Comparison of SGLT2 inhibitors vs. DPP4 inhibitors for patients with metabolic dysfunction associated fatty liver disease and diabetes mellitus.

PURPOSE: This study aimed to examine the potential benefit of sodium-glucose cotransporter 2 (SGLT2) inhibitors for patients with metabolic dysfunction-associated fatty liver disease (MAFLD) and diabetes mellitus (DM) using a real-world database. METHODS: We analyzed individuals with MAFLD and DM newly initiated on SGLT2 or dipeptidyl peptidase 4 (DPP4) inhibitors from a large-scale administrative claims database. The primary outcome was the change in the fatty liver index (FLI) assessed using a linear mixed-effects model from the initiation of SGLT2 or DPP4 inhibitors. A propensity score-matching algorithm was used to compare the change in FLI among SGLT2 and DPP4 inhibitors. RESULTS: After propensity score matching, 6547 well-balanced pairs of SGLT2 and 6547 DPP4 inhibitor users were created. SGLT2 inhibitor use was associated with a greater decline in FLI than DPP4 inhibitor use (difference at 1-year measurement, - 3.8 [95% CI - 4.7 to - 3.0]). The advantage of SGLT2 inhibitor use over DPP4 inhibitor use for improvement in FLI was consistent across subgroups. The relationship between SGLT2 inhibitors and amelioration of FLI was comparable between individual SGLT2 inhibitors. CONCLUSIONS: Our analysis using large-scale real-world data demonstrated the potential advantage of SGLT2 inhibitors over DPP4 inhibitors in patients with MAFLD and DM.

Visible luminescent lanthanide ions and a large π-conjugated ligand system shake hands.

Visible luminescence europium(iii) complexes with large π-conjugated systems were theoretically and experimentally studied. A strategy for extending the π-conjugation of a ligand for use with europium(iii) ions was found on the basis of fragment molecular orbital and density functional theory calculations. Using this method, a novel europium complex was designed and synthesized. Its excited state properties were assessed from the luminescence spectrum, excitation spectrum, luminescence lifetime, and luminescence quantum yield. In this study, the novel photophysics induced by the combination of visible luminescent europium(iii) ions and large π-conjugated systems are described.

Characterization of the immunoglobulin heavy chain complementarity determining region (CDR)-III sequences from human B cell precursor acute lymphoblastic leukemia cells.

Sequence analysis of the immunoglobulin heavy chain complementarity determining region (CDR)-III of B-lineage cells at various stages has provided important insights concerning B cell maturation and selection. Knowledge of human CDR-III sequences has been relatively limited compared with that of the murine system. We analyzed the CDR-III sequences of B cell precursor acute lymphoblastic leukemia (pre-B ALL) cells in 23 newly diagnosed and 10 relapsed patients, in order to elucidate the organization of CDR-III in B cell precursors. We found a very low frequency of somatic mutations in D and JH regions, preferential use of DLR, DXP, DHQ52, and DN elements, and of 3' side JH segments, and no predominant usage of D coding frames. Unusual joinings such as VH-D-D-JH and VH-JH were observed in three, and one sequences, respectively. We compared the CDR-III sequences derived from 10 patients between diagnosis and relapse. Two of them had three spots of mutated nucleotides at relapse, all of which were found in the N region near the D segments. Our data showed the possibility of somatic mutation at relapse, in addition to developmentally regulated rearrangement of the immunoglobulin gene at the stage of B cell precursors.

Efficacy and safety of recombinant human soluble thrombomodulin (ART-123) in disseminated intravascular coagulation: results of a phase III, randomized, double-blind clinical trial.

