Claudia Rosso

Molecular Defects Associated with the Acute Phase CML

Parts of the Bcr/Abl hybrid transcript supposed to be important for its transforming ability were sequenced in a series of CML blast crises, in order to evaluate the possible presence of alterations responsible for the disease transition from the chronic to the acute phase. In addition, the N- and Ki-ras as well as the p53 involvement was investigated by exploring their structure and expression in the same patients. We used traditional types of molecular analysis including Southern and Northern blot, together with methods that allow a rapid detection of point mutations and microdeletions, such as SSCP, single strand conformation polymorphism and direct sequencing. The results obtained may be summarized as follows: no alterations were found in the parts of the Bcr/Abl transcripts investigated in the present study (SH2, SH3 and the region surrounding codon 832); p53 alterations were observed in 5% and N- and Ki-RAS mutations in 5% of the cases examined. These molecular defects are therefore responsible for the clinical progression of the Ph1-positive CML only in a minority of cases.

Polyclonal haemopoieses associated with long-term persistence of the AML1-ETO transcript in patients with FAB M2 acute myeloid leukaemia in continous clinical remission.

Summary. The t(8;21) (q22;q22) translocation is a recurring chromosomal abnormality observed in about 20‐40% of AML patients with subtype FAB M2 (AML‐M2). The molecular facet of this translocation is represented by the formation of a new hybrid gene, the AML1‐ETO, which is regularly transcribed in a chimaeric mRNA and translated into a new fusion protein believed to have a key role in the pathogenesis of this type of leukaemia. We looked for the presence of AML1‐ETO transcripts, by RT‐PCR, in 49 unselected patients affected by AML‐M2 diagnosed at various Italian Institutions. A hybrid transcript was detected in 11 cases (23%). Minimal residual disease status was investigated in three patients in continuous complete remission (CCR) after a median follow‐up of 44 months; at least one sample from each subject was found positive for the AML1‐ETO transcript suggesting a long‐term persistence of t(8;21) leukaemic cells.

ABL proteins in Philadelphia-positive acute leukaemias and chronic myelogenous leukaemia blast crises

Summary. The Philadelphia chromosome (Ph1) is present in 95% of chronic myelogenous leukaemias (CML) and 15% of acute lymphoblastic leukaemias (ALL). This cytogenetic marker is due to a t(9;22) translocation, which causes a rearrangement of the ABL oncogene. In order to better define the relationship between type of genomic rearrangement, variant ABL protein expressed and haematological phenotype, a series of Phl‐positive acute leukaemias, both myeloblastic (AML) and lymphoblastic, and several CML lymphoid blast crises have been analysed at the DNA and protein level.

Molecular cytogenetic analysis discloses complex genetic imbalance in a t(11;21) myelodysplastic syndrome

Three cases of t(11;21)(q24;q11.2) myelodysplastic syndromes (MDS) showed karyotypic evolution resulting in the presence of two der(11)t(11;21) without normal chromosome 11 and with partial trisomy 21q. In one of these, we performed further molecular cytogenetic investigations which showed 1) that this rearrangement led to changes in the dosage and location of both c-ets 1 and c-ets 2 protooncogenes; and 2) that the presence of two 11q + chromosomes did not result from a nondisjunction, but that a second chromosome rearrangement had occurred. The final genetic imbalance resulting from this cytogenetic change involves at least hemizygosity for some sequences on the long arm of chromosome 11, including c-ets 1, plus trisomy for the most part of the long arm of chromosome 21, including c-ets 2.

MOLECULAR EVIDENCE OF TRANSIENT COMPLETE REMISSION AFTER AUTOGRAFTING IN Ph‐/BCR REARRANGED CHRONIC MYELOGENOUS LEUKAEMIA

