyses demonstrated that soluble ULBP1, an NKG2D ligand, down-regulated NKG2D receptors on CD2-positive cells in peripheral blood. Moreover, soluble ULBP1 attenuated the cytotoxic effects of peripheral blood mononuclear cells on K562, which express membranous ULBP1. Our results suggest that soluble NKG2D ligands can be easy-to-measure biomarkers for the prediction of activity of immune-meditated bone marrow injury in BFS and that soluble NKG2D ligands suppress redundant immune-mediated bone marrow injury.

Visualization of soluble carbohydrate distribution in apple fruit flesh utilizing MALDI-TOF MS imaging.

To confirm availability of Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry imaging (MSI) for visualizing distribution of soluble carbohydrates in apple (Malus domestica) fruits a horizontal fruit flesh specimen was cut from a matured 'Fuji' fruit, mounted on a glass slide, lyophilized and then ion intensities of individual soluble carbohydrates were probed around the specimen using a MALDI-TOF MSI apparatus automatically. Contents of soluble carbohydrates in adjacent tissue of the same fruit were also determined using HPLC to compare the distribution of individual carbohydrate based on the ion intensities from MALDI-TOF MSI with those from HPLC. Positive correlation (P < 0.001, R2 > 0.95) was confirmed between the concentration of each standard carbohydrate and the relative ion intensity of MALDI-TOF mass spectrometry (MS), and thus it seems possible to use the ion intensity of MALDI-TOF MS for determining the relative concentration of carbohydrates in a sample. Singly charged ions attached with a potassium ion only were detected from the apple fruit specimen when DHB was used as a matrix for MALDI-TOF MSI. Graded increase of sucrose content from center to cortex side of the fruit flesh was confirmed by both MALDI-TOF MSI and HPLC. When pseudo color images on the distribution of individual carbohydrates based on the results from MALDI-TOF MSI were compared with the content of carbohydrates in the adjacent 16 tissue blocks quantified using HPLC, strong (P < 0.001, R2 = 0.6222) and weak (P < 0.10, R2 = 0.2123) correlation was confirmed between the brightness and the content of sucrose and sorbitol, respectively. These facts indicate that distribution of sucrose and sorbitol in apple fruit tissue can be visualized using MALDI-TOF MSI. Thus, MALDI-TOF MSI will be useful for examining carbohydrate metabolism during the maturing of apple fruit.

Expression of CD56 is an unfavorable prognostic factor for acute promyelocytic leukemia with higher initial white blood cell counts.

Expression of CD56 has recently been introduced as one of the adverse prognostic factors in acute promyelocytic leukemia (APL). However, the clinical significance of CD56 antigen in APL has not been well elucidated. We assessed the clinical significance of CD56 antigen in 239 APL patients prospectively treated with all-trans retinoic acid and chemotherapy according to the Japan Adult Leukemia Study Group APL97 protocol. All patients were prospectively treated by the Japan Adult Leukemia Study Group APL97 protocol. The median follow-up period was 8.5 years. Positive CD56 expression was found in 23 APL patients (9.6%). Expression of CD56 was significantly associated with lower platelet count (P = 0.04), severe disseminated intravascular coagulation (P = 0.04), and coexpression of CD2 (P = 0.03), CD7 (P = 0.04), CD34 (P < 0.01) and/or human leukocyte antigen-DR (P < 0.01). Complete remission rate and overall survival were not different between the two groups. However, cumulative incidence of relapse and event-free survival (EFS) showed an inferior trend in CD56(+) APL (P = 0.08 and P = 0.08, respectively). Among patients with initial white blood cell counts of 3.0 × 10(9)/L or more, EFS and cumulative incidence of relapse in CD56(+) APL were significantly worse (30.8% vs 63.6%, P = 0.008, and 53.8% vs 28.9%, P = 0.03, respectively), and in multivariate analysis, CD56 expression was an unfavorable prognostic factor for EFS (P = 0.04). In conclusion, for APL with higher initial white blood cell counts, CD56 expression should be regarded as an unfavorable prognostic factor.

Long-term outcome and prognostic factors of elderly patients with acute promyelocytic leukemia.

