Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

A randomized controlled trial of plasma real-time PCR and antigenemia assay for monitoring CMV infection after unrelated BMT.

Preemptive therapy is the standard strategy for preventing CMV disease after allogeneic hematopoietic SCT. In this study, unrelated BMT recipients were randomly assigned to a plasma real-time PCR group or an antigenemia group to compare the value of these monitoring tools for CMV reactivation. Ganciclovir (GCV) was started at 5 mg/kg/day when PCR reached 300 copies per ml or when antigenemia reached three positive cells per two slides. A total of 88 patients were randomized into the antigenemia group (n=45) or the PCR group (n=43). A significantly higher number of patients reached the threshold in the antigenemia group than in the PCR group (73.3 vs 44.2%, P=0.0089). However, only three patients (one in the antigenemia group and two in the PCR group) developed early CMV disease. These patients exclusively had colitis and were successfully treated with GCV or foscarnet. The median number of antigenemia-positive cells at the start of GCV was 47 in the PCR group. These findings suggest that antigenemia assay with the current cutoff was too sensitive and led to unnecessary use of GCV. However, the appropriateness of the threshold may be different by the methodology used, and therefore, it is difficult to generalize.

[Skeletal muscle T-cell lymphoma following hemophagocytic syndrome].

A 43-year-old man was admitted to our hospital because of hemophagocytic syndrome (HPS) in August, 1998. A CT scan, gallium scintigraphy, gastrofiberscopy and colonofiberscopy showed no evidence of malignant lymphoma. Virus-associated HPS was suspected because of an increased titer of anti-Epstein-Barr (EB) virus antibody (EBV VCA IgG 2,560x, EBV EA IgG 40x, EBNA 20x). The HPS resolved spontaneously for 40 hospital days, but two weeks into the period of HPS remission, the patient developed pain and marked swelling of the right thigh muscle, and pectoral, biceps brachii, quadriceps femoralis and masseter muscles. Otherwise, CT scan and gallium scintigraphy showed no abnormal findings. A biopsy of the right quadriceps femoralis muscle revealed non-Hodgkin's lymphoma with muscle infiltration. Immunohistologic examination confirmed T-cell type (CD3, CD43, CD45, CD45RO) lymphoma, and Southern blot analysis for T-cell receptor revealed a rearranged band. The lymphoma cells were negative for EBV genome monoclonality. The patient responded well to CHOP therapy and achieved a complete remission. This is considered a very rare case of T-cell lymphoma infiltrating multiple skeletal muscles following an episode of hemophagocytic syndrome.

A case of chronic myeloid leukemia with minor bcr-abl transcript following fluorouracil therapy for esophageal carcinoma.

We report here a very rare case of chronic myeloid leukemia (CML) with a minor bcr-abl transcript, which developed following long-term chemotherapy with fluorouracil for esophageal carcinoma. A 64-year-old male patient was diagnosed with CML. Four years earlier, he had suffered from esophageal carcinoma, which was treated by surgical resection followed by oral administration of fluorouracil (200 mg/day) for 4 years. Molecular analysis of his Philadelphia chromosome (Ph) using reverse-transcriptase polymerase chain reaction (RT-PCR) and subsequent sequencing revealed a minor bcr-abl transcript. The clinical course of this patient was aggressive with a short chronic phase of CML. This is the first reported case of secondary CML with a minor bcr-abl transcript.

Thrombopoietin can influence mature megakaryocytes to undergo further nuclear and cytoplasmic maturation.

OBJECTIVE: We studied the effects of TPO on the nuclear and cytoplasmic maturation of mature megakaryocytes. MATERIALS AND METHODS: We prepared mature megakaryocytes by velocity sedimentation method and investigated the maturational effect of TPO on such mature megakaryocytes from the aspects of DNA ploidy and cytoplasmic features using a flow cytometry and Br-dU incorporation. We also studied the effects of TPO on expression of GpIIb and NF-E2 transcripts by RT-PCR and on proplatelet formation. RESULTS: DNA content increased when megakaryocytes were cultured for 2 days with TPO, resulting in generation of megakaryocytes with a DNA content of 32N or 64N. SCF had a similar, but weaker, effect, while IL-6 did not influence mature megakaryocytes. The proportion of megakaryocytes incorporating Br-dU into their nuclei was 44.3% +/- 6.9% after culture with TPO, 19.5% +/- 4.6% with SCF, 7.5% +/- 3.5% with IL-6, and 5.2% +/- 2.7% in control culture. Flow cytometry showed that TPO did not generate larger megakaryocytes with more complex intracellular structures when compared with cells of the same DNA content class (16N) cultured without TPO. In addition, TPO also did not enhance the expression of NF-E2 transcripts, but it delayed proplatelet formation by cultured megakaryocytes. CONCLUSION: TPO first affected endomitosis by mature megakaryocytes and then altered their cytoplasmic maturation, with both maturation processes remaining proportionate to each other.

[Diagnostic imaging of malignant lymphoma: current usefulness and limitation].

