Pre-transplant imatinib-based therapy improves the outcome of allogeneic hematopoietic stem cell transplantation for BCR-ABL-positive acute lymphoblastic leukemia.

A high complete remission (CR) rate has been reported in newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) following imatinib-based therapy. However, the overall effect of imatinib on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is undetermined. Between 2002 and 2005, 100 newly diagnosed adult patients with Ph+ALL were registered to a phase II study of imatinib-combined chemotherapy (Japan Adult Leukemia Study Group Ph+ALL202 study) and 97 patients achieved CR. We compared clinical outcomes of 51 patients who received allo-HSCT in their first CR (imatinib cohort) with those of 122 historical control patients in the pre-imatinib era (pre-imatinib cohort). The probability of overall survival at 3 years after allo-HSCT was 65% (95% confidence interval (CI), 49-78%) for the imatinib cohort and 44% (95% CI, 35-52%) for the pre-imatinib cohort. Multivariate analysis confirmed that this difference was statistically significant (adjusted hazard ratio, 0.44, P=0.005). Favorable outcomes of the imatinib cohort were also observed for disease-free survival (P=0.007) and relapse (P=0.002), but not for non-relapse mortality (P=0.265). Imatinib-based therapy is a potentially useful strategy for newly diagnosed patients with Ph+ALL, not only providing them more chance to receive allo-HSCT, but also improving the outcome of allo-HSCT.

Induction therapy by frequent administration of doxorubicin with four other drugs, followed by intensive consolidation and maintenance therapy for adult acute lymphoblastic leukemia: the JALSG-ALL93 study.

In order to improve the disappointing prognosis of adult patients with acute lymphoblastic leukemia (ALL), we applied similar induction therapy as that used for acute myeloid leukemia (AML), ie frequent administration of doxorubicin (DOX). DOX 30 mg/m(2) was administered from days 1 to 3 and from days 8 to 10 together with vincristine, prednisolone, cyclophosphamide and L-asparaginase, followed by three courses of consolidation and four courses of intensification. From December 1993 to February 1997, 285 untreated adult patients with de novo ALL were entered. Of 263 evaluable patients (age 15 to 59; median 31), 205 (78%) obtained complete remission (CR). At a median follow-up period of 63 months, the predicted 6-year overall survival (OS) rate of all patients was 33%, and disease-free survival (DFS) rate of CR patients was 30%, respectively. By multivariate analysis, favorable prognostic factors for the achievement of CR were age <40 and WBC <50 000/microl; for longer OS were age <30 and WBC <30 000/microl; and for longer DFS of CR patients were FAB L1 and ALT <50 IU/l. Among 229 patients who had adequate cytogenetic data, 51 (22%) had Philadelphia (Ph) chromosome. Ph-negative chromosome was a common favorable prognostic factor for CR, longer OS and DFS. DFS was not different between early sequential intensification (n = 48) and intermittent intensification (n = 43) during the maintenance phase. Among CR patients under 40 years old, the 6-year survival was not different between the allocated related allo-BMT group (34 patients) and the allocated chemotherapy group (108 patients). However, among patients with Ph-positive ALL, the survival of patients who actually received allo-BMT was superior to that of patients who received chemotherapy (P = 0.046).

Serum thymidine kinase and soluble interleukin-2 receptor predict recurrence of malignant lymphoma.

Before and after therapy, serum thymidine kinase (TK) and soluble interleukin-2 receptor (sIL-2R) were serially determined in 28 patients with malignant lymphoma (ML). In 15 patients achieving and maintaining complete remission (CR) for more than 2 years, serum TK and sIL-2R were unchanged or decreased gradually. In contrast, logarithmic linear increases of TK and sIL-2R were observed in 13 relapsed patients. The increments of the serum markers occurred more than 10 months before the relapse. A significant positive correlation between the slope of the line for TK and that for sIL-2R was noted. The doubling time for TK estimated from the slope also showed a positive correlation with that for sIL-2R. Taken together, serum TK and sIL-2R were shown to be quite sensitive and interrelated serum markers for the recurrence of ML. Slopes of logarithmic linear increase, which are proper and specific for the individual patients, are inversely correlated with the doubling time and reflect proliferation of ML. We conclude that serum TK and sIL-2R are better predictors of relapse than LDH and the international prognostic index (IPI).

Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t(4;12)(p16;p13) chromosomal translocation.

Fusions of the ETV6/TEL gene to receptor or protein tyrosine kinases (TKs), such as PDGFRbeta, JAK2, ABL, ABL2, TRKC, and Syk, have been reported in various hematological malignancies. Expression of the resultant chimeric proteins is believed to lead to constitutive TK activity through activation by the helix-loop-helix (HLH) domain of ETV6. We identified a novel ETV6 partner gene, fibroblast growth factor receptor 3 (FGFR3), in a patient with peripheral T-cell lymphoma (PTCL) with a t(4;12)(p16;p13) translocation. The ETV6-FGFR3 transcript showed a fusion of exon 5 of ETV6 to exon 10 of FGFR3, resulting in an open reading frame for a chimeric protein consisting of the HLH domain of ETV6 and the TK domains of FGFR3. This is the first report of ETV6 and FGFR3 involvement in PTCL.

Reappraisal of the clinical significance of CD7 expression in association with cytogenetics in de novo acute myeloid leukaemia.

Debate exists over whether CD7 expression indicates an unfavourable prognosis in de novo acute myeloid leukaemia (AML). Meanwhile, the type of cytogenetics is a strong prognostic factor in AML. We analysed 256 de novo adult AML cases and found that the proportion of CD7+ cases increased stepwise from the cases with favourable cytogenetics to the cases with intermediate and unfavourable cytogenetics (3 out of 69 cases, 51 out of 140 cases and 25 out of 47 cases respectively, P < 0.0001). CD7-positivity adversely affected the survival only in the cases with unfavourable cytogenetics (P < 0.03). We recommend that CD7 expression in AML be interpreted in association with the cytogenetics.

[CD7(+) acute myeloid leukemia (M0) associated with a mediastinal bulky mass lesion].

A 41-year-old man visited his doctor in May 2000 because of a sore throat and high fever. His symptoms did not improve, despite administration of antibiotics and nonsteroidal anti-inflammatory drugs. Since a chest X-ray examination revealed an anterior mediastinal bulky tumor, he was referred and admitted to our hospital on June 21, 2000. The peripheral white blood cell count was 44,540/microliter with 74% myeloblasts. Bone marrow aspiration revealed a hypercellular marrow with 82% myeloblasts, which were negative for peroxidase and alpha-naphthyl butylate esterase staining. Blast cells were positive for CD7, CD13, CD33, CD34, and HLA-DR, and negative for CD56. A needle biopsy specimen of the mediastinal tumor consisted of myeloblasts. We diagnosed the patient as having CD7 (+) acute myeloid leukemia (AML) (M0) with a bulky mediastinal mass based on the surface marker analysis, although the clinical features resembled myeloid/NK precursor acute leukemia. The patient achieved a complete remission after two courses of induction therapy. We are planning an allogeneic stem cell transplantation during his first remission because of the high risk of relapse.

Effect of a selective Abl tyrosine kinase inhibitor, STI571, on in vitro growth of BCR-ABL-positive acute lymphoblastic leukemia cells.

By employing a new semi-quantitative assay system that includes co-culturing leukemia cells with the mouse bone marrow-derived stromal cell line MS-5, we examined the suppressive effect of a selective inhibitor of ABL tyrosine kinase, STI571, on acute lymphoblastic leukemia (ALL) cells with BCR-ABL fusion. Leukemic blast cells from eight patients with B-precursor ALL, including three patients with BCR-ABL-positive ALL, were cultured on monolayers of MS-5 cells for 3 weeks with or without addition of variable amounts of STI571. In all cases, cobblestone areas (CAs) were formed, showing clear linear cell dose-dependent curves, allowing quantitative assessment of blast cell growth. The progenitor frequencies obtained by this direct CA-forming cell (CAFC) assay were equivalent to ALL progenitor frequencies assessed by the standard limiting dilution assay. The number of CAFCs ranged from 12.3 to 140.3/10(4) cells. In BCR-ABL-positive ALL patients, CA-containing cells were examined by FISH, and all contained BCR-ABL fusion genes. STI571 inhibited CA formation of BCR-ABL-positive ALL cells virtually 100% at 0.1-1.0 micromol/l. None of the five BCR-ABL-negative ALL patients showed this growth inhibition by STI571 at 0.1-1.0 micromol/l. Our results indicate that STI571 selectively inhibits in vitro growth of BCR-ABL-positive ALL cells.

Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human malignancy. An internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence of the FLT3 gene (FLT3/ITD) is found in 20% of patients with acute myeloid leukemia (AML) and is strongly associated with leukocytosis and a poor prognosis. On the other hand, mutations of the c-KIT gene, which have been found in mast cell leukemia and AML, are clustered in 2 distinct regions, the JM domain and D816 within the activation loop. This study was designed to analyze the mutation of D835 of FLT3, which corresponds to D816 of c-KIT, in a large series of human hematologic malignancies. Several kinds of missense mutations were found in 30 of the 429 (7.0%) AML cases, 1 of the 29 (3.4%) myelodysplastic syndrome (MDS) cases, and 1 of the 36 (2.8%) acute lymphocytic leukemia patients. The D835Y mutation was most frequently found (22 of the 32 D835 mutations), followed by the D835V (5), and D835H (1), D835E (1), and D835N (1) mutations. Of note is that D835 mutations occurred independently of FLT3/ITD. An analysis in the 201 patients newly diagnosed with AML (excluding M3) revealed that, in contrast to the FLT3/ITD mutation (n = 46), D835 mutations (n = 8) were not significantly related to the leukocytosis, but tended to worsen disease-free survival. All D835-mutant FLT3 were constitutively tyrosine-phosphorylated and transformed 32D cells, suggesting these mutations were constitutively active. These results demonstrate that the FLT3 gene is the target most frequently mutated to become constitutively active in AML.

[Clinical significance of WHO classification and MDS 2000 classification in myelodysplastic syndromes].

Excluding chronic myelomonocytic leukemia, a total of 92 consecutive patients with myelodysplastic syndrome showing less than 20% blasts in the bone marrow were analyzed. We evaluated the clinical significance of the WHO and MDS 2000 classifications by reviewing each MDS patient according to the classification. The WHO criteria classified the MDS patients into 36 with RA, 22 with RCMD and 33 with RAEB, whereas according to the MDS 2000 criteria there were 19 RAEB-I patients and 15 RAEB-II patients. Based on the WHO classification, the RCMD patients had higher platelet counts and percentages of blasts among BM cells than the RA patients (P = 0.0018, P = 0.0001). Twenty percent of the RA patients, 44.8% of the RCMD patients, and 70.8% of the RAEB patients had cytogenetic abnormalities. Among them, the poor karyotype was present in 6.7% of the RA patients, 21.0% of the RCMD patients and 41.6% of the RAEB patients. The rate of acute leukemia death was 14.3% in the RA patients, 67.7% in the RAEB patients and 50.0% in the RCMD patients. Analysis of survival times revealed significant differences between RA and RCMD patients (P = 0.0482). The clinical features of RCMD patients were intermediate between those of RAEB and RA patients. There was no difference between the clinical features of the RAEB-I and RAEB-II patients in the MDS 2000 classification.

Interstitial deletion of the short arm of chromosome 12 during clonal evolution in myelodysplastic syndrome with t(5;12)(q13;p13) involving the ETV6 gene.

We report here a 65-year-old man with a myelodysplastic syndrome (MDS), refractory anemia with excess of blasts. He had received chemotherapy with tegafur for renal carcinoma. Chromosome analysis of bone marrow cells revealed complex karyotypes; del(5)(q13) was observed in all 20 metaphase spreads, and two related aberrations, add(12)(p11) and add(12)(p13), were detected in 13 and 7 cells, respectively. Fluorescence in situ hybridization (FISH) analysis with chromosome-specific DNAs revealed that these alterations originated from a reciprocal translocation (5;12)(q13;p13). Therefore, del(5)(q13), add(12)(p11), and add(12)(p13) were revised as der(5)t(5;12)(q13;p13), der(12)del(12)(p11p13)t(5;12)(q13;p13), and der(12)t(5;12)(q13;p13), respectively. Fluorescence in situ hybridization with a series of cosmid probes spanning the ETV6 gene showed that the 12p13 breakpoint on the der(12)t(5;12)(q13;p13) was located in intron 1, but the exon 1 signal was deleted. Our results suggest that a fusion gene was generated between the 5'-end of an unidentified partner at 5q13 and the 3'-end of ETV6 by t(5;12)(q13;p13), and that the interstitial deletion (12)(p11p13) occurred following t(5;12) during clonal evolution. del(12)(p11p13), including the rearranged ETV6 gene, may be implicated in the progression of MDS.

