Jastinder Sohal

TEL-AML1 fusion in acute lymphoblastic leukaemia of adults

A number of fusion genes have been identified by study of acquired chromosomal translocations. Their detailed characterization has provided insights into mechanisms of leukaemogenesis and has enabled the development of molecular methods to assist in the diagnosis and monitoring of residual disease after treatment. The TEL‐AML1 fusion gene is associated with a cryptic t(12;21)(p12;q22) translocation, and is the commonest known genetic abnormality in childhood B‐cell precursor acute lymphoblastic leukaemia (ALL), occurring in about 25% of cases. We have used RT‐PCR, followed by Southern blotting and direct sequencing, to establish the incidence of TEL‐AML1 rearrangement in 131 adults with acute leukaemia (101 with ALL and 30 with chronic myeloid leukaemia in blastic crisis). Three patients were positive for TEL‐AML1 transcripts. All three had common‐ALL. All other patients were negative for TEL‐AML1. We conclude that the TEL‐AML1 fusion gene is found in adult ALL, though less commonly than in children.

Identification of four new translocations involving FGFR1 in myeloid disorders

The 8p11 myeloproliferative syndrome (EMS) is associated with three translocations, t(8;13)(p11;q12), t(8;9)(p11;q33), and t(6;8)(q27;p11), that fuse unrelated genes (ZNF198, CEP110, and FOP, respectively) to the entire tyrosine kinase domain of FGFR1. In all cases thus far examined (n = 10), the t(8;13) results in an identical mRNA fusion between ZNF198 exon 17 and FGFR1 exon 9. To determine if consistent fusions are also seen in the variant translocations, we performed RT‐PCR on four cases and sequenced the products. For two patients with a t(8;9), we found that CEP110 exon 15 was fused to FGFR1 exon 9. For two patients with a t(6;8), we found that FOP exon 5 (n = 1) or exon 7 (n = 1) was fused to FGFR1 exon 9. To determine if FGFR1 might be involved in other myeloid disorders with translocations of 8p, we developed a two‐color FISH assay using two differentially labeled PAC clones that flank FGFR1. Disruption of this gene was indicated in a patient with a t(8;17)(p11;q25) and Ph‐negative chronic myeloid leukemia in association with systemic malignant mast cell disease, a patient with acute myeloid leukemia with a t(8;11)(p11;p15), and two cases with T‐cell lymphoma, myeloproliferative disorder, and marrow eosinophilia with a t(8;12)(p11;q15) and ins(12;8)(p11;p11p21), respectively. For the patient with the t(8;11), the chromosome 11 breakpoint was determined to be in the vicinity of NUP98. We conclude that 1) all mRNA fusions in EMS result in splicing to FGFR1 exon 9 but breakpoints in FOP are variable, 2) two‐color FISH can identify patients with EMS, and 3) the t(8;17)(p11;q25), t(8;11)(p11;p15), t(8;12)(p11;q15), and ins(12;8)(p11;p11p21) are novel karyotypic changes that most likely involve FGFR1.

The rate and kinetics of molecular response to donor leucocyte transfusions in chronic myeloid leukaemia patients treated for relapse after allogeneic bone marrow transplantation

We have assessed the molecular response of 30 consecutive patients with chronic myeloid leukaemia (CML) treated for relapse after allogeneic bone marrow transplantation (BMT) by donor leucocyte transfusions (DLT). Response was evaluated by qualitative nested and quantitative competitive RT‐PCR for BCR‐ABL mRNA at various time intervals before and after DLT. The probability of attaining molecular remission at 2 years was 61% (95% CI 42–78%). Disease state at the time of DLT was significantly associated with response: molecular remission was achieved for 9/10 (90%) patients treated early (cytogenetic or molecular relapse) compared to only 8/20 (40%) patients treated late (haematological relapse; P=0.009). The Kaplan–Meier estimates of molecular remission at 2 years post DLT for patients treated in early or late relapse were 86.6% and 47.3% respectively (P=0.004). The median time interval from DLT to molecular remission was 11.0 months (range 2.5–32). Molecular remissions were durable for most (15/17) patients (median follow‐up 21.2 months; range 0–55). Two patterns of molecular response were found: a very rapid decline after an initial lag phase or a more gradual decline over a period of several months. We conclude that molecular monitoring is a sensitive indicator of response to DLT; different kinetics of molecular response may reflect disease heterogeneity or differences in the mode of action of DLT.