BACKGROUND: Soluble thrombomodulin is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C. OBJECTIVES: We conducted a multicenter, double-blind, randomized, parallel-group trial to compare the efficacy and safety of recombinant human soluble thrombomodulin (ART-123) to those of low-dose heparin for the treatment of disseminated intravascular coagulation (DIC) associated with hematologic malignancy or infection. METHODS: DIC patients (n = 234) were assigned to receive ART-123 (0.06 mg kg(-1) for 30 min, once daily) or heparin sodium (8 U kg(-1) h(-1) for 24 h) for 6 days, using a double-dummy method. The primary efficacy endpoint was DIC resolution rate. The secondary endpoints included clinical course of bleeding symptoms and mortality rate at 28 days. RESULTS: DIC was resolved in 66.1% of the ART-123 group, as compared with 49.9% of the heparin group [difference 16.2%; 95% confidence interval (CI) 3.3-29.1]. Patients in the ART-123 group also showed more marked improvement in clinical course of bleeding symptoms (P = 0.0271). The incidence of bleeding-related adverse events up to 7 days after the start of infusion was lower in the ART-123 group than in the heparin group (43.1% vs. 56.5%, P = 0.0487). CONCLUSIONS: When compared with heparin therapy, ART-123 therapy more significantly improves DIC and alleviates bleeding symptoms in DIC patients.

Expression and methylation status of the FHIT gene in acute myeloid leukemia and myelodysplastic syndrome.

To clarify the role of fragile histidine triad (FHIT) in hematological malignancies, we examined the methylation status and the expression level of the FHIT gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells in comparison with the methylation of the p15(INK4B) gene. The FHIT methylation was found in 13 of 94 (13.8%) AML and 22 of 40 (55.0%) MDS cases, but not in normal mononuclear cells (MNCs). Both the frequency and density of methylation increased in the advanced-stages MDS and the relapsed AML cases. Although FHIT and p15(INK4B) methylations were not correlated in MDS and AML, increased FHIT methylation at the relapse in AML was associated with p15(INK4B) methylation. The median expression level in AML was significantly higher than in normal MNCs, although the median expression level in those with methylation was significantly lower than in those without methylation. Furthermore, the methylation level at relapse was significantly higher than at diagnosis in AML. These results suggested that FHIT methylation was accumulated through the disease progression of MDS and AML, and the role of the FHIT gene as a tumor suppressor seemed different in AML and MDS.

Efficacy of gemtuzumab ozogamicin on ATRA- and arsenic-resistant acute promyelocytic leukemia (APL) cells.

Acute promyelocytic leukemia (APL) cells express a considerable level of CD33, which is a target of gemtuzumab ozogamicin (GO), and a significantly lower level of P-glycoprotein (P-gp). In this study, we examined whether GO was effective on all-trans retinoic acid (ATRA)- or arsenic trioxide (ATO)-resistant APL cells. Cells used were an APL cell line in which P-gp was undetectable (NB4), ATRA-resistant NB4 (NB4/RA), NB4 and NB4/RA that had been transfected with MDR-1 cDNA (NB4/MDR and NB4/RA/MDR, respectively), ATO-resistant NB4 (NB4/As) and blast cells from eight patients with clinically ATRA-resistant APL including two patients with ATRA- and ATO-resistant APL. The efficacy of GO was analyzed by (3)H-thymidine incorporation, the dye exclusion test and cell cycle distribution. GO suppressed the growth of NB4, NB4/RA and NB4/As cells in a dose-dependent manner. GO increased the percentage of hypodiploid cells significantly in NB4, NB4/RA and NB4/As cells, and by a limited degree in NB4/MDR and NB4/RA/MDR cells. Similar results were obtained using blast cells from the patients with APL. GO is effective against ATRA- or ATO-resistant APL cells that do not express P-gp, and the mechanism of resistance to GO is not related to the mechanism of resistance to ATRA or ATO in APL cells. Leukemia (2005) 19, 1306-1311. doi:10.1038/sj.leu.2403807; published online 26 May 2005.

Phase II study: treatment of non-Hodgkin's lymphoma with an oral antitumor derivative of bis(2,6-dioxopiperazine).