Molecular analysis at the breakpoint cluster region (bcr) gene level (Groffen et al, 1984) has proved to be a relevant diagnostic tool in Ph’ CML, showing in 30-40% of patients the same bcr rearrangement patterns observed in typical Ph’+ cases (Kurzrock et al, 1986). Moreover, given the specificity of this alteration, molecular probes can be used for monitoring the leukaemic clone throughout the course of the disease, particularly when therapies capable of inducing partial or total Ph’ (or ‘bcr+’) suppression are employed. Among these, interferon and autologous stem cell transplantation are currently under investigation and have been reported to induce, at least in rare instances, complete remission and cytoconversion in CML (Yoffe et al, 1987; Brito-Babapulle et al, 1987). We report here two patients with Ph’-/bcr rearranged CML who received high dose chemotherapy and peripheral blood stem cell autograft. Patient 1. A 44-year-old female presented with splenomegaly and cytological features consistent with CML in chronic phase (Hb 12.7 g/dl, WBC 185x 10y/l, platelet count 383 x 1OY/1). Although a normal karyotype was found on cytogenetic examination, a bcr gene rearrangement was documented in her marrow DNA using a 3’ bcr probe (Fig 1, lane 1). Collection and cryopreservation of PBSC were performed within 1 month from diagnosis according to standard methods (Goldman et al, 1978). Normalization of WBC counts were achieved employing conventional doses of hydroxyurea. High-dose chemotherapy including busulphan 4 mg/kg daily (days 6 to 3 p.0.) and melphalan 60 mg/ mL (on day -2 i.v.) were administered as conditioning regimen 3 months after diagnosis. An autograft of 10 x lo8/ kg body weight thawed nucleated cells was infused on day 0. Bone marrow engraftment along with PMN recovery >0.5 x loy/] were evident at day + 18, whereas thrombocytopenia (< 50 x 10y/l) lasted 180 d. Platelet support was, however, necessary only up to the third week from treatment. Complete regression of the splenomegaly was obtained after pre-transplant chemotherapy. Molecular hybridization of BM DNA with the 3’ bcr probe showed at day

A frequent EcoRI polymorphism in the bcl-2 gene

60 the disappearance of the rearranged band documented at diagnosis (Fig 1, lane 2). A subsequent BM DNA control performed on day

Variant Breakpoint Positions on Chromosome 22 in Ph′-Positive Chronic Myelogenous Leukemias

210, whilst the patient still was in complete remission, revealed again the presence of an abnormal fragment of the same size of the rearranged band seen at the onset, but accounting for only a small proportion of cells (Fig 1, lane 3 ) . At 1 year of follow up after autografting the patient is in haematological remission but a further DNA control performed at day

Minimal residual disease status in transplanted chronic myelogenous leukemia patients: low incidence of polymerase chain reaction positive cases among 48 long disease-free subjects who received unmanipulated allogeneic bone marrow transplants.

330 still showed the same abnormal fragment, of similar intensity of that observed at day

Expression of GATA-1 mRNA in human myeloid leukemic cells

210. Patient 2 . A 32-year-old man presented with splenomegaly, a WBC count of 253 x 10y/l (with a cytologic pattern of typical CML), an Hb level of 9.0 g/dl and a platelet count of 714 x 109/1. Again, a rearrangement within the bcr was detected by Southern blotting analysis in spite of the absence of the Ph’ chromosome. Cryopreservation of PBSC at diagnosis and autografting (at 7 months) were performed following the same procedures. He achieved a PB recovery lasting 13 d for PMN >0.5 x 10y/l and 11 d for platelets > 50 x 10”/1. A moderate spleen enlargement persisted after treatment. At day +60 aPBexaminationrevealed 12.5 x 10y/l WBC (80% neutrophils) and Southern blot analysis showed the presence of an allelic abnormal fragment of the bcr gene exactly identical as size and intensity to the rearranged band detected at diagnosis. A progressive increase of WBC count was subsequently observed and alpha 2b interferon therapy was started 80 d after autografting. The present report, in line with previous observations, confirms that autografting with cryopreserved PBSC following high-dose chemotherapy is a promising therapeutic approach for selected groups of patients (Marcus 6; Goldman, 1986). Restoration of partially or totally Ph’ negative haemopoiesis is likely to be associated with prolonged

Repeated PCR in CML during IFN-α therapy

The frequency and Mendelian inheritance of an EcoRI polymorphism mapping within the bcl-2 locus was evaluated. The high frequency of the two identified alleles makes it a useful marker for genes located on the 18q21 region.