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Fluorescent in situ hybridization (FISH) analysis with a panel of DNA probes for 13q13.1-q14.3 was performed on 20 cases of myeloid malignancies, of which 17 showed a del(13)(q) and three had translocations affecting 13q. By chromosome morphology, deletions consistently involved bands q14 and q21. In addition to confirming the chromosome data, FISH allowed us to delineate a commonly deleted region that was flanked by YAC 833A2 and YAC 854D4. Three cases with 13q translocations unexpectedly showed accompanying cryptic microdeletions of 13q, and in one case the commonly deleted region could be narrowed to a genomic segment, which includes YAC 937C7, RB1, and YAC 745E3. Homozygous deletions were not detected. This region overlaps with the smallest deleted region of 13q14 in chronic lymphocytic leukemia.

A t(5;12)(q33;p13) translocation is a recurrent chromosome abnormality in a subgroup of myeloid malignancies with features of both myeloproliferative disorders and myelodysplastic syndromes (MDSs). The molecular consequence of a t(5;12) is a fusion between the platelet-derived growth factor receptor-B gene on chromosome 5 and a novel ETS-like gene, TEL, on chromosome 12. We report on three patients with a t(5;12)(q33;p13) diagnosed as chronic myelomonocytic leukemia, and one case of a t(10;12)(q24;p13) in a progressive MDS, with eosinophilia and monocytosis. Involvement of the TEL gene in these chromosome translocations was investigated by fluorescence in situ hybridization (FISH) with cosmid probes containing selectively the 5′ end or 3′ end of TEL. Hybridization of these cosmids to the der(5)/der(10) or a der(12), respectively, demonstrated a rearrangement of TEL in both translocations, showing that the t(10;12) is a variant translocation of the t(5;12). Cloning of the fusion cDNA of one case of t(5;12) showed that the breakpoint occurred at the RNA level at exactly the same position as reported by Golub et al (Cell 77:307, 1994). In addition, the TEL gene on chromosome 12 could be localized between two probes previously mapped to 12p13, namely PRB1 and D12S178, leading to a better definition of the position of TEL in this chromosome region. Moreover, in the case involving chromosome 10, the breakpoint occurred between cKTN206 and cKTN312/LYT-10 at 10q24. Clinicohematological data in these studies as well as the restriction mapping of chromosomal breakpoints strongly suggest that (1) common features in MDSs involving the TEL gene are monocytosis and eosinophilia, (2) chromosomes other than no. 5 may be involved and at least a t(10;12)(q24;p13) variant chromosome translocation does exist in these MDSs, and (3) both standard and variant 12p/TEL translocations may be identified by FISH with appropriate probes.

Structural rearrangements including deletions of the short arm of chromosome 12 are frequent cytogenetic findings in various hematologic malignant disorders. Using FISH with a panel of DNA probes we detected loss of a common region of 12p in 22 patients with different hematologic disorders. Nine of them were characterized cytogenetically by a del(12p), seven by unbalanced translocations, and in the remaining cases the loss of the 12p region was masked by translocations and insertions, adding extra material to the short arm of chromosome 12. The smallest commonly deleted region found in all cases analyzed included ETV6, the gene for p27kip1 (CDKN1B), and the D12S178 marker.

Fluorescent in situ hybridization (FISH) analysis with a panel of DNA probes for 13q13.1-q14.3 was performed on 20 cases of myeloid malignancies, of which 17 showed a del(13)(q) and three had translocations affecting 13q. By chromosome morphology, deletions consistently involved bands q14 and q21. In addition to confirming the chromosome data, FISH allowed us to delineate a commonly deleted region that was flanked by YAC 833A2 and YAC 854D4. Three cases with 13q translocations unexpectedly showed accompanying cryptic microdeletions of 13q, and in one case the commonly deleted region could be narrowed to a genomic segment, which includes YAC 937C7, RB1, and YAC 745E3. Homozygous deletions were not detected. This region overlaps with the smallest deleted region of 13q14 in chronic lymphocytic leukemia.

