Noriko Satake

Novel MLL-CBP fusion transcript in therapy-related chronic myelomonocytic leukemia with a t(11;16) (q23;p13) chromosome translocation

CBP, which is located on 16p13 and encodes a transcriptional adaptor/coactivator protein, has been shown to fuse by the t(8;16)(p11;p13) translocation to MOZ on 8p11 in acute myeloid leukemia. We found a t(11;16)(q23;p13) in a child with therapy‐related chronic myelomonocytic leukemia. Subsequent reverse transcriptase‐polymerase chain reaction and direct sequencing analyses revealed the MLL‐CBP fusion transcript in CMML cells. Because 11q23 translocations involving MLL and t(8;16) involving MOZ and CBP have been reported in therapy‐related leukemias, both the MLL and CBP genes may be targets for topoisomerase II inhibitors. Accordingly, we believe that most t(11;16)‐associated leukemias may develop in patients who have been treated with cytotoxic chemotherapy for primary malignant diseases. Genes Chromosom. Cancer 20:60–63, 1997.

Treatment of the mouse model of mucopolysaccharidosis I with retrovirally transduced bone marrow

Mucopolysaccharidosis I is a lysosomal storage disorder caused by mutations in the IDUA gene, resulting in deficiency of alpha-L-iduronidase and accumulation of glycosaminoglycans. Bone marrow transplantation has been the only available therapy, soon to be joined by enzyme replacement. We have tested retroviral gene therapy in a knockout mouse model of the disease. Bone marrow from Idua-/- male donor mice was transduced with human IDUA cDNA in an MND vector and transplanted into 6-8-week-old, lethally irradiated female Idua-/- mice. Sham-treated mice received Idua-/- bone marrow that was either unmodified or transduced with eGFP. Unmodified Idua+/+ (wild type) bone marrow was transplanted for comparison. Recipient mice were sacrificed 2-6 months after transplantation. Three biochemical parameters were used to gauge therapeutic success: appearance of alpha-L-iduronidase activity, reduction of beta-hexosaminidase activity and reduction of soluble glycosaminoglycan accumulation. Transplantation of unmodified +/+ bone marrow was effective in reducing storage in liver and spleen, but not in kidney or brain. The level of alpha-L-iduronidase activity achieved by transplantation of IDUA-transduced bone marrow varied greatly between experiments. But even modest activity resulted in correction of pathology of kidney, bladder epithelium, fibrocartilage, choroid plexus, and thalamus, as seen by light microscopy, while electron microscopy showed the presence of some normal neurons in the cortex. The partial correction of brain pathology is attributed to migration of donor hematopoietic cells, demonstrated by the presence of the Y chromosome and of normal microglia in the brain of mice receiving IDUA cDNA.

Disappearance of AML1-MTG8 (ETO) fusion transcript in acute myeloid leukaemia patients with t(8;21) in long-term remission

Summary. In a study of 23 patients with t(8;21)‐associated acute myeloid leukaemia the AML1‐MTG8 fusion transcript was present in the majority of serial samples obtained from 17 patients followed for up to 34 months after diagnosis, but was absent in samples from all six patients who had been in continuous complete remission for 61 months after allogeneic bone marrow transplantation (BMT), or for 52, 53, 123,182 and 198 months, respectively, after courses of intensive chemotherapy. Previous studies showed that the AML1‐MTG8 fusion transcript was present in most patients with this type of translocation in long‐term remission. Our results indicate that blood cells of patients with t(8;21) in remission of over 10 years may not show the AML1‐MTG8 fusion transcript, and that those of patients who have undergone allogeneic BMT or intensive chemotherapy may become fusion transcript‐negative much earlier. Our study suggests that leukaemic cells with the AML1‐MTG8 fusion transcript may survive for some time after courses of chemotherapy or BMT, but that they may eventually be eradicated by immunologic and other antileukaemic mechanisms.

The der(21)t(12;21) chromosome is always formed in a 12;21 translocation associated with childhood acute lymphoblastic leukaemia

We studied 116 patients (93 children and 23 adults) with acute lymphoblastic leukaemia (ALL) using fluorescence in situ hybridization (FISH) with the yeast artificial chromosome (YAC) clone, 964c10, which includes the recently described ETS‐like gene, TEL, on 12p13. FISH revealed that nine of the patients had a t(12;21), which had not been previously detected. The nine patients were all children, seven boys and two girls, aged 1–10 years (median 3 years), had an early B immunophenotype, and achieved complete remission, although two of them experienced haematological relapse. In addition to the t(12;21), FISH also revealed that three of the nine had a del(12p) in the other homolog of chromosome 12 or in the der(12) chromosome itself, and that two others had 12p translocations in the other chromosome 12 homolog. Although chromosomal rearrangements associated with the t(12;21) were heterogenous and complex, fusion of the sequences from chromosomes 12 and 21 on the der(21)t(12;21) chromosomes was consistent, suggesting that the TEL‐AML1 gene fusion on the der(21) chromosome may be critical in leukaemogenesis and that FISH or reverse transcriptase‐polymerase chain reaction (RT‐PCR) targeted to the chimaeric sequences on the der(21) will be most useful in detecting the t(12:21) or following a patient with the t(12;21), which is one of the most frequent chromosomal rearrangements in both Caucasian and Asian childhood ALL.

