Giovanni Giudici

Delineation of multiple deleted regions in 7q in myeloid disorders

Loss of chromosome material due to deletions of the long arm of chromosome 7, del(7q), is a consistent finding in all types of myeloid disorders, invariably associated with a poor prognosis. Two different segments, 7q22 and 7q32–q33, have been implicated as critical regions of gene loss associated with these disorders. In the present study, we used fluorescence in situ hybridization (FISH) to characterize the 7q22 breakpoint of an apparently balanced t(7;7)(p13;q22) in an acute myeloid leukemia patient. FISH analysis on bone marrow metaphases from this patient revealed that the sequence corresponding to a series of three ordered cosmids from 7q22 was deleted from one of the der(7) chromosomes. These cosmids contain the human homologue of the Drosophila homeobox gene cut (CUTL1) and span a region of approximately 150 kb. Although the proximal boundary of the deleted segment could not be exactly defined, we estimate the size of this deletion to be approximately 500 kb. Subsequently, we carried out FISH studies using the CUTL1 cosmids on a further 16 patients with deletions of 7q and myeloid disorders. The sequence corresponding to at least two of the cosmids was deleted from the del(7q) in 11 out of 14 cases with a proximal breakpoint within 7q22. Further detailed FISH mapping in this series of 17 patients has identified two other nonoverlapping commonly deleted segments at 7q31–q32 and 7q33, respectively. These data confirm and refine other studies, implying that several different genes on 7q may be involved in the pathogenesis of myeloid diseases. Genes Chromosomes Cancer 25:384–392, 1999.

Identification of new partner chromosomes involved in fusions with the ETV6 (TEL) gene in hematologic malignancies.

Several partner genes on different chromosomes have been reported to be fused with the ETV6 gene (located in chromosome band 12p13), with different breakpoints and different frequencies, in various hematologic malignancies, particularly acute myeloid and lymphoid leukemias and myelodysplastic syndromes. By using FISH and molecular analyses, we have analyzed five different pediatric and adult patients carrying cytogenetic abnormalities involving 12p13. Our findings demonstrate that ETV6 was rearranged in all the cases analyzed. In particular, ETV6 was disrupted by translocations with chromosomal bands 7q22, 7q36, 9q11, and 13q12, not previously described as partners of ETV6 in translocations, thus extending its promiscuity in rearranging with different partner genes. Genes Chromosomes Cancer 21:223–229, 1998.

Characterization of the human myeloid leukemia-derived cell line GF-D8 by multiplex fluorescence in situ hybridization, subtelomeric probes, and comparative genomic hybridization.

The human myeloid leukemia cell line GF‐D8 was established from the peripheral blood blasts of a patient with acute myeloid leukemia FAB subtype M1 (AML‐M1). The karyotype, which has not changed significantly over several years of culture, was described initially as 44,XY,‐5,del(7q),inv(7q),add(8q),add(11q),del(12p),‐15,‐17,+mar. With the advent of multicolor fluorescence in situ hybridization (FISH) techniques, the prospect of accurately characterizing this complex karyotype became feasible. In the present study, we applied 24‐color whole‐chromosome painting and analyzed the results using a filter‐based detection system and proprietary software for multiplex FISH (M‐FISH). This resulted in the refinement of the karyotype and the identification of hitherto unsuspected chromosome rearrangements. M‐FISH identified the origin of the add(8q) and add(11q) as well as the small marker chromosome. Both the del(7q) and del(12p) were redefined as unbalanced translocations and an apparently normal chromosome 11 was shown to be t(11;17). Importantly, the del(12p) was shown to be a der(12)t(7;12). Single‐color whole‐chromosome painting studies confirmed these findings, but also identified a cryptic t(Y;12) not seen in the original M‐FISH analysis. We then carried out a FISH screening assay using a complete set of chromosome‐specific subtelomeric probes. This allowed the identification of p and q subtelomeric regions involved in the translocations and indicated amplification of the 8q subtelomeric region. Comparative genomic hybridization (CGH) revealed a highly unbalanced karyotype, as deletions accompanied the majority of translocations, and identified the regions of amplification as 8q22.3‐qter and 11q21‐qter. Finally, conventional FISH with centromeric and unique sequence probes was necessary to elucidate all of the rearrangements. Genes Chromosomes Cancer 24:213–221, 1999.

Familial partial monosomy 7 and myelodysplasia: different parental origin of the monosomy 7 suggests action of a mutator gene.