Studies focused on elderly acute promyelocytic leukemia (APL) are relatively limited. To evaluate prognostic impact in elderly APL, we compared the long-term outcome of elderly APL patients (60-70 years) with younger patients (15-59 years) treated with all-trans retinoic acid combined with anthracycline and cytarabine in the Japan Adult Leukemia Study Group (JALSG) APL97 study. Of 283 evaluable patients, 46 (16.3%) were elderly who had more frequent lower platelet (P = 0.04), lower albumin (P = 0.006) and performance status 3 (P = 0.02), higher induction death rate due to differentiation syndrome (P = 0.03), and non-relapse mortality (NRM) during consolidation therapy (P = 0.001). Overall survival was significantly inferior in elderly patients (P = 0.005), but disease-free survival and cumulative incidence of relapse were not. Better therapeutic approaches should be considered to reduce NRM during induction and consolidation therapy in elderly APL. This study was registered at http://www.umin.ac.jp/ctrj/ under C000000206.

Successful treatment of Aspergillus liver abscesses in a patient with acute monoblastic leukemia using combination antifungal therapy including micafungin as a key drug.

While anti-cancer chemotherapy has improved the survival of patients with hematologic malignancies, it has also exposed such patients to the risk of life-threatening infection due to neutropenia. In intensive chemotherapy for leukemia, invasive aspergillosis resulting in death is infrequently observed. In such cases, aggressive diagnostic and therapeutic intervention is required. Herein, we report a case of Aspergillus liver abscesses in a patient with acute monoblastic leukemia. The patient presented with febrile neutropenia and concomitantly with an elevated serum beta-D: -glucan level during chemotherapy. The abscesses were finally diagnosed by liver biopsy. Although antifungal monotherapy of voriconazole or liposomal amphotericin B, both of which are recommended for invasive aspergillosis, showed a poor response, when combined with micafungin, an echinocandin, both had a highly favorable effect against the infection. Therefore, our clinical experience suggests that the serum test is useful for the rapid diagnosis of invasive aspergillosis, especially in deep tissues, and that combination antifungal therapy with micafungin should be considered when initial monotherapy for fungal infection shows an insufficient effect.

Low-dose cytarabine and aclarubicin in combination with granulocyte colony-stimulating factor for elderly patients with previously untreated acute myeloid leukemia.

We evaluated the efficacy of low-dose cytarabine and aclarubicin combined with granulocyte colony-stimulating factor (CAG) in elderly patients with previously untreated acute myeloid leukemia. Patients aged between 60 and 70 years who were not eligible for standard chemotherapy protocols and patients aged over 70 years were all registered. Thirty-three of 68 patients (49%) achieved remission. Median disease-free survival was 10 months and overall survival was nine months. Performance status after chemotherapy in patients who achieved remission was generally favorable. The present study demonstrates that CAG therapy is efficacious and well tolerated in the majority of elderly patients.

Hairy cell leukemia responsive to anti-thymocyte globulin used as immunosuppressive therapy for aplastic anemia.

Hairy cell leukemia (HCL) is occasionally misdiagnosed as aplastic anemia when only a few leukemic cells are present in the circulation. Here, we describe a patient with HCL who initially presented with pancytopenia and received a diagnosis of aplastic anemia. The patient was treated with immunosuppressive therapy including cyclosporine A and anti-thymocyte globulin (ATG). No blood cell transfusion was required for approximately 3 years after ATG therapy. She was referred to our hospital because of an abdominal mass and requiring periodic blood transfusions. A bone marrow biopsy at this time revealed proliferation of lymphocytes with a fried egg appearance and an increase in reticulin fibers that are typical findings of HCL. It is notable that our patient with a presumably long history of HCL and an increase in marrow reticulin fibers showed good recovery of hematopoiesis after cladribine therapy. Some HCL patients may receive an initial diagnosis of aplastic anemia and may show a good response to ATG masking the underlying HCL.

NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes.