Identification of breakpoint cluster regions at 1p36.3 and 3q21 in hematologic malignancies with t(1;3)(p36;q21).

The reciprocal translocation t(1;3)(p36;q21) is associated with myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) characterized by trilineage dysplasia, in particular dysmegakaryocytopoiesis, and a poor prognosis. As yet no molecular genetic analyses of the t(1;3) have been reported. In four patients with t(1;3), all of whom had AML-M4, which evolved from MDS, the breakpoints at 3q21 clustered within a 60-kb region centromeric to the breakpoint of the inv(3)(q21q26), whereas the breakpoints at 1p36 clustered within a 90-kb region at 1p36.3. The presence of novel clusters in both the 3q21 and 1p36 breakpoints (BCRs) suggests a common, underlying molecular mechanism for the development of t(1;3)-positive MDS/AML. The Ribophorin I (RPN1) gene close to the BCR at 3q21 was highly expressed without gross structural changes, whereas the GR6 gene located within the BCR at 3q21 was not expressed. No other highly expressed genes were isolated in a 150-kb region at 3q21. Thus, it is likely that a gene at 1p36.3 is activated by the translocation of the 3q21 region or a gene important for transformation lies on 3q21, outside the 150-kb region. Further characterization of the BCRs at 1p36.3 and 3q21 should provide important insights into the molecular genetic mechanisms involved in the genesis of t(1;3)-positive MDS/AML. Genes Chromosomes Cancer 27:229-238, 2000.

New system for assessing the prognosis of refractory anemia patients.

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies >/=3% were classified as high risk (12 patients); of patients without pseudo-Pelger-Huët anomalies >/=3%, those with intermediate/poor karyotype according to IPSS, Hb /=10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

Fusion of TEL/ETV6 to a novel ACS2 in myelodysplastic syndrome and acute myelogenous leukemia with t(5;12)(q31;p13).

We identified a novel human long fatty acyl CoA synthetase 2 gene, ACS2, as a new ETV6 fusion partner gene in a recurrent t(5;12)(q31;p13) translocation in a patient with refractory anemia with excess blasts (RAEB) with basophilia, a patient with acute myelogenous leukemia (AML) with eosinophilia, and a patient with acute eosinophilic leukemia (AEL). ACS2 is expressed in the brain and bone marrow and is highly conserved in man and rats. The resulting ETV6/ACS2 fusion transcripts showed an out-frame fusion of exon 1 of ETV6 to exon 1 of ACS2 in the AEL case, an out-frame fusion of exon 1 of ETV6 to exon 11 of ACS2 in the AML case, and a short in-frame fusion of ETV6 exon 1 to the 3' untranslated region of ACS2 in the RAEB case. Reciprocal ACS2/ETV6 transcripts were identified in two of the cases. Fluorescence in situ hybridization (FISH) analysis with ETV6 cosmids on 12p13, and BACs and P1s on 5q31, demonstrated that the 5q31 breakpoints of the AML and AEL cases involved the 5' portion of the ACS2 gene, and that the 5q31, breakpoint of the RAEB case involved the 3' portion of the ACS2 gene. None of the resulting chimeric transcripts except for the ACS2/ETV6 transcript in the RAEB case led to a fusion protein. Disruption of the second ETV6 allele by t(12;19) was detected in the AML case by FISH analysis. These observations suggest that the disruption of ETV6 and/or ACS2 may lead to the pathogenesis of hematologic malignancies with t(5;12)(q31;p13).

[A neutropenic acute myeloid leukemia patient complicated with chronic otitis media due to Aspergillus niger and yeast-like fungi caused by superinfection].