Between 1998 and 2002, 509 patients with AML (median age 46 yrs, range 15–60) were enrolled in the multicenter LAM99P study of the Italian GIMEMA group. To better evaluate the clinical impact of genetic characterization, all patients received a uniform protocol and diagnostic samples were centralised for cytogenetic and molecular studies. Therapy consisted of HU pre-treatment (2g/m2 for 5 days) followed by induction with DNR (50 mg/m2 d 1, 3, 5), cytarabine (100 mg/m2 d 1–10) and etoposide (100 mg/m2 d 1–5) and consolidation with cytarabine (500 mg/m2/q12 hrs d 1–6) and DNR (50 mg/m2 d 4–6). After consolidation, eligible patients with an identical HLA donor were to receive allogeneic SCT and the remaining peripheral blood autologous SCT. Cytogenetic and molecular genetic characterization (including analysis of major fusion genes, FLT3 and NPM status) was available in 397 (78%) patients. Compared to previous GIMEMA studies, the possibility to collect samples during the 5d of HU pretreatment considerably improved genetic characterization and in particular centralised karyotyping by overcoming the problem of sampling and shipment over the w-end. After induction, 269/397 (68%) patients achieved CR. For induction response, conventional K identified 3 distinct risk groups as follows: low risk (inv. 16 and t8;21), intermediate (normal K and other anomalies not comprised in the high risk group) and high risk (t3;3, inv.3, t9;22, 11q23, 5/7 abnormalities complex K,) with CR rates of 92%, 67% and 39%, respectively (P<.0001). NPM mutations were significantly associated with older age, higher WBC, normal K and FLT3-ITD. CR rates in NPM+ (mutated) vs. NPM- (wildtype) groups were 76% vs. 60% for the whole population and 81% vs, 61% for patients in the normal K group (P<.001 for both comparisons). Multivariate analysis for CR indicated that low risk K and NPM+ were independent factors favorably affecting CR achievement while FLT3 status had no significant impact on CR. The analysis of prognostic factors for DFS and OS was carried out in 269 patients in CR (median follow-up of 39 mos.) and multivariate analysis performed after adjusting for unfavorable factors (WBC count). Multivariate analysis of variables influencing OS showed the following: low vs intermediate K, P=.0005; high vs intermediate K, P<.0001; FLT3+ vs. FLT3−, P=.06. Multivariate analysis for DFS showed: low risk vs. intermediate risk K, p=.01; high risk vs. intermediate risk K, p= .03; FLT3+ vs. FLT3-, p=.0002. NPM status did not significantly influence DFS in either the whole population or in the normal K group. In particular, there was no difference in the DFS rates among patients NPM+ and NPM- in the normal K/FLT3- group while in the normal K/FLT3+ group there was a trend (p=.06) for lower relapse rate for NPM+ patients as compared to NPM- ones. These results highlight the relevance of combining cytogenetic and molecular studies in the diagnostic work up of AML and confirm the impact of karyotype on all outcome estimates as well as of FLT3 status on DFS. As to NPM mutations, these appear a favorable predictor of CR achievement. Further investigations in large clinical trials are needed to assess the prognostic value of NPM mutations on outcome in AML with normal karyotype.

Between 1998 and 2002, 509 patients with AML (median age 46 yrs, range 15–60) were enrolled in the multicenter LAM99P study of the Italian GIMEMA group. To better evaluate the clinical impact of genetic characterization, all patients received a uniform protocol and diagnostic samples were centralised for cytogenetic and molecular studies. Therapy consisted of HU pre-treatment (2g/m2for 5 days) followed by induction with DNR (50 mg/m2 d 1, 3, 5), cytarabine (100 mg/m2 d 1–10) and etoposide (100 mg/m2 d 1–5) and consolidation with cytarabine (500 mg/m2/q12 hrs d 1–6) and DNR (50 mg/m2 d 4–6). After consolidation, eligible patients with an identical HLA donor were to receive allogeneic SCT and the remaining peripheral blood autologous SCT. Cytogenetic and molecular genetic characterization (including analysis of major fusion genes, FLT3 and NPM status) was available in 397 (78%) patients. Compared to previous GIMEMA studies, the possibility to collect samples during the 5d of HU pretreatment considerably improved genetic characterization and in particular centralised karyotyping by overcoming the problem of sampling and shipment over the w-end. After induction, 269/397 (68%) patients achieved CR. For induction response, conventional K identified 3 distinct risk groups as follows: low risk (inv. 16 and t8;21), intermediate (normal K and other anomalies not comprised in the high risk group) and high risk (t3;3, inv.3, t9;22, 11q23, 5/7 abnormalities complex K,) with CR rates of 92%, 67% and 39%, respectively (P<.0001). NPM mutations were significantly associated with older age, higher WBC, normal K and FLT3-ITD. CR rates in NPM+ (mutated) vs. NPM- (wildtype) groups were 76% vs. 60% for the whole population and 81% vs, 61% for patients in the normal K group (P<.001 for both comparisons). Multivariate analysis for CR indicated that low risk K and NPM+ were independent factors favorably affecting CR achievement while FLT3 status had no significant impact on CR. The analysis of prognostic factors for DFS and OS was carried out in 269 patients in CR (median follow-up of 39 mos.) and multivariate analysis performed after adjusting for unfavorable factors (WBC count). Multivariate analysis of variables influencing OS showed the following: low vs intermediate K, P=.0005; high vs intermediate K, P<.0001; FLT3+ vs. FLT3−, P=.06. Multivariate analysis for DFS showed: low risk vs. intermediate risk K, p=.01; high risk vs. intermediate risk K, p= .03; FLT3+ vs. FLT3-, p=.0002. NPM status did not significantly influence DFS in either the whole population or in the normal K group. In particular, there was no difference in the DFS rates among patients NPM+ and NPM- in the normal K/FLT3- group while in the normal K/FLT3+ group there was a trend (p=.06) for lower relapse rate for NPM+ patients as compared to NPM- ones. These results highlight the relevance of combining cytogenetic and molecular studies in the diagnostic work up of AML and confirm the impact of karyotype on all outcome estimates as well as of FLT3 status on DFS. As to NPM mutations, these appear a favorable predictor of CR achievement. Further investigations in large clinical trials are needed to assess the prognostic value of NPM mutations on outcome in AML with normal karyotype.