It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplastic anaemia (AA) and myelodysplastic syndromes (MDS). However, the molecular events in immune-mediated marrow injury have not been elucidated. We recently reported an abnormal expression of stress-inducible NKG2D (natural-killer group 2, member D) ligands, such as ULBP (UL16-binding protein) and MICA/B (major histocompatibility complex class I chain-related molecules A/B), on granulocytes in some PNH patients and the granulocyte killing by autologous lymphocytes in vitro. The present study found that the expression of NKG2D ligands was common to both granulocytes and BM cells of patients with PNH, AA, and MDS, indicating their exposure to some incitement to induce the ligands. The haematopoietic colony formation in vitro by the patients' marrow cells significantly improved when their BM cells were pretreated with antibodies against NKG2D receptor, suggesting that the antibodies rescued haematopoietic cells expressing NKG2D ligands from damage by autologous lymphocytes with NKG2D. Clinical courses of patients with PNH and AA showed a close association of the expression of NKG2D ligands with BM failure and a favourable response to immunosuppressive therapy. We therefore propose that NKG2D-mediated immunity may underlie the BM failure in PNH and its-related marrow diseases.

Relationship between an effective dose of imatinib, body surface area, and trough drug levels in patients with chronic myeloid leukemia.

The standard dose of imatinib for the treatment of chronic-phase chronic myeloid leukemia (CML) is 400 mg/day. Some patients receive reduced doses of imatinib because of serious adverse effects. Recently, the effective plasma threshold for trough imatinib levels was demonstrated to be 1,002 ng/mL. In this study, we evaluated the association of an imatinib dose with trough plasma concentrations and clinical outcomes in 31 patients with chronic-phase CML who were treated at Kumamoto University Hospital. Twenty-seven patients were optimally treated with various doses of imatinib. The mean (+/-SD) trough plasma concentrations of imatinib were 1.40 +/- 0.57 microg/mL in 13 patients receiving 400 mg/day and 1.15 +/- 0.44 microg/mL in 9 patients receiving 300 mg/day as an effective dose. Mean trough levels of the two groups were not significantly different and exceeded the effective plasma threshold. Body surface area (BSA) was significantly smaller in patients receiving the reduced dose compared with those receiving the standard dose (p = 0.001). The effective imatinib dose was associated with age and gender as well as BSA. A reduced dose of 300 mg/day of imatinib may be sufficient for the treatment of CML patients with smaller body size, particularly when intolerability arises.

Micafungin-induced immune hemolysis attacks.

The echinocandins provide an attractive new option for prophylactic and empirical treatment of invasive fungal infections in patients with neutropenia after intensive cytotoxic chemotherapy or hematopoietic stem cell transplantation. We present two patients with hematological diseases who experienced massive intravascular hemolysis followed by renal failure after administration of micafungin. In indirect antiglobulin test, significant agglutination was observed when red blood cells were exposed to the mixture of micafungin and either of the patients' plasma samples, indicating that production of antibodies directed against both micafungin and red blood cell membrane induced hemolysis attack. Micafungin-mediated immune hemolysis represents an uncommon but life-threatening adverse reaction leading to renal failure.

[Aspergillosis of masticator space including mandibular bone responding to itraconazole treatment in a diabetic adult: case report].

Aspergillosis of the bone is rare and resistant to treatment. We report a case of Aspergillus infection of the masticator space including mandibular bone in a diabetic adult. After extraction of a posterior tooth, the patient began to suffer from facial pain. The pain worsened in spite of antibiotic treatment. The results of serum tests and biopsy showed an invasive aspergillosis of the left masticator space including the mandibular bone six months after the onset. Although invasive aspergillosis can be fatal, the infection in our case responded to itraconazole treatment. Even in diabetes mellitus, invasive aspergillosis may occur after surgical interventions such as tooth extraction.

A randomized study with or without intensified maintenance chemotherapy in patients with acute promyelocytic leukemia who have become negative for PML-RARalpha transcript after consolidation therapy: the Japan Adult Leukemia Study Group (JALSG) APL97 study.