There have been few reports describing otomycosis in association with compromised hosts. So we report a neutropenic acute myeloid leukemia (AML) patient complicated with otomycosis caused by superinfection. A 51-year-old male was admitted because a third relapse of AML in March 1998. Two years ago, he was diagnosed as having chronic otitis media involving the VII cranial nerve due to Pseudomonas aeruginosa coinciding with AML. Then, he had suffered from a right-sided earache and otic discharge in accord with every myelosuppression, which improved on treatment with otic administration of ofloxacin. After 1 course of induction chemotherapy, he developed a spiking fever with severe earache and otic discharge at a nadir period of WBC. Ear swab cultures yielded Aspergillus niger and yeast-like fungi. So, he was treated with intravenous administration of amphotericin B (AMPH-B): initial dose was 5 mg/day and was gradually increased to 30 mg/day. Thereafter, the otic symptoms subsided and never recurred. Subsequently, he was given another antifungal agent, itraconazole. Although induction chemotherapies resulted in failure, he did not suffer otic symptoms until his death due to cerebral bleeding in January 1999. For neutropenic patients without rapid hematological improvement, we recommend intensive antifungal therapy as the first-line of therapy for otomycosis rather than local therapy.

[Atypical chronic myeloid leukemia presenting with trilineage dysplasia and IgG (lambda) type monoclonal gammopathy].

A 78-year-old man was diagnosed as leukocytosis in February 1994. Physical examination revealed marked hepatosplenomegaly. A peripheral blood examination disclosed 95,090/microliter leukocytes without hiatus leukemicus, 6.5 g/dl Hb, and 15.0 x 10(4)/microliter platelets. The neutrophil alkaline phosphatase score was 27, and serum VB12 was above 1,600pg/ml. IgG was identified as monoclonal immunoglobulin of type lambda. Bone marrow specimens demonstrated marked granulocytic hyperplasia. Neither the Philadelphia chromosome (Ph1) nor BCR gene rearrangement was detected; hence, the diagnosis of Ph1 (-) chronic myeloid leukemia (CML) was made. The patient was treated with hydroxyurea and low-dose VP-16 with no improvement, and died of pneumonia and sepsis in June 1995. This case was considered to be consistent with atypical CML (aCML) according to the FAB classification because monocytosis was not observed. It seems likely and interesting that the coexistent monoclonal gammopathy and aCML might have arisen from common abnormal hematopoietic stem cells.

Therapy-related AML after successful chemotherapy with low dose etoposide for virus-associated hemophagocytic syndrome.

A 19-year-old male patient with virus associated hemophagocytic syndrome (VAHS) began receiving chemotherapy including etoposide (cumulative dose of 900 mg/m2 intravenously) and Ara-C (cumulative dose of 360 mg/m2 intravenously) in July 1994. He achieved complete remission, but developed acute myelomonocytic leukemia (AML, FAB M4) with t(9;11)(p22;q23) in March 1997 and a rearrangement of the MLL gene was also recognized. The MLL gene rearrangement is closely associated with secondary leukemia with an 11q23 translocation. It is highly likely that this case of AML was caused by the cytostatic treatment the patient received, including etoposide for VAHS.

Alterations in the colorectal carcinoma gene and protein in a novel human myeloid leukemia cell line with trisomy 18 established from overt leukemia after myelodysplastic syndrome.

A new human myeloid leukemia cell line (OIH-1), with alterations in chromosome 18 and the deleted in the colorectal carcinoma (DCC) gene and its product, was established from the peripheral blood (PB) of a patient with acute myeloblastic leukemia (AML) after myelodysplastic syndrome (MDS). Serial cytogenetics showed the presence of two clones, one with i(18)(q11) and another with trisomy 18. Southern blot analysis of OIH-1 cells with i(18)(q11) showed an extremely reduced intensity of 20- and 14-kb EcoRI fragments, suggesting the allelic loss of the DCC gene. Immunoprecipitation (IP) analysis by the murine monoclonal antibody (MoAb) AF5, specific for the DCC extracellular domain, failed to detect normal 180-kDa DCC protein, however extra 85-kDa protein was detected. However, Southern blot analysis of the latter clone of OIH-1 with trisomy 18 showed normal structure of the DCC gene. IP analysis with AF5 or G92-13 (specific for the extracellular domain) did not detect the DCC protein, but a 150-kDa protein other than the DCC-specific 180-kDa protein was detected with G97-449, specific for the cytoplasmic domain of the DCC protein. RT-PCR analysis showed the expression of the DCC mRNA in OIH-1 cells carrying each type of chromosome 18 abnormalities. These alterations in the DCC gene and protein may contribute to progression of malignancy for OIH-1 cells. The OIH-1 cell line may be useful for studying the role of the DCC gene in leukemogenesis of MDS or AML.