BACKGROUND: Although razoxane (ICRF-159), a derivative of bis(2,6-dioxopiperazine), has shown significant antitumor activity in several murine tumors, inadequate bioavailability has limited its clinical efficacy. Sobuzoxane (MST-16), another derivative of bis(2,6-dioxopiperazine), has shown activity against a broad spectrum of murine tumors and human tumor xenografts in nude mice and a lack of cross-resistance to vincristine, doxorubicin, cyclophosphamide, fluorouracil, etoposide, and mitomycin C. These findings suggest that MST-16 has a mode of cytocidal activity different from that of other antitumor agents. PURPOSE: The present late phase II study was conducted to evaluate the clinical efficacy and toxicity of MST-16 in non-Hodgkin's lymphoma (NHL). METHODS: As part of a multi-institutional cooperative study, we conducted a study of MST-16 in 27 patients with NHL who were assessable for drug efficacy and toxicity. MST-16, a bis(2,6-dioxopiperazine) analogue and an inhibitor of topoisomerase II, was administered orally at a dose of 1600 mg/m2 a day for 5-7 days at intervals of 2-3 weeks. RESULTS: Response consisted of one complete remission and seven partial remissions in 27 assessable patients. Response was achieved at a median of 13 days (range, 9-62 days) after initiation of therapy and lasted a median of 46 days (range, 29-155 days). Major toxic effects were leukopenia in 70% of the patients, thrombocytopenia in 44%, and gastrointestinal disorders in 37%. CONCLUSIONS: MST-16 was shown to be effective in NHL and deserves further clinical trial, since the drug shows little cross-resistance to available antitumor drugs. IMPLICATIONS: Phase II clinical studies of MST-16 in treatment of breast cancer, gastric cancer, and adult T-cell leukemia and lymphoma are also being conducted in Japan. Future trials of combination chemotherapy using MST-16 with other antitumor drugs are warranted in view of the additive effects observed in studies of MOLT-3 cells and studies of L1210 leukemia in mice.

Accelerated degradation of PML-retinoic acid receptor alpha (PML-RARA) oncoprotein by all-trans-retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway.

Acute promyelocytic leukemia (APL) is associated with a chromosomal translocation t(15;17) and successfully differentiated by all-trans-retinoic acid (ATRA) in vivo as well as in vitro. The PML-retinoic acid receptor alpha (RARA) oncoprotein, which is generated by the translocation, blocks the differentiation, and ATRA is thought to modulate the dominant negative function of PML-RARA. However, the molecular effect of ATRA on PML-RARA is unknown. In this study, we showed by means of immunoblotting that the expression of PML-RARA decreased within 12 h in APL cells treated with ATRA at concentrations greater than 0.1 microM. The decrease of PML-RARA was associated with restoration of the normal subcellular PML localization. PML-RARA transcripts were not down-regulated by ATRA. However, lactacystin, a specific inhibitor of the proteasome, almost completely inhibited the decrease of PML-RARA. These data indicate that the PML-RARA degradation is accelerated by pharmacological concentrations of ATRA, suggesting that ATRA allows APL cells to differentiate by relieving the differentiation block.

A study on thapsigargin-induced calcium ion and cation influx pathways in vascular endothelial cells.

To investigate Ca2+/cation entry pathway in vascular endothelial cells, we examined the effects of thapsigargin on [Ca2+]i and Mn2+ entry in cultured porcine aortic endothelial cells. Thapsigargin inhibits the activity of endoplasmic reticulum (ER, intracellular Ca2+ pool) Ca(2+)-ATPase, and stimulates Ca2+ entry from extracellular space by depleting intracellular Ca2+ pool. Cultured endothelial cells were loaded with fura-2/AM, and [Ca2+]i was measured by the ratios of fluorescence at 340/380 nm excitation, and Mn2+ entry was observed by the quenching of fluorescence at 360 nm excitation. Thapsigargin elevated [Ca2+]i in a time- and dose-dependent manner. The increase in [Ca2+]i caused by thapsigargin was lowered in Ca(2+)-free solution containing 3 mM EGTA. Verapamil (10(-5) M) and equimolar replacement of extracellular NaCl by LiCl had no effects on the maximum elevation of [Ca2+]i by thapsigargin. The increase in [Ca2+]i by thapsigargin was significantly inhibited by either NiCl2 (10(-3) M) or membrane depolarization using 50 mM KCl. Thapsigargin stimulated Mn2+ entry concomitantly with the increase in [Ca2+]i. Mn2+ entry was augmented in Ca(2+)-free solution. These results suggested that (1) the increase in [Ca2+]i by thapsigargin consisted of both Ca2+ release from ER and Ca2+ entry from extracellular space, and (2) thapsigargin also stimulated Mn2+ entry, which was interfered with in the presence of extracellular Ca2+.

Behenoyl cytosine arabinoside, daunorubicin, 6-mercaptopurine, and prednisolone combination therapy for acute myelogenous leukemia in adults and prognostic factors related to remission duration and survival length.

