Identification of insulin-like growth factor 2 mRNA-binding protein 3 as a radioresistance factor in squamous esophageal cancer cells.

Identification of reliable markers of radiosensitivity and the key molecules that donate susceptibility to anticancer treatments to esophageal cancer cells would be highly desirable. We found that the mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was higher in radioresistant TE-5 and TE-9 cells than in radiosensitive TE-12 cloneA1 cells. Conversely, knocking down expression of IGF2BP3 mRNA in TE-5 and TE-9 cells using small interfering RNA significantly enhanced their radiosensitivity. Furthermore, patients with squamous cell esophageal cancers strongly expressing IGF2BP3 tended to respond poorly to chemoradiation. These data suggest that IGF2BP3 may be a key marker of radiosensitivity that diminishes the susceptibility of squamous cell esophageal cancer cells to radiotherapy. IGF2BP3 may, thus, be a useful target for improving radiotherapy for patients with esophageal squamous cell carcinoma.

Risk and prognostic factors for Japanese patients with chronic graft-versus-host disease after bone marrow transplantation.

The incidence and prognostic factors for chronic graft-versus-host disease (cGVHD) were evaluated for 255 Japanese patients who survived more than 100 days after bone marrow transplantation, and of whom 119 (47%) developed cGVHD. Prior acute GVHD (grade 2-4) and use of an unrelated donor were significantly associated with the onset of cGVHD. Presence of cGVHD did not have an impact on mortality (hazard ratio (HR) = 0.89; 95% confidence interval (CI), 0.59-1.3). Three factors at diagnosis were associated with cGVHD-specific survival: presence of infection (HR = 4.1; 95% CI, 1.6-10.3), continuing use of corticosteroids at the onset of cGVHD (HR = 3.9; 95% CI, 1.7-9.1), and a Karnofsky performance score <80 (HR = 4.7; 95% CI, 2.0-11.3). The probability of cGVHD-specific survival at 4 years was 79% (95% CI, 70-86%). The severity and death rate of Japanese patients with cGVHD was lower than those for populations in Western countries, which might be the result of greater genetic homogeneity of Japanese ethnics. Our patients could not be accurately classified when the proposed prognostic models from Western countries were used, thus indicating the need for a different model to identify high-risk patients.

Cytomegalovirus antigenemia and outcome of patients treated with pre-emptive ganciclovir: retrospective analysis of 241 consecutive patients undergoing allogeneic hematopoietic stem cell transplantation.

CMV disease remains a major infectious complication after allogeneic hematopoietic stem cell transplantation (HSCT). To investigate the relationship between CMV antigenemia, treatment with ganciclovir (GCV), and outcome, we retrospectively analyzed 241 consecutive patients at risk for CMV infection who underwent allogeneic HSCT. Antigenemia-guided pre-emptive strategy with GCV was used for all patients. CMV antigenemia developed in 169 patients (70.1%), and CMV disease in 18 patients (7.5%). Multivariate analysis showed that acute GVHD (grades II-IV) was the only risk factor for developing antigenemia, and acute GVHD and advanced age for CMV disease. GCV use, as well as acute GVHD and advanced age, significantly increased the risk for bacterial and fungal infection after engraftment. Those who developed CMV antigenemia had a poorer outcome than those who did not (log-rank, P=0.0269), although the development of CMV disease worsened the outcome with only borderline significance (log-rank, P=0.0526). In conclusion, detection of antigenemia proved to be a poor prognostic factor for HSCT patients, which may be attributed to a combination of factors, including CMV disease itself, the effect of treatment, and a host status that allows for reactivation of CMV. Optimal pre-emptive strategy needs to be determined.

Male siblings with dyserythropoiesis, microcephaly and intrauterine growth retardation.

