Masami Nagai

Combined spectral karyotyping and DAPI banding analysis of chromosome abnormalities in myelodysplastic syndrome.

Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy‐related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G‐banding. To locate the chromosomal breakpoints, DAPI‐counterstained band images from all metaphases were transformed to G‐band–like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G‐banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G‐banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of ‐5/5q‐ and ‐7/7q‐) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with ‐5/5q‐ and in 4 of 8 with ‐7/7q‐, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G‐banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336–345, 1999.

Decreased expression of p33ING1 mRNA in lymphoid malignancies

The ING1 is a newly cloned putative tumor‐suppressor gene, which is involved in the p53 signaling pathway. We found decreased expression of ING1 mRNA in 4 of 5 T‐cell lines and 5 of 11 B‐cell lines including two Burkitt lymphomas and two myelomas. These observations suggest that decreased ING1 expression might play an important role in the development or progression of some lymphoid tumors. Polymerase chain reaction‐SSCP and sequencing analyses found neither point mutations nor small deletions in the ING1 gene, suggesting that decreased expression is due to transcriptional or post‐transcriptional mechanisms. Am. J. Hematol. 62:118–119, 1999.

Analysis of the p16INK4A, p15INK4B and p18INK4C genes in multiple myeloma

To study the structural integrity of the cyclin‐dependent kinase inhibitors known as INK4A (p16), INK4B (p15) and INK4C (p18) in multiple myeloma, we examined 20 primary myeloma samples (including one case of plasma cell leukaemia) using polymerase chain reaction–single strand conformation polymorphism, and 17 samples were examined by Southern blot analysis. The plasma cell leukaemia sample had homozygous deletions of the p15 and p16 genes (6%). One myeloma case had a p15 gene homozygous deletion (6%) with an intact p16 gene. This sample also had a p18 homozygous deletion, suggesting that the deletion of both genes may be important in either the development or progression of myeloma. No point mutations of these INK4 genes were found in the 20 samples. This is the first report that indicates that deletions of p15, p16 and p18 genes occur in some individuals with multiple myeloma (2/17 cases).

Methylation of the p16INK4A gene in multiple myeloma

The p16INK4A (p16) binds to both cyclin D‐CDK4 and cyclin D‐CDK6 and inhibits the progression of the cell cycle from G1 to S phase. Loss of expression of this protein can occur by several mechanisms including structural alterations. Recent studies have suggested that the loss of expression of p16 can occur by hypermethylation of the gene. The methylation status of the p16 gene in multiple myeloma was examined in three myeloma cell lines (U266, RPMI8226 and IM9) and 16 primary myeloma samples using methylation‐specific polymerase chain reaction (MSP). The U266 and RPMI8226 cell lines contained a completely methylated p16 gene and the IM9 line had a partially methylated p16 gene. Identical results were obtained by another polymerase chain reaction (PCR)‐based methylation assay system as well as Southern blotting after using a methylation‐sensitive restriction enzyme. The U266 cell line expressed no p16, and the IM9 had weak expression as determined by reverse transcript (RT‐)PCR. The U266 cells began to express, and IM9 increased the accumulation of, the p16 RNA after treatment with the demethylating agent 5′‐aza‐2‐deoxycytidine (10−6–10−5 M). This suggested that the levels of methylation of the p16 gene detected by the MSP technique correlated with the regulation of transcription of this gene. Examination of the primary myeloma samples showed that eight of 16 (50%) contained a methylated p16 gene. We have previously found that alterations of the p16 gene, such as deletions and point mutations, are rare in primary multiple myeloma; none of the 16 samples included in this study had p16 gene alterations. Our results suggest that methylation of the p16 gene may contribute to the development and/or progression of multiple myeloma.

Microsatellite instability during the progression of acute myelocytic leukaemia

We studied microsatellite instability (MSI) at the onset and during progression of 17 individuals with acute myelocytic leukaemia (AML). These included two cases of M0, eight with M1 and seven with M4, according to the FAB classification. The DNA from diagnostic, remission and relapsed stages of their disease was analysed at 69 loci. Two MSI were found in the diagnostic and remission phase paired samples (12%), and eight MSI were identified in six of the relapsed phase samples (35%). These results indicate that mismatch repair errors such as MSI are unimportant at the onset of AML, but might have importance during the progression of the disease.

Specific skin manifestations in CD56 positive acute myeloid leukemia

We found 16 CD56+ cases (29.6%) among 54 acute myeloid leukemia (AML) patients; they showed significantly Frequent cutaneous involvement compared to CD56‐ cases (43.8% vs.15.8%, p<0.05). Four of the CD56+ AML cases with specific skin manifestations were reviewed histologieally. In all cases, cutaneous leukemic cells were seen in the dermis and subcutaneous tissue with accentuation around the adnexa/nerve, but sparing the epidermis. In addition, angiocentric/angiodestructive and prominent cohesive tumor cell growth were seen in two rases, respectively. These findings suggest that the expression of CD56 may often be associated with the cutaneous involvement in AML, and that the above histological findings should remind us of the possibility of specific skin manifestations in CD56+ AML.

Myeloid- and lymphoid-specific breakpoint cluster regions in chromosome band 13q14 in acute leukemia.

Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B‐cell and T‐cell precursor acute lymphoblastic leukemia (BCP‐, TCP‐ALL), in differentiated and undifferentiated acute myeloblastic leukemia (AML), and in chronic myeloid leukemia (CML) at progression to blast crisis. The nature of these translocations and their pathologic consequences remain unknown. To begin to define the gene(s) involved on chromosome 13, we have performed fluorescence in situ hybridization (FISH) using a panel of YACs from the region, on a series of 10 cases of acute leukemia with t(12;13)(p12;q14) and 1 case each with “variant” translocations including t(12;13)(q21;q14), t(10;13)(q24;q14) and t(9;13)(p21;q14). In 8/13 cases/cell lines, the 13q14 break fell within a single 1.4 Mb CEPH MegaYAC. This YAC fell immediately telomeric of the forkhead (FKHR) gene, which is disrupted in the t(2;13)(q35;q14) seen in pediatric alveolar rhabdomyosarcoma. Seven of the 8 cases with breaks in this YAC were AML. In 4/13 cases, the 13q14 break fell within a 1.7‐Mb YAC located about 3 Mb telomeric of the retinoblastoma (RB1) gene: all 4 cases were ALL. One case of myelodysplastic syndrome exhibited a break within 13q12, adjacent to the BRCA2 gene. These data indicate the presence of myeloid‐ and lymphoid‐specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia. Genes Chromosomes Cancer 25:222–229, 1999.

Diffuse Large B-cell Lymphoma Presenting with Hypercalcemia and Multiple Osteolysis

Osteolysis and hypercalcemia are observed in 5 – 15%, and 10%, respectively, of malignant lymphoma patients during their clinical course. However, both osteolysis and hypercalcemia are uncommon at onset of the disease. We encountered a 24-year-old male non-Hodgkins lymphoma patient who had multiple osteolytic lesion from the onset of the disease and repeated episodes of hypercalcemia during the clinical course. The patient died with refractory disease. We studied the expression of chemokines which might affect bone resorption using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Increased expressions of MIP-1α, MIP-1β and RANKL, which are osteoclast-activating factors, were observed in the RNA derived from the patients lymphoma cells. The secretion of osteoclast-activating factors such as MIP-1α by the tumor cells (and/or bone marrow stromal cells) might be involved in the etiology of osteolysis and hypercalcemia in some malignant lymphoma cases.

Molecular-cytogenetic Characterization of Non-Hodgkin's Lymphoma with Double and Cryptic Translocations of the Immunoglobulin Heavy Chain Gene

The present study aimed to characterize the clinical and molecular-cytogenetic features of non-Hodgkins lymphoma (NHL) with double translocation of the immunoglobulin heavy chain (IGH) gene. G-banding analysis, fluorescence in situ hybridization (FISH) with the IGH (Cgamma and VH) and oncogene (c-MYC, BCL1, BCL2, and BCL6) probes, and long-distance polymerase chain reaction (LD-PCR) were performed on 6 patients with B-cell lymphoma, one with angioimmunoblastic T-cell lymphoma, and one with acute lymphoblastic leukemia (ALL) with B-cell phenotype. G-banding analysis detected two different 14q32 translocations, t(14,18) and add (14)(q32) in a patient with ALL. Two distinct partners of double IGH translocation identified by FISH were as follows: c-MYC + BCL2 in 3 patients, c-MYC + BCL1 in 2, c-MYC + BCL6 in one, BCL2 + 9q22 in one, and 1q21 + 6q27 in one. Colocalization of BCL1 and c-MYC probes was demonstrated in a patient with mantle cell lymphoma. LD-PCR detected c-MYC/Cmu, c-MYC/Calpha and BCL6/Cmu, and c-MYC/Calpha fusion in each one patient. Seven of 8 patients showed high serum LDH. Central nervous system and leukemic involvement was observed in 5 and 6 patients, respectively. Median survival time of patients with c-MYC/IGH translocation was 9 months. The results defined a clinical subset of B-cell lymphoma/leukemia showing extremely poor prognosis. C-MYC/IGH translocation is possibly an evolutionary alteration following the primary IGH translocation with BCL1, BCL2, or BCL6. Furthermore, FISH identified one novel (9q22) and one cryptic chromosomal breakpoints (6q27) involved in IGH translocation.

A novel mature B‐cell line (DOBIL‐6) producing both parathyroid hormone‐related protein and interleukin‐6 from a myeloma patient presenting with hypercalcaemia

A novel human EBV‐negative B‐cell line, designated DOBIL‐6, was established from a patient with non‐secretary myeloma. The DOBIL‐6 cell has cytoplasmic γ protein and expresses CD19, 20, 38, 45RO, VLA‐4 and PCA‐1 antigens, but lacks CD10, 45RA and VLA5 antigens. Chromosome analysis showed that DOBIL‐6 cells had many complex structural abnormalities, including t(11;14) (q13;q32), which were consistent with that of the fresh tumour cells. Interestingly, abundant interleukin‐6 (IL‐6) and parathyroid hormone‐related protein (PTHrP) accumulated in the culture supernatant of DOBIL‐6 cells. Hypercalcaemia and splenomegaly associated with plasma cell proliferations which resulted in the expansion of the light zones in the follicles were observed in DOBIL‐6 transplanted nude mice. RT‐PCR analysis detected mRNA for PTHrP, and IL‐6 as well as its receptor (GP80) in DOBIL‐6 cells. Treatment of the DOBIL‐6 cells with neutralizing anti‐IL‐6 antibody inhibited their growth in a dose‐dependent manner, whereas the addition of exogenous IL‐6 stimulated it in serum‐depleted conditions. These findings suggest that both IL‐6 and PTHrP are produced in DOBIL‐6 cells, and that IL‐6 promotes its growth by an autocrine mechanism. Since IL‐6 is known to stimulate not only the growth of B‐cell neoplasms but also osteoclastic bone resorption by cooperating with PTHrP, this simultaneous production of IL‐6 and PTHrP might be synergistically linked and play a role in the development of hypercalcaemia of the patient. The DOBIL‐6 cell is a useful tool to clarify the mechanism of hypercalcaemia associated with mature B‐cell neoplasms.