Two sisters are reported, both with a myelodysplastic syndrome (MDS) associated with partial monosomy 7. A trisomy 8 was also present in one of them, who later developed an acute myeloid leukemia (AML) of the M0 FAB-type and died, whereas the other died with no evolution into AML. Besides FISH studies, microsatellite analysis was performed on both sisters to gather information on the parental origin of the chromosome 7 involved in partial monosomy and of the extra chromosome 8. The chromosomes 7 involved were of different parental origin in the two sisters, thus confirming that familial monosomy 7 is not explained by a germ-line mutation of a putative tumor-suppressor gene. Similar results were obtained in two other families out of the 12 reported in the literature. Noteworthy is the association with a mendelian disease in 3 out of 12 monosomy 7 families, which suggest that a mutator gene, capable of inducing both karyotype instability and a mendelian disorder, might act to induce chromosome 7 anomalies in the marrow. We postulate that, in fact, an inherited mutation in any of a group of mutator genes causes familial monosomy 7 also in the absence of a recognized mendelian disease, and that marrow chromosome 7 anomalies, in turn, lead to MDS/AML.

Identification of a novel molecular partner of the E2A gene in childhood leukemia

The ‘promiscuous’ E2A gene, at 19p13.3, is fused with two different molecular partners, PBX1 and HLF, following two chromosome translocations recurrent in childhood pre-B ALL. We have identified a novel gene, FB1, by virtue of its fusion with E2A and by a combination of molecular techniques. FB1 was localized on 19q13.4, suggesting that the novel chimera originated by a cryptic rearrangement of chromosome 19. Two FB1 transcripts, of 1.2 kb and 1.1 kb, are differentially expressed at low level in a variety of human tissues, including hemopoietic cell lines from different lineages. Accordingly, FB1 cDNA displays high homology with a number of cDNA clones from different human tissues. High homology was found also with cDNA clones from mouse and rat, suggesting that the sequence might be conserved at least among mammals. The function of the putative FB1 protein, however, is currently unknown as database sequence comparisons have failed to reveal strong homology with known proteins. The E2A/FB1 fusion appears to be a recurrent feature of pre-B ALLs, suggesting that it might have a role in the development and/or progression of leukemogenesis.

Reverse transcriptase/polymerase chain reaction follow‐up and minimal residual disease detection in t(1;19)‐positive acute lymphoblastic leukaemia

The t(1;19) is the most frequent recurring chromosomal translocation in childhood acute lymphoblastic leukaemia (ALL). In most cases typical chimaeric E2A‐PBX1 transcripts are expressed as a consequence of this rearrangement, allowing the molecular detection of the t(1;19) at the RNA level. This translocation has been associated with a poor clinical outcome, although intensified chemotherapy has been reported to nullify its adverse prognostic impact. We therefore used reverse transcriptase/polymerase chain reaction (RT‐PCR) to detect residual leukaemic cells at successive times during treatment and to monitor the response to chemotherapy in six t(1;19)‐positive ALL paediatric patients. Five of these patients rapidly achieved molecular remission and no evidence of minimal residual disease (MRD) was found in the remission bone marrows beyond the third month of treatment. One patient still displayed residual leukaemic cells at the end of therapy, although she has been in continuous complete clinical remission (CCR) for 84 months. However, this patient is peculiar in our series in that two different types of chimaeric E2A‐PBX1 transcripts were expressed in her leukaemic cells, only one being detectable in remission.

Molecular Diagnosis and Clinical Relevance of t(9;22), t(4;ll) and t(l;19) Chromosome Abnormalities in a Consecutive Group of 141 Adult Patients with Acute Lymphoblastic Leukemia

Over a time period of five years leukemic blast samples from 141 consecutive patients with adult ALL were referred to our laboratory, for molecular evaluation of chromosome abnormalities. The t(9;22), t(4;11) and t(1;19) which are most commonly found in adult ALL with a B-precursor phenotype were molecularly analyzed by similar RT-PCR based protocols. BCR-ABL transcripts generated by the t(9;22) translocation were demonstrated in 36 patients (25%) and were restricted to the 109 patients with B precursor ALL (33% of this group). Of 83 patients showing a, common phenotype (CD10+), 34 were BCR-ABL positive (41%) whereas only 2 out of 26 with Null ALL (HLADr+, CD19+, CD10) were positive. Interestingly, the percent of BCR-ABL positive CD1O+ ALL increases significantly with age being 20% in patients less than 30 years old and more than 50% in older patients. None of the T-ALL (24 patients) and B-ALL (8 patients) were positive. The majority of cases (67%) showed the p190 gene subtype. The cytogenetic diagnosis of Philadelphia chromosome was always confirmed by the molecular analysis and this approach allowed for the detection of the presence of the BCR-ABL rearrangement in 26 patients when a negative result or no metaphases were obtained. The complete remission rate was similar among BCR-ABL positive and negative patients but a shorter remission duration was observed in those showing molecular evidence of t(9;22) and this finding was significantly evident in CD1O+ ALL patients. By means of comparison, in most of the same adult ALL patients, we analyzed the yet unrecognized prevalence of the t(4;11) and t(1;19) translocations by the molecular analysis of their chromosomal breakpoints. Rearrangements of the ALL-1 gene on 11q23 band and ALL- l1AF.4 fusion transcripts specific for the t(4;11) were demonstrated in 7 out of the 21 Null ALL investigated, with no additional positive cases found among the other ALL subgroups. Overall the clinical behavior of t(4; 11) positive patients was dismal with a very short CR duration. Chimeric E2A-PBX1 transcripts generated by the t(1;19) were found in only two of the 87 B-precursor ALL analyzed. The presented results provide further evidence for the utility of RT-PCR based methods for the molecular diagnosis of chromosome translocations in ALL. The identification of such abnormalities can significantly contribute to the identification of more appropriate therapeutic options for standard and high risk ALL patients