BCR-ABL-positive progenitors in chronic myeloid leukaemia patients in complete cytogenetic remission after treatment with interferon-α

To determine the source of residual disease detected in patients with chronic myeloid leukaemia (CML) in complete cytogenetic remission (n = 8) after treatment with interferon‐α (IFN‐α), we have tested CFU‐GM colonies grown from bone marrow mononuclear cells or from plastic‐adherent (PΔ) cells for BCR‐ABL mRNA using a nested multiplex RT‐PCR. We compared our results with those obtained by analysis of colonies from newly diagnosed patients (n = 4) and patients achieving no cytogenetic response (n = 1) or incomplete cytogenetic response to treatment with IFN‐α (n = 5). A total of 1239 informative colonies were analysed. A small proportion of BCR‐ABL‐positive colonies was detected in all eight patients in complete cytogenetic remission, suggesting the persistence of leukaemia that could potentially lead to relapse. The overall proportion of BCR‐ABL‐positive colonies in patients achieving a cytogenetic response to IFN‐α correlated with the levels of BCR‐ABL transcripts detected in the peripheral blood by competitive RT‐PCR (P = 0.004). We conclude that residual disease detected in the peripheral blood of complete cytogenetic responders to IFN‐α is at least partly derived from clonogenic myeloid cells. It is probable that the leukaemia clone in CML is only very rarely or never entirely eradicated by treatment with IFN‐α.

Assignment of ZNF262 to human chromosome band 1p34→p32 by in situ hybridization

ZNF262 (previously known as KIAA0425) was originally cloned as a large human cDNA that is expressed in the brain (Suyama et al., 1999). ZNF262 is a member of a widely expressed new family of MYM domain containing proteins that includes ZNF198, the chromosome 13q11→q12 gene that is fused to FGFR1 by the t(8;13) in the 8p11 myeloproliferative syndrome (Macdonald et al., 1995, Reiter et al., 1998, Smedley et al., 1998) and ZNF261 (KIAA0385/DXS6673E), which maps to Xq13.1 (van der Maarel et al., 1996). Here we show that ZNF262 maps to chromosome 1p34→p32, thus excluding it from involvement in any of the other known translocations variants seen in the 8p11 myeloproliferative syndrome. Abnormalities of this region are seen, however, in other hematological malignancies and ZNF262 is a candidate for involvement in translocations such as the t(1;11)(p32;q23) in acute lymphoblastic leukemia (Harrison et al., 1998). Northern blotting indicated that ZNF262, in common with the other family members, is widely expressed (not shown). Materials and methods

A case of myelofibrosis with a t(4;13)(q25;q12): evidence for involvement of a second 13q12 locus in chronic myeloproliferative disorders.

Aberrations of 13q are frequently found in myeloproliferative disorders (MPD). In a case of primary proliferative polycythaemia which transformed to myelofibrosis, karyotype analysis at transformation revealed an acquired t(4;13)(q25;q12). FISH and Southern analysis demonstrated that the ZNF198 gene, disrupted in the t(8;13)(p11;q12) myeloproliferative syndrome, was unaffected by the t(4;13). FISH analysis mapped the 13q12 breakpoint to the genomic region flanked by the genetic markers D13S1126 and D13S1121. Physical mapping estimated this region to be <80 kbp. Our results suggest the possibility of a novel gene, distinct from ZNF198, that is located at chromosome 13q12 and involved in the pathogenesis of myelofibrosis.

Cloning of ZNF237, a novel member of the MYM gene family that maps to human chromosome 13q11→q12

We have cloned a novel, widely expressed human gene, ZNF237, that shows extensive similarity to the N-terminal region of ZNF198. Two alternatively spliced regions were identified by RT-PCR; the major splice variant is predicted to encode a 383 amino acid protein that contains a single diverged MYM domain. ZNF237 maps to 13q11→q12, immediately proximal to ZNF198.