Fifty-one consecutive previously untreated adult patients with acute myelogenous leukemia (AML) were treated with BHAC-DMP (N4-behenoyl-I-beta-D-arabinofuranosyl-cytosine, daunorubicin, 6-mercaptopurine, and prednisolone) therapy. Forty-two patients (82.4%) achieved complete remission (CR). The Kaplan-Meier analysis revealed a probability for remaining in remission of 14% and for survival of 23% at 6 years. Pretreatment factors related to the achievement of CR, such as age, French-American-British (FAB) classification and WBC at the start of treatment, were not identified. Factors related to the CR duration and survival time of the patients who had achieved CR were first analyzed by a univariate analysis with the generalized Wilcoxon test. WBC count at the start of treatment, percent of blasts in the marrow at 1 and 2 weeks after the initiation of therapy, days required until CR, number of courses of induction therapy required until CR, and days required for the disappearance of circulating blasts were identified as statistically significant prognostic factors. When these characteristics were further analyzed by the Cox multivariate regression model, the percent of blasts in the bone marrow at 2 weeks was the most important prognostic factor with a statistical significance, and WBC count at the start of treatment and days required until CR (or number of courses required to achieve CR) were also important factors, with borderline significance.

An early phase II study of CPT-11: a new derivative of camptothecin, for the treatment of leukemia and lymphoma.

An early phase II study of a new camptothecin analog and an inhibitor of topoisomerase I, CPT-11, was conducted in 62 patients with refractory leukemia and lymphoma by four different treatment schedules in a multiinstitutional cooperative study. CPT-11 therapy resulted in four complete remissions (CRs) and three partial remissions (PRs) in 29 assessable non-Hodgkin's lymphoma (NHL) patients, one PR in three Hodgkin's disease (HD), one CR and one PR in 11 acute lymphoblastic leukemia (ALL), and one PR in 15 acute myelogenous leukemia (AML) patients. Single infusion of 200 mg/m2 every 3 to 4 weeks produced no response in both leukemia and lymphoma patients. Sixty-minute infusions of 40 mg/m2/d for 5 days every 3 to 4 weeks or for 3 days weekly produced four CRs (17%) and four PRs (17%) in 24 patients with malignant lymphoma. Sixty-minute infusions of 20 mg/m2 twice a day for 7 days every 3 to 4 weeks resulted in one CR and two PRs in 12 patients with acute leukemia. No response was seen in an acute leukemia patient by another treatment schedule. CPT-11 was effective in two (15%) of 13 primarily refractory leukemia and lymphoma cases, in two of four relapsed cases, and in seven (17%) of 41 relapsed and refractory cases. Major side effects were leukopenia (91%) and gastrointestinal (GI) (76%). CPT-11 was shown to be effective against refractory leukemia and lymphoma, and thus deserves further clinical study; the novel antitumor activity mode of this drug predicts no cross-resistance to presently available antitumor drugs.

Human urinary macrophage colony-stimulating factor reduces the incidence and duration of febrile neutropenia and shortens the period required to finish three courses of intensive consolidation therapy in acute myeloid leukemia: a double-blind controlled study.

PURPOSE: To determine whether macrophage colony-stimulating factor (M-CSF) reduces the incidence and duration of febrile neutropenia during three courses of intensive consolidation therapy and whether it shortens time to complete consolidation therapy. PATIENTS AND METHODS: In 198 adult patients with acute myeloid leukemia (AML) in complete remission (CR), M-CSF (8 x 10(6) U/d) or placebo was administered from 1 day after the end of each consolidation chemotherapy for 14 days. RESULTS: The duration and incidence of febrile neutropenia was significantly reduced by 34% (P = .00285) and 17% (P = .02065), respectively, in 88 assessable patients in the M-CSF group compared with those in 94 assessable patients in the placebo group. Patients in the M-CSF group had 565 days and 133 episodes of febrile neutropenia during 7,901 days at risk, while patients in the placebo group had 977 days and 185 episodes during 9,077 days at risk. The median period required to finish the three courses of consolidation therapy was 93 days in the M-CSF group, which was significantly shorter than 110 days in placebo group (P = .0050). In the M-CSF group, the recovery of neutrophils and platelets was significantly faster (P = .0348 and P = 0.0364, respectively), the administration of systemic antimicrobial agents tended to be less (P = .0839), and the frequency of platelet transfusion (P = .0259) and the total volume of transfused platelets (P = .0292) were significantly less. However, there was no significant difference in the disease-free survival. CONCLUSION: M-CSF significantly reduced the incidence and duration of febrile neutropenia during the intensive consolidation therapy, and shortened the time to complete consolidation chemotherapy in AML.