EWS-ERG fusion transcript produced by chromosomal insertion in a Ewing sarcoma

The EWS gene is fused in Ewing sarcoma‐like tumors by a chromosomal translocation to one of the four ETS‐family genes: FLII, ERG, ETVI, and EIAF. The orientation of EWS and FLII on chromosomes 22 and 11, respectively, is 5′ centromeric and 3′ telomeric, whereas that of ERG on chromosome 21 is the reverse. Although 10% of Ewing‐family tumors express the EWS‐ERG fusion transcript, there have been no reports on tumors with t(21;22)(q22;q12) identified by banding cytogenetics. We found the karyotype 50,XY,+8,+8,+12,+mar in all metaphase cells from a tumor. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis performed on the tumor and direct sequencing of the products identified the EWS‐ERG fusion transcript. Subsequent two‐color fluorescence in situ hybridization (FISH) analysis with EWS and ERG clones showed the fused signals on the der(21) chromosome, but no ERG signals on the chromosome 22 homologs. Thus, our RT‐PCR and FISH analyses indicated that the chromosome 22 fragment containing the 5′ portion of EWS had been inverted and inserted into chromosome 21 and had fused to the 3′ portion of ERG. This subtle chromosome aberration could not be identified by routine cytogenetics. A chromosomal inversion/insertion has also been described in acute leukemia with the MLL‐AF10 fusion gene, and this may be a common pathway for producing fusion of reverse‐oriented genes in leukemias and solid tumors. Genes Chromosom. Cancer 18:228–231, 1997.

Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line.

Background Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable. Patients and Methods The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose L-PAM. Results CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0–1,000 &mgr;mol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to L-PAM (LD 90 = 42 &mgr;mol/L; peak plasma concentration in the patient equals 3.9 &mgr;mol/L) and moderately resistant to BSO (LD 90 = 509 &mgr;mol/L; steady-state concentration in the patient equals 397 &mgr;mol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3–4 logs of cell kill, combination index <1) at clinically achievable levels of BSO (100–400 &mgr;mol/L) and levels of L-PAM (10–40 &mgr;mol/L) clinically achievable only with hematopoietic stem cell support. Conclusions The in vitro results obtained for CHLA-171 suggest that BSO/L-PAM therapy may be optimally effective for drug-resistant neuroblastoma using myeloablative doses of L-PAM.

Minimal residual disease with TEL-AML1 fusion transcript in childhood acute lymphoblastic leukaemia with t(12;21).

We analysed the TEL‐AML1 transcript using reverse transcription‐polymerase chain reaction (RT‐PCR) in order to detect minimal residual disease (MRD) in seven children with t(12;21)‐associated B‐lineage ALL. Leukaemic cells with the TEL‐AML1 transcript appear to be very sensitive to chemotherapy, and may be eradicated in most patients if adequate chemotherapy is given. However, a small number of patients with t(12;21) ALL may relapse under the currently used chemotherapy, and we believe that RT‐PCR for detecting MRD with the transcript is a suitable tool for monitoring the efficacy of chemotherapy or impending relapse in these patients. We analysed the TEL‐AML1 transcript using reverse transcription‐polymerase chain reaction (RT‐PCR) in order to detect minimal residual disease (MRD) in seven children with t(12;21)‐associated B‐lineage ALL. Two sets of primers, TEL exon 5 and AML1 exon 3 or 4, detected two types of transcript in four patients and two other types in two other patients. The two different translocation breakpoints in the AML1 gene with or without splicing out of AML1 exon 3 seemed to result in these four types of transcript in leukaemia samples.

STX11 mutations and clinical phenotypes of familial hemophagocytic lymphohistiocytosis in North America

Mutations in STX11 are responsible for Familial Hemophagocytic Lymphohistiocytosis (FHLH) type 4, a rare primary immunodeficiency which has previously been observed only in patients of Kurdish, Turkish, and Lebanese ethnic background.

Synergism of buthionine sulfoximine and melphalan against neuroblastoma cell lines derived after disease progression

BACKGROUND Despite intensive-alkylator based regimens, >50% of patients with high-risk neuroblastoma (NB) die from recurrent disease that is probably due, in part, to acquired alkylator resistance. PROCEDURE Using buthionine sulfoximine (BSO)-mediated, glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance, we examined six NB cell lines established after progressive disease following either standard chemotherapy, BSO/L-PAM therapy, or myeloablative therapy and autologous hematopoietic stem cell transplant (AHSCT). RESULTS Four of the six cell lines (three p53-nonfunctional and one p53-functional) showed high-level L-PAM resistance. CONCLUSIONS Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index >1) for all cell lines tested. In L-PAM-resistant cell lines, the minimal cytotoxicity observed for BSO combined with nonmyeloablative concentrations of L-PAM was markedly enhanced (>4 logs total cell kill) when BSO was combined with myeloablative concentrations of L-PAM. In alkylator-resistant NB, the optimal use of BSO may require dose escalation of L-PAM to levels requiring AHSCT.

Power dependent oxygenation state transition of red blood cells in a single beam optical trap

Laser tweezers Raman spectroscopy (LTRS) was used to demonstrate that a red blood cell (RBC) in a single beam optical trap transitions from an oxygenated to a partially deoxygenated state with increasing trapping power. Continuous switching between the two states is possible by repeatedly cycling between low and high trapping powers. Alterations in the hemoglobin conformation and interactions due to cell folding in the trap are proposed to be responsible for the transition. This study demonstrates that mechanically induced biochemical changes by optical forces need to be considered when applying single beam optical tweezers for cell analysis. LTRS holds promise as a functional assay to characterize normal and diseased RBCs based on their biochemical response to the forces of a single beam optical trap.