Male siblings with intrauterine growth retardation, hydrops, mild liver dysfunction, chronic diarrhoea, failure to thrive and microcephaly are reported. In both patients, the intrauterine growth retardation was detected in the second trimester of pregnancy. Relatively severe early onset neonatal jaundice, microcytosis, anisocytosis and abnormal iron metabolism were also seen. Bone marrow examination in the second sibling showed marked ringed sideroblasts and multilobulated erythroblasts in late developmental stages. The brain was very small with enlarged cerebrospinal fluid space, a reduced number of gyri and a thin cortex. The clinical and laboratory findings in these patients appear to be unique.

Clonality assay of hematopoietic disorders: significance of the buccal epithelium as non-hematopoietic control and of 95% rejection limit as a novel criterion for monoclonality.

In clonality assays using X chromosome inactivation patterns (XCIPs), several factors such as constitutive and acquired XCIP skewing, lack of appropriate controls for hematopoietic diseases including multilineage disorders, and ambiguous criteria for monoclonality, have complicated determination of clonality. To address these issues, we studied the significance of the buccal epithelium as a non-hematopoietic control and the usefulness of the 95% rejection limit as a criterion for monoclonality. Sixty-nine females informative for human androgen receptor gene (HUMARA) were divided into "young," "middle-aged" and "elderly" groups. When XCIP correlation between the buccal epithelium, peripheral granulocytes, and peripheral lymphocytes was analyzed, the buccal epithelium showed a good correlation with granulocytes and lymphocytes in "young" and "middle-aged" groups, whereas the correlation was poor for the "elderly" group. For all age groups, there was an excellent correlation between granulocytes and lymphocytes. When we performed clonality assay for seven "young" and "middle-aged" patients with various leukemic phases using buccal epithelium as a non-hematopoietic control, all cases were accurately evaluated with the aid of a novel criterion, the 95% rejection limit. Our findings suggest that the buccal epithelium may constitute an effective control, especially when a non-hematopoietic control is required, and that the 95% rejection limit may serve as a statistically-defined criterion for monoclonality.

Pregnancy-associated cytotoxic lymphoma: a report of 4 cases.

The clinicopathological and biological significance of Hodgkin's disease and non-Hodgkin's lymphoma, which are infrequently encountered in women of childbearing age, remains to be clarified. We recently reviewed 4 cases of non-Hodgkin's lymphoma of the T/natural killer (T/NK)-cell phenotype, all of which were associated with pregnancy and characterized by the expression of the cytotoxic granule-associated proteins T-cell intracellular antigen-1 and/or granzyme B. The 4 cases selected had presented between November 1993 and May 1999. The criteria for selection were that the onset of clinical manifestations occurred during pregnancy or within 6 months after delivery. The patients comprised 1 patient with p80/anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL), 1 with p80/ALK-negative ALCL, and 2 with peripheral T/NK-cell lymphomas of unspecified type. The diseases followed aggressive clinical courses: 3 patients died within 6.5 months after diagnosis, and only 1 was still alive with the disease 17 months after diagnosis. The diseases appeared to progress rapidly after delivery. Maternal immunity and hormonal changes during pregnancy may be closely related to the biological behavior of these unusual tumors. This study is, to the best of our knowledge, the first to address pregnancy-associated cytotoxic lymphoma.

Posttreatment nadir M-protein level is a stronger discriminator of survival following plateau attainment than is percent reduction in M-protein in patients with IgG myeloma.

We conducted a retrospective study of patients with IgG or IgA myeloma who attained plateau to evaluate the relationships between survival and posttreatment nadir M-protein levels and between survival and the response to treatment evaluated by the percent reduction in M-protein. Of the 127 patients comprising 92 IgG and 35 IgA myeloma patients with disease stages II or III, 51 (40.2%) attained plateau. For IgG myeloma patients who attained plateau, survival time was not affected by the percent reduction in M-protein (median survival, 59.5 months for responding patients versus 54.4 months for nonresponding patients, P = .6910). Posttreatment nadir M-protein level, however, did affect survival time (median survival, 61.2 months for <3000 mg/dL versus 25.7 months for >3000 mg/dL, P = .0439). These findings suggest that the posttreatment nadir M-protein level is a stronger discriminator of survival following plateau attainment than the percent reduction of M-protein in patients with IgG myeloma.