Analysis of prognostic factors in newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Japan Adult Leukemia Study Group.

PURPOSE: We conducted a multicenter study of differentiation therapy with all-trans retinoic acid (ATRA) followed by intensive chemotherapy in patients with newly diagnosed acute promyelocytic leukemia (APL) and analyzed the prognostic factors for predicting complete remission (CR), event-free survival (EFS), and disease-free survival (DFS). PATIENTS AND METHODS: All patients received ATRA until CR. If patients had an initial leukocyte count greater than 3.0 x 10(9)/L, they received daunorubicin (DNR) and behenoyl cytarabine (BHAC). During therapy, if patients showed blast and promyelocyte counts greater than 1.0 x 10(9)/L, they received additional DNR and BHAC. After achieving CR, patients received three courses of consolidation and six courses of maintenance/intensification chemotherapy. RESULTS: Of 198 registered, 196 were assessable (age range, 15 to 86 years; median, 46) and 173 (88%) achieved CR. Multivariate analysis showed that no or minor purpura at diagnosis (P = .0046) and age less than 30 years (P = .0076) were favorable factors for achievement of CR. Predicted 4-year overall survival and EFS rates were 74% and 54%, respectively, and the 4-year predicted DFS rate for 173 CR patients was 62%. Multivariate analysis showed that age less than 30 years (P = .0003) and initial leukocyte count less than 10 x 10(9)/L (P = .0296) were prognostic factors for longer EFS, and initial leukocyte count less than 10.0 x 10(9)/L was a sole significant prognostic factor for longer DFS (P = .0001). CONCLUSION: Our results show that age, hemorrhagic diathesis, and initial leukocyte count are prognostic factors for APL treated with ATRA followed by intensive chemotherapy.

Randomized trials between behenoyl cytarabine and cytarabine in combination induction and consolidation therapy, and with or without ubenimex after maintenance/intensification therapy in adult acute myeloid leukemia. The Japan Leukemia Study Group.

PURPOSE: We analyzed complete remission (CR), disease-free survival (DFS), and event-free survival (EFS) rates in two groups of patients treated with either N4-behenoyl-1-beta-D-arabinosylcytosine (BHAC) or cytarabine, and analyzed DFS with or without ubenimex, a biologic response modifier. PATIENTS AND METHODS: Newly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either BHAC or cytarabine as remission-induction combination chemotherapy and two courses of consolidation therapy. After maintenance/intensification therapy, patients in CR were randomized to receive either ubenimex and no drug. RESULTS: Of 341 patients registered, 326 were assessable. The age of assessable patients ranged from 15 to 82 years (median, 48). The overall CR rate was 77%: 72% in the BHAC group and 81% in the cytarabine group, and there was a significant difference between the two groups (P = .035, chi 2 test). The predicted 55-month EFS rate of all patients was 30%: 23% in the BHAC group and 35% in the cytarabine group, with a significant difference between groups (P = .0253). The predicted 55-month DFS rate of all CR patients was 38% and that of CR patients less than 50 years of age was 47%. There was no significant difference in DFS between the ubenimex group and the group that did not receive ubenimex. CONCLUSION: Analyses of our clinical trial showed that the use of BHAC in remission-induction therapy and in consolidation therapy resulted in poorer CR and EFS rates in adult AML patients compared with the use of cytarabine at the doses and schedules tested. Immunotherapy with ubenimex after the end of all chemotherapy did not improve DFS.

Clinicopathologic characteristics of leukemia in Japanese children and young adults.