The recent developments of imaging modalities allow us to make a diagnosis of malignant lymphoma, to determine its staging, and to evaluate its response to treatments noninvasively. Especially, CT and MR imagings can visualize almost all organs involved by lymphoma with high spatial and contrast resolutions. However, it is difficult to detect the small diseased lymph nodes and splenic lesions. The tumor viability after treatments can not be accurately evaluated with imaging studies. The coverage of the whole lymph nodes during one examination is not easily achieved, either. The multidetector CT system, fast MR imaging techniques, new MR contrast media, and PET are expected to overcome these difficulties.

Immunological reconstitution after cord blood transplantation for an adult patient.

We transplanted 4.1x10(7) unrelated umbilical cord blood cells into a 27-year-old patient suffering from transformed acute myelocytic leukemia. The thawing method was the same as described by Rubinstein et al (Proc. Natl. Acad. Sci. USA 1995; 92: 10119-10122). ANC reached over 500x10(9)/l on day 19, and the patient was free from RBC and platelet transfusion on days 26 and 38, respectively. Cytogenetic and molecular analysis after transplant revealed complete chimerism. The CD3+CD4+ lymphocyte count became greater than 100x10(9)/l at 5 weeks after transplantation. The CD3+CD8+ count became greater than 500x10(9)/l at 7 weeks and thereafter progressively increased in spite of administration of CYA. This immunological reconstitution pattern after umbilical cord blood transplantation was different from that after bone marrow transplantation, and resistance of CD3+CD8+ lymphocytes to CYA was the distinguishing characteristic. The rapid hematological recovery and immunological reconstitution may be attributed to the high dose of transfused nucleated cells and umbilical cord blood transplantation may become a promising method for treatment for such cases.

Lymphoblastic lymphoma of natural killer cell origin, presenting as pancreatic tumour.

AIMS: We describe the first case of lymphoblastic lymphoma (LBL) of natural killer (NK) cell origin initially presenting as a pancreatic tumour, and review this type of lymphoma. METHODS AND RESULTS: A 38-year-old woman with abdominal pain was found to have a pancreatic mass. Twenty days later, she developed diffuse lung infiltrates and leucoerythroblastosis of the peripheral blood, and her bone marrow was diffusely infiltrated with blasts. The blasts were positive for CD56, CD16 and P-glycoprotein, but lacked cytoplasmic azurophilic granules, T. B and myeloid cell markers on the cell surface, rearrangement of the genes for T-cell receptor and immunoglobulin, and the Epstein-Barr virus (EBV) genome. The diagnosis was LBL of NK cell origin. She received aggressive therapy, but died of the lymphoma. In contrast to ordinary NK-cell lymphoma, this case and reported cases of LBL of NK-cell origin showed the following common characteristics. The tumour cells often lack cell surface CD2, cytoplasmic CD3, cytoplasmic azurophilic granules, and EBV genome. The prognosis was extremely poor. CONCLUSIONS: LBL of NK-cell origin should be included in the differential diagnosis of pancreatic tumours. To fully characterize this type of lymphoma, further cases must be evaluated.

IL-6 and SCF exert different effects on megakaryocyte maturation.

Megakaryocytic maturation has two aspects, nuclear maturation and cytoplasmic maturation, both of which are affected by several thrombopoietic cytokines. We cultured murine bone marrow cells separated by BSA velocity sedimentation which contained more immature megakaryocytes with lower ploidy in order to study the effects of rhIL-6 and rmSCF on the two aspects of megakaryocyte maturation. During culture with rhIL-6 or rmSCF for 4 d, the megakaryocytes underwent endomitosis, resulting in DNA polyploidization. These two cytokines had different effects on cytoplasmic maturation. Flow cytometry showed that the megakaryocytes cultured with rhIL-6 became larger and had a more complex intracellular structure than those cultured with rmSCF. Morphological studies showed that the megakaryocytes cultured with rhIL-6 were larger and more granular, whereas those cultured with rmSCF were smaller and more basophilic. rhIL-6 promoted cytoplasmic process formation by the megakaryocytes, but rmSCF did not. Analysis of transcripts of PF-4 and GPIIb by RT-PCR suggested that rmSCF kept more megakaryocytes at the protein synthesis stage than did rhIL-6. Based on our findings, these cytokines exert different effects on megakaryocytic cytoplasmic maturational events.

Acute lymphoblastic leukemia with isolated adrenocorticotropic hormone deficiency.

A 57-year-old female was admitted to our hospital because of Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). On admission, disturbance of consciousness and hyponatremia were recognized. The patient's endocrinological data showed low levels of adrenocorticotropic hormone (ACTH) (less than 5 pg/ml) and cortisol (5.9 microg/dl). Other anterior pituitary hormones were normal. Plasma ACTH and cortisol did not respond to the corticotropin releasing factor (CRF) test. A diagnosis of isolated ACTH deficiency was made. This is a rare case of isolated ACTH deficiency complicated with hematological malignancies.

Autologous bone marrow transplantation for patients with advanced chronic myelogenous leukemia.