Unique genotypic features of infant acute lymphoblastic leukaemia at presentation and at relapse

Summary Acute lymphoblastic leukaemia (ALL) of infants aged less than 1 year represents a group of patients with peculiar biological features, poor response to therapy and unfavourable prognosis. In order better to characterize this type of leukaemia, we have investigated the immunoglobulin (Ig) and T‐cell receptor (TCR) genes configuration of 21 infants with ALL, and compared the genotypic features with the phenotypic and karyotypic data, as well as with the clinical outcome. All cases had a pre‐B phenotype; 12 (57%) of them were pre‐pre‐B ALL (CD10‐, CD 19 +). Six of the 16 cases evaluated (38%) displayed chromosomal abnormalities: five had the typical translocation t(4;11)(q21;23). Eleven cases presented with a white blood cell count greater than 100.109/1. The clinical course was unfavourable in 14 patients. The genotype of this group of ALL revealed several peculiarities. (1) Of the 21 cases, six (29%) displayed a multiple rearrangement pattern at the IgH locus. (2) In three cases (15%), the light chain genes were rearranged. (3) The TCR β and γ genes were rearranged in only one case (one case at the TCR β and one at the TCR γ locus). (4) The TCR chain was rearranged in eight cases (40%) and rarely deleted; the rearrangements observed were those most frequently observed in B cell‐precursor ALL. Two cases were evaluated both at presentation and at relapse. While the immunophenotype had remained unmodified, comparison of Ig heavy chain gene rearrangements revealed clonal variations in both cases. Taken together, these findings further underline the biological peculiarities of infant ALL compared to ALL which occurs in older children and in adults, and stress the need of differentiated and aggressive therapeutic approach for these patients.

Transcriptional and post‐transcriptional regulation of IL‐1β, IL‐6 and TNF‐α genes in chronic lymphocytic leukaemia

The present study was designed to define the mechanisms of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and tumour necrosis factor (TNF‐α) gene regulation in chronic lymphocytic leukaemia of B cell origin (B‐CLL). By nuclear run‐on analysis, all B‐CLL cases displayed high levels of nuclear transcription of the IL‐6 and TNF‐α genes, whereas IL‐1β gene transcription was only barely detectable. Upon in vitro culture for 1 h, B‐CLL cells from different patients were substantially heterogeneous in terms of expression of steady state mRNA levels of IL‐1β, IL‐6 and TNF‐α even though the pattern of nuclear transcription of these cytokines was only marginally affected by in vitro culture. mRNA stability was then examined and cytokine gene transcripts showed a half life of more than 2 h in cultured B‐CLL cells and treatment with cycloheximide (CHX) did not affect cytokine transcript levels in B‐CLL cells. These results indicate that: steady state levels of each mRNA do not reflect the rate of nuclear transcription of these cytokines in fresh or cultured B‐CLL cells, that purification and in vitro culture of leukaemic cells may amplify cytokine gene expression in B‐CLL, and that cytokine gene transcripts are relatively stable in B‐CLL.

Reciprocal translocation t(12;13)(p13;q14) in acute nonlymphoblastic leukemia: Report and cytogenetic analysis of two cases☆

Two cases of acute nonlymphoblastic leukemia with a reciprocal translocation t(12;13)(p13;q14) are described. Both patients were male adults with a diagnosis of M0 FAB type. Beside standard cytogenetic analysis, we applied fluorescence in situ hybridization (FISH) in order to investigate the position of the RB gene with respect to the breakpoint at 13q14. Our results showed that the RB gene was proximal to the breakpoint, but, apparently, not split in either case.