Ectopic expression of MAFB gene in human myeloma cells carrying (14;20)(q32;q11) chromosomal translocations.

Chromosome 14q +, which represents a chromosomal rearrangement involving the immunoglobulin heavy chain gene (IgH) locus, is a genetic hallmark of human multiple myeloma (MM). Here, we report the identification of (14;20)(q32;q11) chromosomal translocations found in MM cells. Double color fluorescence in situ hybridization analyses pinpointed the breakpoints at the 20q11 locus in two MM cell lines within a length of at most 680 kb between the KIAA0823 and MAFB gene loci. Among the transcribed sequences in the vicinity of the breakpoints, an ectopic expression of the MAFB gene, which is located at 450 - 680 kb telomeric to one of the breakpoints and encodes a member of the MAF family basic region / leucine zipper transcription factor, was demonstrated to be associated with t(14;20). This finding, together with that of a previous study describing its transforming activity, suggests that the MAFB gene may be one of the targets deregulated by regulatory elements of the IgH gene as a result of t(14;20).

Improvement of splenomegaly and pancytopenia by enzyme replacement therapy against type 1 Gaucher disease: a report of sibling cases.

Gaucher disease is a genetic lipid storage disease and represents a potentially serious health problem. It arises from a deficiency of glucocerebrosidase activity with secondary accumulation of large quantities of glucocerebroside. Symptoms are usually multisystemic, often debilitating or disabling, and sometimes disfiguring, and they can lead to death. We report objective clinical response's to repeated infusion of human placental and recombinant glucocerebrosidase in 2 patients with type 1 Gaucher disease and increased hemoglobin levels and platelet counts. Splenic volume decreased during the period of enzyme administration. Enzyme replacement therapy has improved the treatment of type 1 Gaucher disease by safely and effectively arresting, decreasing, or normalizing many of its major signs and symptoms. Consideration by physicians must be given to Gaucher disease, and appropriate treatments must be given when confronted with cryptogenic pancytopenia or hepatosplenomegaly.

CT-pathologic correlation in a case of solitary plasmacytoma of the rib.

We report a case of solitary plasmacytoma of rib origin in a 46-year-old man. CT revealed a focal expanding mass in the right fifth rib. The mass showed ridging, cortical thickening, and fine erosion on the inner aspect of the cortex and had partly penetrated the bone. On both T1- and T2-weighted MR images, the mass showed low to intermediate intensity, and contrast studies showed homogeneous enhancement. Whole body bone scintigraphy revealed abnormal solitary accumulation, and therefore surgical resection was performed. Macroscopically the specimen revealed destruction of the cortex caused by tumor invasion, corresponding to the CT findings. Microscopic findings included a large number of plasma cells, leading to a histopathological diagnosis of plasmacytoma.

A novel human multiple myeloma-derived cell line, NCU-MM-1, carrying t(2;11)(q11;q23) and t(8;22)(q24;q11) chromosomal translocations with overexpression of c-Myc protein.

A novel cell line, designated as NCU-MM-1, was established from a 66-year-old female patient with multiple myeloma (MM) that had shown rapid progression from solitary plasmacytoma to plasma cell leukemia. Interestingly, cytogenetic analysis including fluorescence in situ hybridization analysis disclosed that this cell line carried 2 kinds of chromosomal translocations involving immunoglobulin light chain (IgL) gene loci without the presence of 14q32 translocations (14q+). The Ig lambda locus juxtaposed to the c-MYC locus at 8q24 on the derivative (8) chromosome and a concomitant overexpression of the c-Myc protein was observed. On the derivative (11) chromosome, the Ig kappa locus was also fused to the chromosome 11q23 locus, which is known to be a nonrandom translocation breakpoint in mature B-cell malignancies. The NCU-MM-1 cell line may thus be useful not only for the identification of the responsible proto-oncogene(s) mapped to 11q23, deregulated by the Ig kappa enhancer sequences, but also for clarification of the molecular origin of MM lacking 14q+ chromosomes because IgL rearrangements can physiologically begin to occur in the pre-B-cell stage.

