Gianluca Barba

Genetic profile of T-cell acute lymphoblastic leukemias with MYC translocations.

MYC translocations represent a genetic subtype of T-lineage acute lymphoblastic leukemia (T-ALL), which occurs at an incidence of ∼6%, assessed within a cohort of 196 T-ALL patients (64 adults and 132 children). The translocations were of 2 types; those rearranged with the T-cell receptor loci and those with other partners. MYC translocations were significantly associated with the TAL/LMO subtype of T-ALL (P = .018) and trisomies 6 (P < .001) and 7 (P < .001). Within the TAL/LMO subtype, gene expression profiling identified 148 differentially expressed genes between patients with and without MYC translocations; specifically, 77 were upregulated and 71 downregulated in those with MYC translocations. The poor prognostic marker, CD44, was among the upregulated genes. MYC translocations occurred as secondary abnormalities, present in subclones in one-half of the cases. Longitudinal studies indicated an association with induction failure and relapse.

Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults

Background Molecular lesions in T-cell acute lymphoblastic leukemias affect regulators of cell cycle, proliferation, differentiation, survival and apoptosis in multi-step pathogenic pathways. Full genetic characterization is needed to identify events concurring in the development of these leukemias. Design and Methods We designed a combined interphase fluorescence in situ hybridization strategy to study 25 oncogenes/tumor suppressor genes in T-cell acute lymphoblastic leukemias and applied it in 23 adult patients for whom immunophenotyping, karyotyping, molecular studies, and gene expression profiling data were available. The results were confirmed and integrated with those of multiplex-polymerase chain reaction analysis and gene expression profiling in another 129 adults with T-cell acute lymphoblastic leukemias. Results The combined hybridization was abnormal in 21/23 patients (91%), and revealed multiple genomic changes in 13 (56%). It found abnormalities known to be associated with T-cell acute lymphoblastic leukemias, i.e. CDKN2A-B/9p21 and GRIK2/6q16 deletions, TCR and TLX3 rearrangements, SIL-TAL1, CALM-AF10, MLL-translocations, del(17)(q12)/NF1 and other cryptic genomic imbalances, i.e. 9q34, 11p, 12p, and 17q11 duplication, del(5)(q35), del(7)(q34), del(9)(q34), del(12)(p13), and del(14)(q11). It revealed new cytogenetic mechanisms for TCRB-driven oncogene activation and C-MYB duplication. In two cases with cryptic del(9)(q34), fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction detected the TAF_INUP214 fusion and gene expression profiling identified a signature characterized by HOXA and NUP214 upregulation and TAF_I, FNBP1, C9orf78, and USP20 down-regulation. Multiplex-polymerase chain reaction analysis and gene expression profiling of 129 further cases found five additional cases of TAF_I-NUP214-positive T-cell acute lymphoblastic leukemia. Conclusions Our combined interphase fluorescence in situ hybridization strategy greatly improved the detection of genetic abnormalities in adult T-cell acute lymphoblastic leukemias. It identified new tumor suppressor genes/oncogenes involved in leukemogenesis and highlighted concurrent involvement of genes. The estimated incidence of TAF_I-NUP214, a new recurrent fusion in adult T-cell acute lymphoblastic leukemias, was 4.6% (7/152).

Comparative genomic hybridization identifies 17q11.2∼q12 duplication as an early event in cutaneous T-cell lymphomas

Mycosis fungoides (MF) and Sézary syndrome (SS) are primary cutaneous T-cell lymphomas (CTCL), a heterogeneous group of extranodal non-Hodgkin lymphomas. In the three cases of MF and four of SS studied, comparative genomic hybridization detected chromosomal imbalances in all SS cases and in one MF case. In all five abnormal cases, the long arm of chromosome 17 was completely or partially duplicated; in three of these five cases, it was the sole genomic event. Notably, a minimal common duplicated region at 17q11.2 approximately q12, corresponded to the mapping of HER2/neu and STAT family genes. The only recurrent loss involved chromosome 10, with deletion of the entire long arm in one case and deletion of band 10q23 in another. Sporadic imbalances included gains at chromosome arms 1q, 2q, 7p, 7q, and 12p. Genomic duplication at 17q11.2 approximately q12 emerged as a primary karyotypic abnormality common to both MF and SS, which suggests that this is an early clonal event.

Different genomic imbalances in low- and high-grade HCV-related lymphomas.

Chronic infection with hepatitis C virus (HCV) is related to monoclonal B-cell lymphoproliferative disorders including a benign monoclonal lymphoproliferation such as type II mixed cryoglobulinemia, and B-cell non-Hodgkins lymphomas (NHL).

Multiple EWSR1-WT1 and WT1-EWSR1 copies in two cases of desmoplastic round cell tumor

To provide new insights into the genomic profile of desmoplastic round cell tumors (DSRCT), we applied fluorescence in situ hybridization (FISH) and metaphase comparative genomic hybridization (M-CGH) to two newly diagnosed cases. FISH detected multiple subclones bearing one to three copies of der(11)t(11;22)(p13;q12) and/or der(22)t(11;22)(p13;q12) in both patients. This peculiar genomic imbalance might result from derivative chromosome duplication due to non-disjunction and/or mitotic recombination between normal and derivative chromosomes 11 and 22. Concomitant loss of normal chromosomes (i.e., 11 in patient 1 and 22 in patient 2) caused loss of the WT1 or EWSR1 wild-type allele. M-CGH identified other genomic imbalances: gain at chromosome 3 in both cases and chromosome 5 polysomy in patient 1. Common genomic events (i.e., trisomy 3 and extra EWSR1-WT1 and WT1-EWSR1 copies) probably contributed to disease pathogenesis and/or evolution of DSRCT. Our study demonstrated that an integrated molecular cytogenetic approach identified EWSR1-WT1 cooperating molecular events and genetic markers for prognosis. Thus, FISH and M-CGH might well be applied in a large series of patients to elucidate the genomic background of DSRCT.