To examine the efficacy of intensified maintenance chemotherapy, we conducted a prospective multicenter trial in adult patients with newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Of the 302 registered, 283 patients were assessable and 267 (94%) achieved complete remission. Predicted 6-year overall survival in all assessable patients and disease-free survival in patients who achieved complete remission were 83.9% and 68.5%, respectively. A total of 175 patients negative for PML-RARalpha at the end of consolidation were randomly assigned to receive either intensified maintenance chemotherapy (n = 89) or observation (n = 86). Predicted 6-year disease-free survival was 79.8% for the observation group and 63.1% for the chemotherapy group, showing no statistically significant difference between the 2 groups (P = .20). Predicted 6-year survival of patients assigned to the observation was 98.8%, which was significantly higher than 86.2% in those allocated to the intensified maintenance (P = .014). These results indicate that the intensified maintenance chemotherapy did not improve disease-free survival, but rather conferred a significantly poorer chance of survival in acute promyelocytic leukemia patients who have become negative for the PML-RARalpha fusion transcript after 3 courses of intensive consolidation therapy.

Severe hemorrhagic complications during remission induction therapy for acute promyelocytic leukemia: incidence, risk factors, and influence on outcome.

BACKGROUND: Even after the introduction of all-trans retinoic acid (ATRA), early hemorrhagic death remains a major cause of remission induction failure for acute promyelocytic leukemia (APL). METHODS: To investigate severe hemorrhagic complications during remission induction therapy with respect to incidence, risk factors, and influence on outcome. Results were analyzed for 279 patients enrolled in the APL97 study conducted by the Japan Adult Leukemia Study Group (JALSG). RESULTS: Severe hemorrhage occurred in 18 patients (6.5%). Although most of them were receiving frequent transfusions, the targeted levels of platelet counts (30 x 10(9)/L) and plasma fibrinogen (1.5 g/L) for this study were reached at the day of bleeding in only 71% and 40%, respectively. Nine of them succumbed to an early death, while the remaining nine patients eventually achieved complete remission (CR). The 5-yr event-free survival rate was 68.1% for those who did not suffer severe hemorrhage, and 31.1% for those who did (P < 0.0001). For patients who achieved CR, on the other hand, there was no difference in disease-free survival between patients with and without severe hemorrhage (P = 0.6043). Risk factor analysis identified three pretreatment variables associated with severe hemorrhage: initial fibrinogen level, white blood cell count, and performance status. Additionally, patients with severe hemorrhage were more easily prone to develop retinoic acid syndrome or pneumonia than patients without hemorrhage. CONCLUSIONS: These results indicate that fatal hemorrhage represents a major obstacle in curing APL, and that patients with such high-risk features may benefit from more aggressive supportive care.

Torsades de pointes upon fluconazole administration in a patient with acute myeloblastic leukemia.

Prolonged QT syndrome often causes torsades de pointes (Tdp), a potentially lethal arrhythmia. A 55-year-old woman with M4Eo who was receiving consolidation chemotherapy had an episode of prolonged QT and Tdp following fluconazole (FCZ) administration. Intravenous supplementation of magnesium sulfate and multiple attempts at electrocardioversion led to recovery from the arrhythmia. FCZ appears to contribute to the development of QT prolongation, in particular with low concentrations of serum potassium or magnesium. Although mechanisms of Tdp development in patients with QT prolongation remain to be determined, it is possible that FCZ administration leads to manifestation of Tdp. Special cautions should be exercised upon the emergence of QT prolongation following FCZ administration.

Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP.

The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of GPI-linked membrane proteins. GPI-deficient blood cells often expand in patients with aplastic anemia who sustain immune-mediated marrow injury putatively induced by cytotoxic cells, hence suggesting that the injury allows PNH clones to expand selectively. We previously reported that leukemic K562 cells preferentially survived natural killer (NK) cell-mediated cytotoxicity in vitro when they acquired PIGA mutations. We herein show that the survival is ascribable to the deficiency of stress-inducible GPI-linked membrane proteins ULBP1 and ULBP2, which activate NK and T cells. The ULBPs were detected on GPI-expressing but not on GPI-deficient K562 cells. In the presence of antibodies to either the ULBPs or their receptor NKG2D on NK cells, GPI-expressing cells were as less NK sensitive as GPI-deficient cells. NK cells therefore spared ULBP-deficient cells in vitro. The ULBPs were identified only on GPI-expressing blood cells of a proportion of patients with PNH but none of healthy individuals. Granulocytes of the patients partly underwent killing by autologous cytotoxic cells, implying ULBP-associated blood cell injury. In this setting, the lack of ULBPs may allow immunoselection of PNH clones.