MUM1/IRF4 expression as a frequent event in mature lymphoid malignancies.

MUM1/IRF4 is a myeloma-associated oncogene transcriptionally activated as a result of t(6;14)(p25,q32) chromosomal translocation and by virtue of its juxtaposition to the immunoglobulin heavy chain gene (IgH) locus. When this oncogene becomes non-functional, no activated B/T lymphocytes and Ig secreting plasma cells are observed, suggesting that MUM1/IRF4 is crucial for lymphoid development. Its expression was analyzed in both reactive lymphoid and lymphoma tissues by means of an immunohistochemical technique using specific goat antiserum against MUM1/IRF4. This analysis detected a 50 kDa MUM1 product whose localization was restricted to the nuclei of the lymphocytes. The MUM1+ cells in reactive lymph nodes were found to consist of plasma cells and a small fraction (approximately 7.9%) of B cells harboring CD20+CD38+, which were located in the light zone of the germinal center. MUM1 expression in peripheral blood B/T lymphocytes was upregulated by mitogenic stimuli, suggesting that MUM1 positivity represents the activated state of the B/T cells. In B cell non-Hodgkin's lymphoma (NHL), MUM1 expression was observed in 73.2% (30/41) of diffuse large B cell lymphoma (DLBCL), 20% (1/5) of marginal zone lymphoma (MZL) and 43% (3/7) of small lymphocytic lymphoma (SLL) cases, whereas it was not seen in any cases of mantle cell lymphoma (MCL) or follicle center lymphoma (FCL). Also, MUM1 was stained at high intensity in various types of T cell lymphomas including adult T cell leukemia/lymphoma (ATL/L) and anaplastic large cell lymphoma (ALCL) and in the majority of Hodgkin's diseases. Our results suggest that a major proportion of lymphomas comprise either physiologically or aberrantly activated neoplastic lymphocytes expressing the MUM1 protein.

No significant association between HA-1 incompatibility and incidence of acute graft-versus-host disease after HLA-identical sibling bone marrow transplantation in Japanese patients.

We retrospectively examined HA-1 typing with polymerase chain reaction using sequence-specific primers in 120 samples from 60 HLA-A2-positive Japanese bone marrow transplantation recipients who received short-term methotrexate and cyclosporin A for graft-versus-host disease (GVHD) prophylaxis and their HLA-identical sibling donors. HA-1-incompatible pairs were observed in 22% of the samples. The probability of developing acute GVHD (grade II to IV) in HA-1-incompatible and -compatible patients was 0% and 19%, respectively (P = .10). In a comparison between HA-1-incompatible and -compatible patients with standard-risk leukemia, in whom age, patient/donor sex, and use of a total body irradiation-containing regimen were equivalent, the probability of developing acute GVHD (grade II to IV) was 0% and 10%, respectively (P = .38). No evidence of recurrent leukemia was observed in the HA-1-incompatible patients with standard-risk leukemia, compared with 37% in HA-1-compatible patients (P = .11). In conclusion, HA-1 incompatibility may not be a risk factor for grade II to IV acute GVHD in Japanese patients who receive methotrexate and cyclosporin A and undergo bone marrow transplantation from HLA-identical sibling donors.

Detection of MUM1/IRF4-IgH fusion in multiple myeloma.

