Kiyoshi Okamoto

Clonality in chronic myeloproliferative disorders defined by X-chromosome linked probes: demonstration of heterogeneity in lineage involvement

Summary The restriction fragment length polymorphisms (RFLP) of the X‐chromosome phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyltransferase (HPRT) genes were used to study the clonal basis of the chronic myeloproliferative disorders (CMPD). Analyses were performed on granulocyte and T‐lymphocyte fractions obtained from 24 females; 13 had essential thrombocythaemia (ET), eight polycythaemia vera (PV) and three myelofibrosis with myeloid metaplasia (MMM). All 24 of these patients had monoclonal patterns of X‐inactivation in the granulocyte fraction. For the T‐lymphocyte fraction, non‐clonal patterns of Xinactivation were observed in 8/13 patients with ET, 7/8 with PV and 1/3 with MMM, while the remaining eight subjects were found to have monoclonal patterns of N inactivation. Our findings suggest that the majority of the CMPD in these patients originated from a relatively committed progenitor cell without the capacity to differentiate into T cells, and convincingly demonstrated heterogeneity of lineage involvement.

Clonality in myelodysplastic syndromes: demonstration of pluripotent stem cell origin using X‐linked restriction fragment length polymorphisms

Restriction fragment length polymorphisms (RFLP) of the X‐chromosome genes phosphoglycerate kinase (PGK) and hypoxanthine phorphoribosyltransferase (HPRT) were used to determine the clonal nature of myelodysplastic syndromes (MDS) in 22 patients. These included eight with refractory anaemia (RA), four with RA with ring sideroblasts (RARS), six with RA with an excess of blasts (RAEB), three with RAEB in transformation (RAEB‐T), and one with chronic myelomonocytic leukaemia (CMML). Monoclonal X‐inactivation patterns were observed in 19/22 patients. The remaining three cases, one each with RA, RARS and RAEB, were of polyclonal composition. Separated T‐lymphocyte and granulocyte fraction analyses in six patients of the former cases revealed that T‐lymphocyte as well as granulocyte fractions showed a monoclonal pattern of X‐inactivation. These results support the view that the majority of MDS arise from a pluripotent stem cell capable of myeloid and lymphoid differentiation.

Severe thrombocytopenia suggesting immunological mechanisms in two cases of vivax malaria

Case 1: A 27‐year‐old woman, referred to our hospital because of relapsing fever after travel to Thailand, was given a diagnosis of vivax malaria. Clinical investigation revealed thrombocytopenia, elevated platelet‐associated IgG (PAIgG), and negative antibody against Plasmodium vivax antigen. After antimalarial treatment, the levels of both the platelets and PAIgG returned to normal. Case 2: A 28‐year‐old Sri Lankan man was admitted to our hospital with a complaint of fever. The patient had thrombocytopenia, elevated PAIgG, and positive antibody against Plasmodium vivax antigen. He contracted malaria in Sri Lanka about 6 months prior to this admission. After treatment, the platelet count and PAIgG level returned to normal. In these two cases, high levels of PAIgG may have been involved in the development of the thrombocytopenia. In the first patient, in particular, the thrombocytopenia was thought to be induced by some immunological mechanism prior to the detection of antimaralial antibodies in serum. Am. J. Hematol. 56:183–186, 1997.

A novel acute lymphoid leukaemia type BCR/ABL transcript in chronic myelogenous leukaemia

Using a reverse transcription‐polymerase chain reaction (RT‐PCR), we identified a patient with typical clinical features of chronic myelogenous leukaemia (CML) in the chronic phase who showed no amplification of the CML‐type BCR/ABL transcript. RT‐PCR with primers detecting the acute lymphoid leukaemia (ALL)‐type transcript disclosed a novel fragment co‐amplified with an ALL‐type fragment. Sequencing revealed the novel transcript to be a chimaeric mRNA produced by fusion of a segment of BCR exon 2 (e2) to ABL exon 2 (a2), with a 21 base‐pair insertion of ABL intron 1b sequence between them. This transcript has not been reported previously.

Analysis of the distribution of CAG repeats and X-chromosome inactivation status of HUMARA gene in healthy female subjects using improved fluorescence-based assay.

We investigated the polymorphic CAG-repeat distribution and the X-inactivation status of the human androgen receptor (HUMARA) gene in 58 female Japanese volunteers. Polymerase chain reaction amplification was performed using a fluorescent-dye-labeled primer under conditions specific for GC-rich targets, and fragments were analyzed. To estimate the length of these fragments, FAM-labeled (blue fluorescent) products were simultaneously compared with ROM-labeled size markers (red) that were created by sequencing various HUMARA fragments. The number of polymorphic CAG repeats of HUMARA in 116 alleles from 58 female subjects ranged from 15 to 28. Of the 58 volunteers, 51 (88.0%) were heterozygous. In 96% of the heterozygous female subjects, the allelic differences were no greater than 6 repeats. X-chromosome inactivation was calculated as the ratio of the area of the smaller peak to the sum of the areas of the smaller and larger peaks. The average ratio was 0.38 (range, 0.09-0.50). Preferential use of 1 allele, by more than 75% (ratio, <0.25), was observed in 5 volunteers (10.9%). The clonal nature of a patient with chronic myelogenous leukemia was easily identified. This method is sensitive enough to discriminate a difference of 1 triplet repeat.