The aim of this study is to clarify the clinicopathologic characteristics of adolescent leukemia in Japan by retrospective analysis. Patients with acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS), consecutively diagnosed from 1986 to 1999, were enrolled. A total of 3,856 patients from 1 to 15 years of age and 1,803 patients from 15 to 29 years of age were eligible for this study. Demographically, the frequency of AML found was almost constant during the teenage years, whereas the frequency of ALL gradually decreased. The relative frequency of CML and MDS apparently started to increase in patients in their late teens. The relative frequency of M3 and t(15;17) gradually increased during adolescence. Among patients aged 1 to 4 years, M7 was the most frequent FAB subtype. Among patients aged 5 to 9 years, M2 and t(8;21) was the most frequent subtype. The percentage of T cell ALL increased in patients 5 to 9 years old, reaching 31.2% in the 20- to 24-year-old age group. The percentage of patients with hyperdiploidy over 50 chromosomes was highest (17.0%) in patients aged 1 to 4 and decreased to 3.9% in the older teens. The percentage of patients with the Ph1 chromosome increased from 9.9% in teens to 30.0% in patients in their late twenties. When comparing event-free survival (EFS) rates for ALL according to age, the estimated 7-year EFS rate was highest for patients aged 1 to 9 years (65.9%) and intermediate for patients aged 10 to 15 years (48.4%). However, the EFS rate was significantly worse for patients aged 15 to 19 years (19.4%) and 20 to 29 years (17.0%) (P = 0.024). On the other hand, the EFS rate for AML decreased with increasing age, although without statistical significance. The overall survival rates are approximate among all age groups. The results of the study indicate that there are considerable variations in biologic features of leukemia between children and young adults. The prognosis for adolescent leukemia may be improved by introducing pediatric trials, which take into account the prognostic biological features.

Treatment of acute promyelocytic leukemia: strategy toward further increase of cure rate.

Acute promyelocytic leukemia (APL) has become a curable disease by all-trans retinoic acid (ATRA)-based induction therapy followed by two or three courses of consolidation chemotherapy. Currently around 90% of newly diagnosed patients with APL achieve complete remission (CR) and over 70% of patients are curable. To further increase the CR and cure rates, detection and diagnosis of this disease at its early stage is very important, hopefully before the appearance of APL-associated coagulopathy. In induction therapy, concomitant chemotherapy is indispensable, except for patients with low initial leukocyte counts. Prophylactic use of intrathecal methotrexate and cytarabine should be done, particularly for patients with hyperleukocytosis. If patients relapse hematologically or even molecularly, arsenic trioxide will be the treatment of choice under careful electrocardiogram monitoring. Am80, liposomal ATRA, gemtuzumab ozogamicin or ATRA in combination with cytotoxic drugs may be used at this stage or later. Allogeneic SCT will be the treatment of choice after patients of age <50 years have relapsed, provided that they have HLA-identical family donors or DNA-identical unrelated donors.

Comparable gene structure of the immunoglobulin heavy chain variable region between multiple myeloma and normal bone marrow lymphocytes.

To characterize multiple myeloma (MM) from the viewpoint of the immunoglobulin (Ig) gene structure, we compared the transcripts of the Ig heavy chain variable region from 23 MM samples with 221 clones of the gamma, alpha and mu chain transcripts amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) from normal bone marrow (BM) cells. The usage of D and JH gene segments and the length of the N regions were the same between MM and the normal gamma, alpha and mu transcripts. Compared with the known germline VH genes, the frame work regions (FWRs) and complementarity determining regions (CDRs) of the VH segments mutated at rates of 8.3 +/- 4.7% and 15.9 +/- 7.7%, respectively, which were the same as the normal gamma and alpha (gamma/alpha) transcripts and higher than the normal mu transcripts. The replacement/silent (R/S) ratios of the mutations in FWRs and CDRs were 1.9 +/- 1.3 and 2.7 +/- 1.8, respectively, which were the same as the gamma/alpha and mu transcripts. On the other hand, we detected the clone-specific mu transcripts by RT-PCR using the primers corresponding to the each respective CDR-III and the constant region of the mu chain in three of the studied six MM samples, suggesting the involvement of a pre-switched B cell in some cases of MM. These findings suggested that the cellular origin of MM is heterogeneous, but that the Ig structure in MM reflects normal B cell maturation to plasma cell through mutation and selection.

Molecular analysis of the t(15;17) translocation in de novo and secondary acute promyelocytic leukemia.