MUM1 (multiple myeloma oncogene 1)/IRF4 (interferon regulatory factor 4) gene has been identified as an oncogene transcriptionally activated by t(6;14)(p25;q32) chromosomal translocation in multiple myeloma (MM). The significance of this alteration in MM remains unknown, as it is not detectable by means of conventional cytogenetic analysis. To address this issue, we established diagnostic procedures based on pulsed-field gel electrophoresis (PFGE) analysis and double color fluorescence in situ hybridization (DCFISH) using DNA probes derived from the MUM1 and the immunoglobulin heavy chain (IgH) gene loci. Among a panel of 17 MM cell lines, three (17.6%) showed fusions between these two loci, which resulted in the juxtaposition of the MUM1 to the IgH 3' alpha-enhancer region by virtue of t(6;14) or insertion of the IgH sequences into the vicinity of the MUM1 gene and in the concomitant overexpression of the MUM1 mRNA. With similar results, fusions between MUM1 and IgH loci were observed by means of interphase DCFISH in eight (21.1%) out of the 38 MM cases, although no definite relationships between MUM1 status and specific clinical findings could be established.

Prognostic significance of CD44v6 in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma in the Revised European-American Lymphoma Classification encompasses various morphologic subtypes of diffuse large-cell lymphomas of B-cell origin. The category is biologically and clinically heterogeneous, even though it constitutes approximately 30% of all non-Hodgkin's lymphomas. Clinically, the International Prognostic Index that identifies high-risk group in aggressive non-Hodgkin's lymphomas is widely accepted. Lacking, however, are biologic or molecular prognostic markers that might aid in understanding the pathogenesis and designing specific therapies. CD44 isoforms are involved in tumor dissemination and might be associated with aggressive behavior of non-Hodgkin's lymphomas. We studied immunohistochemical expression of CD44s and CD44v6 in the tumors and examined their clinical significance in a cohort of patients with primary nodal diffuse large B-cell lymphoma who were uniformly evaluated and treated with doxorubicin-containing chemotherapy (n = 42). In contrast to CD44s signals, CD44v6 signals were weak in routinely processed non-Hodgkin's lymphoma sections. Therefore, we used a highly sensitive catalyzed reporter deposition system and successfully detected CD44v6 signals in diffuse large B-cell lymphomas. Overexpression of the isoform was verified by Southern blot of reverse transcription polymerase chain reaction products. CD44s and CD44v6 were positive in 17 (40%) of 42 and 13 (31%) of 42, respectively. CD44v6 was detected predominantly in lymphoma cells, whereas CD44s was often positive for nonneoplastic small lymphocytes as well. In univariate regression analysis, the B symptoms, being in the International Prognostic Index high-risk group, and CD44v6 expression emerged as significant parameters for poorer overall survival, but CD44s expression did not achieve statistical significance. When multivariate regression analysis was performed using the former three parameters, only CD44v6 expression remained significant (P = .017; relative risk = 3.48), indicating that CD44v6 is a molecule particularly important for predicting worse prognosis. CD44v6, which can be detected in the archival materials, might be a biologically and clinically useful marker in identifying the high-risk group in the diffuse large B-cell lymphoma category of the Revised European-American Lymphoma Classification.

Multiple myeloma preceding the development of chronic myelogenous leukemia.

A case of a 70-year-old man who first developed multiple myeloma and then chronic myelogenous leukemia (CML) within a 3-year period is documented. The patient, with monoclonal hypergammopathy, was diagnosed with smoldering myeloma with IgG-kappa and Bence Jones protein kappa paraproteinemia. No chemotherapy was given for the myeloma until progressive leukocytosis developed after approximately 3 years. This was found to be due to Philadelphia chromosome positive CML. A reverse transcription-polymerase chain reaction assay did not reveal BCR/ABL mRNAs when the myeloma was first diagnosed. The occurrence of 2 distinct hematologic malignancies in the same patient suggests either a different clonal evolution from a common pluripotent malignant stem cell since the CML stem cell also involves the B-lymphoid lineage, a coincident complication of the 2 hematological malignancies, or the coexistence of 2 distinct malignancies due to the same genetic background and/or exposure to similar carcinogenic agents. The literature provides support for the existence of a relationship between multiple myelomas and CML.

Microsatellite instability as a potential marker for poor prognosis in adult T cell leukemia/lymphoma.