Detection of c-myc oncogene amplification in a CML blastic phase patient with double minute chromosomes

Double minute chromosomes (dmin) are relatively rare in leukemias. Cytogenetic analysis of blood cells from a woman with blastic phase chronic myelogenous leukemia (BC-CML) showed numerous dmin chromosomes and complex abnormalities including a Philadelphia (ph(1))-chromosome. Oncogene amplification in hematopoietic malignancies is also rare. Using PCR, we retrospectively investigated the extent of c-myc gene amplification in DNA extracted from stored blood smears from the patient. To qualify the PCR products, the beta-globin gene was used as the internal reference gene and it was co-amplified with the c-myc gene. The extent of amplified c-myc was about 6.8-fold. This finding suggests that the c-myc gene was amplified in dmin and that the gene amplification contributes to the progression to acute leukemia or rapid growth of leukemic cells.

The majority of T lymphocytes are polyclonal during the chronic phase of chronic myelogenous leukemia

To clarify the extent of cell lineage involvement in chronic myelogenous leukemia (CML), we investigated the bcr gene rearrangement and clonality using the X-chromosome-linked restriction fragment length polymorphism (RFLP) methylation method in T lymphocytes and granulocytes. We examined the granulocyte and T-cell fractions from the peripheral blood of seven female patients with CML during the chronic phase; patients were heterozygous for RFLPs at the phosphoglycerate kinase (PGK) or the hypoxanthine phosphoribosyltransferase (HPRT) gene. RFLP-methylation analysis of granulocytes demonstrated a monoclonal pattern in six of the seven patients and a rearrangedbcr gene in all seven patients. In contrast, T lymphocytes exhibited a polyclonal pattern in six cases; in one case, a faint band was observed following methyl-sensitive enzyme cleavage. Thebcr gene analysis in T lymphocytes showed the germline in every case. Our results indicate that the majority of T lymphocytes are polyclonal during the chronic phase of CML and confirm previous reports based on glucose-6-phosphate dehydrogenase, cytogenetic, andbcr rearrangement analyses.

ALL- and CML-type BCR:ABL mRNA transcripts in chronic myelogenous leukemia and related disorders

Using the reverse transcription polymerase chain reaction, we investigated acute lymphoid leukemia (ALL)-type, and chronic myelogenous leukemia (CML)-type BCR/ABL mRNA expression in a total of 66 patients with chronic myeloproliferative disorder (CMPD). Thirty-six of 37 patients with CML were positive for CML-type mRNA. Thirteen of the 25 CML had ALL-type mRNA expression. The patients with ET, PV, MF, and CMML did not have any detectable BCR/ABL expression. The most remarkable finding was that two patients, a Ph1-positive CML patient and a patient with a presumptive diagnosis of essential thrombocythemia (ET), showed only ALL-type chimeric mRNA expression.

Clinical Outcome in Three Patients with Myelodysplastic Syndrome Showing Polyclonal Hematopoiesis

The clinical outcome of 3 myelodysplastic syndrome (MDS) patients with polyclonal hematopoiesis is reported. All patients were heterozygous for the phosphoglycerate kinase (PGK) gene. The presence of polyclonal hematopoiesis was determined by the X-chromosome-linked restriction fragment length polymorphism-methylation method using the PGK gene as a marker. The patients were initially diagnosed as having refractory anemia (RA), RA with ring sideroblasts (RARS), and RA with an excess of blasts (RAEB), respectively. Their pancytopenia persisted during the follow-up period of 11.4 years for the RA patient, 19.5 years for the RARS patient and 0.8 years for the RAEB patient. Although the RARS patient continues to be in good health, leukemic transformation occurred in the other 2 patients. A karyotype change from 46,XX to 45,XX,t(3;21),-7 was observed at the time of disease progression in the RA patient. The coexistence of a monoclonal MDS clone and normal bone marrow cells is thought to be the most probable reason for the polyclonal hematopoiesis of these patients.

Nonsecretion of myeloma protein in spite of an increase in tumor burden by chemotherapy.

SummaryA unique case of IgA κ myeloma is presented. While the myeloma cells had secreted a large quantity of IgA κ monoclonal protein, they were induced to stop secreting the monoclonal protein by cyclophosphamide and vincristine, in spite of a remarkable increase in tumor burden. The absence of intracytoplasmic IgAκ was clearly evidenced by the immunofluorescence technique using anti-IgA and anti-κ monoclonal antibodies.