We report on seven chronic myelogenous leukemia (CML) patients who received autologous bone marrow transplantation (ABMT) using bone marrow (BM) cells while at the chronic phase (CP) under the various treatments. Of the seven patients, four progressed to accelerated phase (AP) in 83-248 weeks after onset and three patients entered blastic crisis (BC) in 84-171 weeks after onset. All patients received high-dose chemoradiotherapy followed by infusion with 11.3 +/- 12.1 x 10(7) (average +/- S.D.) of bone marrow mononuclear cells (BM-MNCs)/kg IFN-alpha was resumed shortly after platelet recovery. Of the four patients in AP, one developed a recurrence of blastoma in 7 weeks, one progressed to second AP in 138 weeks after ABMT and two patients have survived the second CP for 159 and 330 weeks since ABMT, respectively. One of them achieved the complete disappearance of Ph1-positive metaphases for 33 weeks after ABMT. Of patients who received AMBT in BC, three relapsed within 8 weeks and died in 9, 17 and 58 weeks after ABMT, respectively. Hematological recovery was delayed in four patients. Therefore, we retrospectively re-evaluated the number of BM-MNCs collected through 50 procedures from 40 patients with CML-CP. The total MNCs obtained from 30 collections under IFN-alpha treatment was 27.4 +/- 30.9 x 10(8) cells (average +/- S.D.), being significantly lower than that obtained from 20 collections in pre-treatment state or with single chemotherapy other than IFN-alpha treatment (81.8 +/- 68.2 x 10(8) cells) (P < 0.005). The total number of MNCs correlated to white blood cell (WBC) count at BM collection (P < 0.01), which was also lower in the IFN-alpha(+) group than in the IFN-alpha(-) group (7.2 +/- 5.7 and 25.6 +/- 32.3 x 10(9)/l; P < 0.005). Our findings suggested that ABMT with the use of a sufficient number of progenitor cells might be helpful to CML patients in early AP and reach in extended periods of second CP. In addition, we suggest that BM collection is required before the start of IFN-alpha therapy because the total number of BM-MNCs correlated to the WBC count, which might be lower in IFN-alpha treatment.

[Bronchiolitis obliterans organizing pneumonia (BOOP) after allogeneic bone marrow transplantation].

We report a patient who underwent allogeneic bone marrow transplantation for ALL. The patient presented low grade fever, cough and dyspnea at day 3 after after bone marrow transplantation. Imaging studies showed bilateral patchy infiltrates on chest X-ray and chest CT. Though treated by antibiotics, chest auscultation recognized marked bilateral inspiratory crackles. Transbronchial lung biopsy performed on day 34 showed bronchiolitis obliterans and an organizing interstitial pneumonia. The patient was treated with methylprednisolone and follow up computed tomography findings improved.

A role of GAGs in ECM on morphogenesis of megakaryocytes.

The morphogenesis of megakaryocytes that results in the formation of cytoplasmic processes is thought to be the final maturation step before liberation of platelets. We studied the in vitro effects of glycosaminoglycans (GAGs) which are abundant in the bone marrow extracellular matrices, on the morphogenesis of murine megakaryocytes and compared them with those of thrombopoietic cytokines. Heparin, heparan sulfate, chondroitin-6 sulfate, and dermatan sulfate promoted the formation of megakaryocytic processes. Hyaluronic acid failed to support this phenomenon, suggesting that sulfated GAGs in extracellular matrices are involved in the morphogenesis of megakaryocytes. Sulfated GAGs began to act on megakaryocytes with a higher ploidy (16N-32N) from 6 to 24h after incubation, whereas neither rhIL-6 nor rhIL-11 affected this early phase. Our findings indicate that sulfated GAGs promote the morphogenesis of murine megakaryocytes and participate in thrombopoiesis in a different manner from that of cytokines such as rhIL-6 and rhIL-11.

A novel aspect of the maturational step of megakaryocytes in thrombopoiesis: bundle formation of microtubules in megakaryocytes.

We demonstrated bundle formation of microtubules both in the cytoplasmic processes and in the cytoplasm near the nucleus of cultured megakaryocytes by means of electron and immunofluorescent microscopy. To determine whether this bundle formation was related to megakaryocyte maturation, we studied the effects of recombinant human interleukin-6 (rhIL-6) in vitro and in vivo on this event. About 75% of the megakaryocytes in the bone marrow had no fibrous microtubule structures in the cytoplasm (type I), and about 25% had bundles of microtubules (type II). The formation of these bundles was promoted by rhIL-6 both in vitro and in vivo. Considerably more megakaryocytes cultured with recombinant murine (rm) IL-3 and rhIL-6 became type II than those cultured with rmIL-3 alone. Megakaryocytes from mice given rhIL-6 (10 microg/animal/d) subcutaneously also began to form bundles in proportion to an increase in platelet counts. After the administration of rhIL-6, about half of the megakaryocytes contained microtubule bundles in their cytoplasm. These results indicate that microtubule-bundle formation is one maturational event in megakaryocyte development and that rhIL-6 could accelerate this event.