Microsatellite instability (MSI) represents a replication error resulting from the dysfunction of mismatch repair gene products. In this study, MSI was analyzed in 18 patients with various subtypes of adult T cell leukemia/lymphoma (ATL/L). Using six different microsatellite loci, we defined MSI as positive when replication errors were observed in at least two loci. The MSI was positive in four cases (22.2%)with acute type ATL, who tended to show more prognostically unfavorable factors and shorter overall survival. These results suggest that genomic instability may be associated with tumor progression rather than the development of ATL/L itself. In addition, the presence of the MSI at initial presentation could appear to warrant consideration as an additional prognostically unfavorable factor.

Microsatellite instability is rare in the clinical course of myelodysplastic syndrome studied with DNA from fresh and paraffin-embedded tissues.

Microsatellite instability (MSI) has been reported to occur in various types of malignant neoplasms. We performed a polymerase-chain-reaction-based assay for MSI between the initial and the most recently available ("latest") samples from 23 patients with myelodysplastic syndrome (MDS). Of these patients, 15 were informative at more than three microsatellite loci. Seven patients showed an increase in leukemic cells while 8 patients did not during the interval between the two analyses. Only 1 of the patients, who had refractory anemia with excess blasts, which changed to acute myelogenous leukemia, showed microsatellite alteration at the analysis times. Among all 23 patients, two alterations were detected in the 42 informative paired samples that showed an increase in leukemic cells (4.8%), while none was detected in the 59 paired samples without such an increase. In total, therefore only two alterations were detected among 101 informative paired samples (2%). This indicates that MSI is rare in the clinical course of MDS irrespective of disease status, and is consequently not a critical genetic event for disease progression in most MDS patients.

Detection of bcr/abl fusion transcripts by semiquantitative multiplex RT-PCR combined with a colormetric assay in Ph positive leukemia.

We studied the feasibility of the clinical application of a new bcr/abl analysis system, C-TRAK t(9;22), consisting of a multiplex RT-PCR and a colormetric assay. With this system, bcr/abl transcripts could be detected in all of 24 cytogenetic Philadelphia chromosome (Ph) positive leukemia patients and in none of eight Ph negative patients. Multiple bcr/abl transcripts could be detected in three of the 24 Ph positive patients, the fusion of bcr exon 1 to abl exon 2 (e1a2 junction) dominated that of bcr exon 13 to abl exon 2 (b2a2 junction) in two cases and that of bcr exon 14 to abl exon 2 (b3a2 junction) and b2a2 dominated e1a2 in one case. This system was sensitive enough to be able to detect even one bcr/abl transcript-producing cell in 50000 bcr/abl negative background cells, thus making it suitable for semiquantitative evaluation. Minimal residual disease (MRD) was monitored in one Ph positive leukemia patient who underwent allogenic bone marrow transplantation (allo-BMT). After allo-BMT, a weak positivity of the bcr/abl transcript continued with no clinical relapse; this result was consistent with that of a conventional nested PCR assay using ethidium bromide staining. Including all the procedures for RNA extraction, it took only about 10 h to detect the bcr/abl transcripts. Our findings indicate that this bcr/abl analysis system provides a quick and sensitive method for screening bcr/abl transcripts and possibly for monitoring MRD in Ph positive leukemia patients.

Idiopathic CD4+ T-lymphocytopenia in a non-Hodgkin's lymphoma patient.

We report a case of idiopathic CD4+ T-lymphocytopenia with malignant lymphoma (diffuse large, B-cell type) for which there was no evidence of human immunodeficiency virus type 1 or type 2 infection and no other known causes of immunodeficiency. She had never suffered from any opportunistic infection until the diagnosis of malignant lymphoma was made, and the CD4+ T-lymphocytopenia persisted after complete remission of the lymphoma. As the clinical features and immune status of the patient differed from those associated with the acquired immunodeficiency syndrome (AIDS)-related syndrome, we conclude that immunodeficiency in this case did not contribute to the opportunistic infection but may have been associated with the genesis of malignant lymphoma.