V Montefusco

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.

Quantification of BCR-ABL transcripts in CML patients in cytogenetic remission after interferon-α-based therapy

We measured using a competitive quantitative polymerase chain reaction-capillary electrophoresis (PCR-CE)-based assay, the levels of bcr-abl transcripts in 44 patients with chronic myeloid leukemia (CML) after interferon-α (IFN-α) therapy, who achieved a major (10 patients, MCR group) or complete (34 patients, CCR group) cytogenetic response. All 34 CCR patients had molecular evidence of residual disease detected in bone marrow samples at the time of best karyotypic response. The median number of bcr-abl transcripts of 34 evaluable patients in the CCR group at the time of complete cytogenetic remission was 4/μg RNA (range 3–4600), while the median number of bcr-abl transcripts of 10 patients in the MCR group at the time of best cytogenetic response was 4490/μg RNA (range 600–23u2009900) (Pu2009=u20090.000024). In nine CCR and five MCR patients we were able to quantify the amount of bcr-abl transcript both at diagnosis and after interferon therapy: no statistical difference (Pu2009=u20090.18) was found between the two groups at diagnosis (median bcr-abl transcripts/μg RNA was 30u2009000 vs 39u2009650, respectively). During IFN-α therapy, the two groups were evaluable at the time of major karyotypic conversion: at this point, there was a statistical difference of expression of bcr-abl transcript between the CCR group (17 patients) (median 2700; range 76–40u2009000) and the MCR group (10 patients) (median 4490; range 600–23u2009900), respectively (Pu2009=u20090.046). No differences of bcr-abl amount of transcript were found in patients with CCR obtained either by IFN-α therapy alone (20 patients) vs IFN-α plus ABMT (13 patients) (Pu2009=u20090.47). We firstly demonstrated that although the CCR and MCR groups were clinically, cytogenetically and molecularly indistinguishable at diagnosis, the two groups could be recognized successfully during interferon therapy based on the level of bcr-abl transcript. Bone Marrow Transplantation (2000) 25, 729–736.

Concomitant expression of the rare E1/A3 and B2/A3 types of BCR/ABL transcript in a chronic myeloid leukemia (CML) patient.

Concamitant expression of the rare E1/A3 and B2/A3 types of BCR/ABL transcript in a chronic myeloid leukemia (CML) patient

Translisin recognition site sequences flank translocation breakpoints in a Philadelphia chromosome positive chronic myeloid leukemia patient expressing a novel type of chimeric BCR-ABL transcript (E8-INT-A2)

Translisin recognition site sequences flank translocation breakpoints in a Philadelphia chromosome positive chronic myeloid leukemia patient expressing a novel type of chimeric BCR-ABL transcript (E8-INT-A2)

Quantitative Evaluation of BCR-ABL Amount of Transcript Post Mobilization with G-CSF of Peripheral Blood Stem Cells from Chronic Myeloid Leukemia Patients in Cytogenetic Response

We studied nine patients affected by chronic myeloid leukemia (CML Ph+ and bcr-abl positive) and treated with alpha-interferon (α-INF) in order to: first, to evaluate the feasibility of a mobilization of peripheral blood stem cells induced by granulocyte-colony-stimulating factor (G-CSF) and the contamination by Ph+ cells and second, to quantify the amount of bcr-abl leukemia associated transcript by a quantitative assay during mobilization procedures, and post mobilization follow-up. Eight achieved a complete karyotypic remission before mobilization obtained with discontinuation of a-INF for few days and G-CSF at a dosage of 15μg/kg/day for 5-7 consecutive days. By quantitative-competitive polymerase chain reaction (QC-PCR) assay, all the leukaphereses and bone marrow samples during post mobilization follow up were studied to determine the amount of bcr-abl transcript. Karyotypic and molecular analysis on evaluable leukapheresis showed that all the harvests were Ph negative and bcr-abl positive: in seven cases the levels of bcr-abl transcript were higher or equal to the pre-apheresis status. In three out of four patients, who underwent more than one leukapheresis procedure, we noticed a decreasing amount of bcr-abl contamination from the first to the last apheresis. Our results suggest that in patients who achieved a complete or major cytogenetic conversion with α-INF, it is possible to obtain a sufficient amount of PBSC for autografting by leukapheresis following priming G-CSF therapy and that the amount of neoplastic transcript does not seem to increase.