Masaru Shibano

CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG‐A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG‐A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG‐A gene were all different and included two single‐base insertions, one single‐base deletion, two two‐base deletions, and one each of eight‐base insertion and nine‐ and ten‐base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI‐anchoring defect and that the PIG‐A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG‐A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.

Natural killer cell-derived large granular lymphocyte lymphoma of lung developed in a patient with hypersensitivity to mosquito bites and reactivated Epstein-Barr virus infection

A 17‐year‐old female developed natural killer (NK) cell‐derived large granular lymphocyte (LGL) lymphoma of the lung. She had a past history of hypersensitivity to mosquito bites (HMB). After an eight‐year chronic, active Epstein‐Barr virus (EBV) infection, she developed multiple lung lesions and pleural effusion. In the effusion, 60% of the cells were LGL. They were CD2+, 3−, 16+, 56+, 57+, 45RO+/RA + weak, and possessed strong NK activity. No rearrangement of T‐cell–receptor genes was detected. From all these results, a diagnosis of NK‐LGL lymphoma of the lung was made. EB virus DNA was detected in cells infiltrating the pleural effusion. The clonality of the LGLs was determined by Southern blot hybridization with the terminal repeat sequence of EB virus as a probe, and by chromosomal abnormalities. The patient died from respiratory failure. Necropsy of the lung revealed diffuse lymphoma composed of polymorphic cells with typical angiocentric lesions. Reportedly, lymphomas of NK lineage show predominantly extranodal involvement, and primary lung lesions are rare. In the pleural effusion of the present case, abnormally high levels of soluble Fas ligand, interleukin‐10 and interferon γ were detected. This hypercytokinemia, reflecting the microenvironment of lymphoma cells, may play a role in the progression of the lymphoma and organ injury in the lung. Am. J. Hematol. 59:309–315, 1998.

Increased frequency of somatic mutations at glycophorin A loci in patients with aplastic anaemia, myelodysplastic syndrome and paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 106 erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (≧ 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG‐A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.

Analysis of PIG‐A gene in a patient who developed reciprocal translocation of chromosome 12 and paroxysmal nocturnal hemoglobinuria during follow‐up of aplastic anemia

The relationships between paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndrome (MDS) are not clear. Here we describe a patient, J20, who developed a reciprocal translocation of chromosome 12 and PNH during follow‐up of AA. All metaphases in CD59‐deficient bone marrow mononuclear cells had the translocation, whereas none of the CD59‐sufficient cells had it, indicating that the PNH clone coincided with a cell population bearing the chromosomal aberration. We found a somatic single‐base deletion mutation in the PIG‐A gene of this patients peripheral blood cells. This is the first patient with PNH with a PNH clone containing a chromosomal translocation.

Phenotypical heterogeneity of CD4 + CD8 + double-positive chronic T lymphoid leukemia

Chronic T lymphoid leukemias are defined as leukemias of post-thymic T cells. The CD4+CD8+ double-positive (DP) phenotype is seen in a few cases. Since DP generally occurs in thymic T cells, whether the DP T leukemia cells represent thymic or peripheral T cells has been a matter of controversy. To address this issue, we studied phenotypical features in eight cases of DP T cell leukemia. Thymic DP T cells and peripheral CD8+ T cells have CD8 of αβ subunit, while CD8αα is induced in CD4+ T cells on activation with IL-4. We found that two patients with DP T large granular lymphocyte leukemia (LGLL) showed dim expression of CD8αα, identical to the phenotype on IL-4-activated DP-T cells. The leukemic cells of these patients expressed IL-4 mRNA and produced high levels of IL-4. These findings suggest that they may be derived from peripheral CD4+ T cells. Three patients with adult T cell leukemia/lymphoma (ATLL) showed CD8αα, suggestive of an activated peripheral T cell origin. One case expressed CD8αα dim and IL-4 mRNA, while the other two cases expressed no IL-4 mRNA and showed CD8αα bright phenotype, features not found in normal T cell populations. Three patients with T-prolymphocytic leukemia (T-PLL) expressed CD8αα. The DP phenotype is relatively common in T-PLL, and CD4+CD8αβ+ is characteristic of thymic T cells. The DP T-PLL cells did not express TdT,CD1 or recombination activating gene-1 (RAG-1), which is down-regulated at the late stage of thymic T cell development. On the basis of these findings, we propose a late thymic origin for DP T-PLL. The phenotype of DP T cells differed for each entity and appeared to correlate with minor normal DP T cell population.

Assessment of alkaline phosphatase on the surface membrane of neutrophils by immunofluorescence

Expression of alkaline phosphatase (ALP) on the surface membrane of neutrophils (mNAP) was studied by immunofluorescence using an anti‐ALP monoclonal antibody. Fluorescent intensity distribution of mNAP was analyzed using FACS (fluorescence‐activated cell sorter). The mean fluorescent intensity (MFI) of the mNAP in this assay was well correlated with the neutrophil ALP (NAP) score demonstrated cytochemically (r = 0.832). mNAP levels in various hematological disorders were evaluated by % mNAP+ cells and MFI. The levels in aplastic anemia and polycythemia vera were significantly higher, and in chronic myelocytic leukemia and paroxysmal nocturnal hemoglobinuria (PNH), the levels were significantly lower compared with the levels in healthy volunteers. Two‐color immunofluorescence with anti‐ALP and anti‐CD16 showed that the PNH clone was essentially negative for mNAP, whereas residual normal neutrophils (CD16+) had levels slightly higher than those in normal individuals. Highly reproducible results were obtained in the blood samples which were stored at 4°C for at least 24 hr without any treatment prior to immunofluorescent staining. No degradation of fluorescent intensity was seen 4 days after staining and fixation. The mNAP assay is simple, without subjective evaluation for quantification, and is useful for differential diagnosis of hematological disorders. Am. J. Hematol. 60:12–18, 1999.

Primary Marginal Zone Lymphoma in the Posterior Mediastinum with Pleural Involvement

We herein report a case of primary marginal zone lymphoma (MZL) of the posterior mediastinum in an 84-year-old woman. Computed tomography of the chest showed a posterior mediastinal mass in the right thoracic paravertebral region with right pleural effusion. Pathological findings of a surgical biopsy from the posterior mediastinum, along with immunohistochemical and flow cytometric results, indicated MZL. The patient was treated with chemotherapy and radiation therapy for the mediastinal lesion and achieved complete remission. A relapse occurred 3 months after the initial treatment regimen. However, a second relapse has not occurred more than 2 years after second-line chemotherapy. This is the first case of MZL originating in the posterior mediastinum.