Rescue of genomic information in adult acute lymphoblastic leukaemia (ALL) with normal/failed cytogenetics: a GIMEMA centralized biological study.

Metaphase (M‐) and array (A‐) Comparative Genomic Hybridization (CGH) were used to investigate 40 cases of T‐ and 32 of B‐cell acute lymphoblastic leukaemia (ALL) with normal/failed cytogenetics. M‐CGH was performed in all cases and A–CGH in 10/12 T‐ALL cases with uncertain/normal M‐CGH results. M‐CGH was abnormal in 38/72 cases, with a total of 110 imbalances (60 gains, 50 losses). 25/40 patients with T‐ALL (62·5%) showed 77 imbalances, with at least 1 genomic imbalance and a mean of 3 aberrations/patient (range 1–12). 13/32 patients with B‐ALL (40·6%) presented 34 imbalances, with a mean of 2·6 imbalances (range 1–8). A‐CGH detected 4 more T‐ALL cases with genomic imbalances. A‐CGH identified NF1/17q11·2 deletion and interphase fluorescence in situ hybridization provided a 10·8% estimated overall incidence of NF1/17q11·2 deletion in T‐ALL. In all but one case (6/7) with NF1 deletion, denaturing high‐performance liquid chromatography and direct sequencing detected NOTCH1 gene mutations. Three or more imbalances in CGH‐positive cases were significantly associated with resistance to treatment and death during or after induction therapy. We suggest that the work‐up for ALL at diagnosis should include CGH investigations, particularly when cytogenetics is uninformative, because they may provide potentially valuable information with prognostic and therapeutic implications.

DDX3X-MLLT10 fusion in adults with NOTCH1 positive T-cell acute lymphoblastic leukemia.

MLLT10 (also known as AF10 ), at chromosome 10 band p12, is emerging as a promiscuous gene. Six partners have been reported to date: PICALM(CALM )/11q14, MLL /11q23, NAP1L1 /12q21, HNRNPH1 /5q35, DDX3X /Xp11.3 and NUP98 /11p15.[1][1],[2][2] All fusions retain the MLLT10 octapeptide motif-leucine-

NUP98/11p15 translocations affect CD34+ cells in myeloid and T lymphoid leukemias.

We assessed lineage involvement by NUP98 translocations in myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML), and T-cell acute lymphoblastic leukaemia (T-ALL). Single cell analysis by FICTION (Fluorescence Immunophenotype and Interphase Cytogenetics as a Tool for Investigation of Neoplasms) showed that, despite diverse partners, i.e. NSD1, DDX10, RAP1GDS1, and LNP1, NUP98 translocations always affected a CD34+/CD133+ hematopoietic precursor. Interestingly the abnormal clone included myelomonocytes, erythroid cells, B- and T- lymphocytes in MDS/AML and only CD7+/CD3+ cells in T-ALL. The NUP98-RAP1GDS1 affected different hematopoietic lineages in AML and T-ALL. Additional specific genomic events, were identified, namely FLT3 and CEBPA mutations in MDS/AML, and NOTCH1 mutations and MYB duplication in T-ALL.

FISH analysis reveals frequent co-occurrence of 4q24/TET2 and 5q and/or 7q deletions.

We investigated TET2 deletion in 418 patients with hematological malignancies. Overall interphase FISH detected complete or partial TET2 monoallelic deletion (TET2(del)) in 20/418 cases (4.7%). TET2(del) was very rare in lymphoid malignancies (1/242 cases; 0.4%). Among 19 positive myeloid malignancies TET2(del) was associated with a 4q24 karyotypic abnormality in 18 cases. In AML, TET2(del) occurred in CD34-positive hematopoietic precursors and preceded established genomic abnormalities, such as 5q- and -7/7q-, which were the most frequent associated changes (Fishers exact test P=0.000).

Clustering of genomic breakpoints at the MLL locus in therapy-related acute leukemia with t(4;11)(q21;q23)

Genomic characterization of translocation breakpoints is relevant to identify possible mechanisms underlying their origin. The consistent association of anthracylines (e.g., epirubicin and idarubicin) in inducing therapy‐related acute leukemias (t‐AL) with mixed lineage leukemia (MLL) gene rearrangement suggests that MLL translocations are causative events for t‐AL. Using asymmetric multiplex PCR strategy followed by direct DNA sequencing, we characterized the genomic breakpoints of the MLL and AFF1 genes in two patients who developed t‐AL with t(4;11)(q21;q23). Chemotherapeutic treatment of the primary disease in both patients included topoisomerase II (topo II) targeting agents. In one case, the MLL breakpoint was located in intron 9 at nucleotide position chr11:118354284 while the AFF1 breakpoint was in intron 3 at nucleotide position chr4:87992070. The breakpoint junction sequences revealed an insertion of two nucleotides at the MLL‐AFF1 junction. In the other patient, the MLL breakpoint was located in intron 11 at nucleotide position chr11:118359130‐32 and the AFF1 break was in intron 3 at nucleotide position chr4:87996215‐17. The MLL breakpoint found in the latter patient was identical to that of two previously reported cases, strongly suggesting the presence of a preferential site of DNA cleavage in the presence of topo II inhibitor. In addition, microhomologies at the breakpoint junctions were indicative of DNA repair by the non‐homologous end joining (NHEJ) pathway. This study further supports the evidence that MLL breakpoints in therapy‐related acute leukemia with MLL‐AFF1 are clustered in the telomeric half of the breakpoint cluster region that contains topo II recognition sites.