To study mechanism of chromosomal translocation, we analyzed the breakpoints (b/p) of the PML and RARA genes in 120 and 5 patients with de novo and secondary (therapy-related) acute promyelocytic leukemia (APL), respectively. In de novo APL, the b/p in the PML gene were clustered in introns 3 (bcr 3; 30%) and around intron 6 (bcr 1 and 2: 70%). The b/p of the RARA gene were widely distributed in intron 2. In studied 8 de novo APL patients, no consensus sequence-motif was found around the b/p, but there were identical stretches of one to seven nucleotides between the PML and RARA genes in the joining regions, suggesting non-selective DNA double strand cleavage followed by single strand base-pairing within identical short stretches as a molecular mechanism of the translocation. In 4 secondary APL patients after chemotherapy including etoposide against Langerhans cell histiocytosis, the b/p of the PML gene were located in intron 6, and those of the RARA gene were in a restricted region within intron 2, 1 kb EcoRI-BamHI fragment, while in an APL patient after chemotherapy without etoposide against breast cancer, the b/p of the PML and RARA genes were located in intron 6 and another region within intron 2, respectively. These data suggest that a different mechanism was associated with the t(15;17) translocation in etoposide-related APL.

Comparison of the immunoglobulin gene transcripts between immature B lineage acute lymphoblastic leukemia and the normal phenotypic counterparts in the bone marrow.

Immature B lineage acute lymphoblastic leukemia (ALL) is divided into two subtypes, 'pre-B' and 'early pre-B' ALL, by the presence or absence of cytoplasmic immunoglobulin (cIg). To study their clonal origin, we compared mu-chain transcripts in six cIg+ and eight cIg- ALL samples (CD10+/- CD19+ surface Ig-) with those in the normal phenotypic counterparts (CD10+ CD19+ surface Ig-) sorted from the bone marrow (BM). Northern blot analysis showed that the cIg+ ALL samples expressed greater amounts of mu-chain transcripts than the cIg- ALL samples. In the ALL samples and their counterparts, sequence analysis of mu-chain transcripts revealed infrequent somatic mutations of the V(H) genes and the similar usage of D and J(H) gene segments, but the length of complementarity determining region (CDR)-3 in the ALL samples was longer than that in the counterparts (50.0 +/- 15.5 vs 40.8 +/- 12.7 bp, P = 0.01). The mu-chain transcripts in the six cIg+ ALL samples and the counterparts (119/120 clones) had productive sequences, whereas those in the eight cIg- ALL samples had nonsense codons and/or frame shifts in their CDR-3. Our data suggest that a phenotype of ALL, 'pre-B' or 'early pre-B', is associated with V(H)-D-J(H) gene recombinatorial events, and that the CD10+ CD19+ surface Ig- population in the BM is not simply the cellular origin of ALL.

Characterization of the immunoglobulin light chain variable region gene expressed in multiple myeloma.

We studied the organization, diversification and clinical significance of the immunoglobulin light chain (IgL) variable region genes expressed in 17 kappa-chain and 16 lambda-chain producing multiple myeloma (MM) samples. The V genes from 31 MM samples had over 84.9% homology to the known germline Vkappa/lambda genes, whereas one Vkappa and one Vlambda gene had only 75.5% and 65.9% homology, respectively. While all five Jkappa segments were equally used, only Jlambda-1 or Jlambda-2/3 was used among seven Jlambda segments. N nucleotide addition was found at two Vkappa-Jkappa and five Vlambda-Jlambda junctions. The lambda-chain complementarity determining region (CDR)-3 was longer and more variable than the kappa-chain CDR-3 mainly due to junctional flexibility of Vlambda and Jlambda segments. Somatic mutations were more frequent in the Jlambda than the Jkappa segments, and were distributed in the CDR-3 as well as the frame work region (FWR)-4. Those of the Jkappa segments, however, were limited to FWR-4. In FWR-4, replacement mutations were clustered at codon 106 of kappa-chain and 103 of lambda-chain. Thus nucleotide mutation or conservation was dependent on position, indicating a structural necessity of IgL for the development of myeloma cells in addition to a non-random distribution of mutations. There was no characteristic IgL sequence according to the isotype of M-protein, clinical stage or renal complication.