Carole Barin

Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogénétique Hématologique study

PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Dismal prognostic value of monosomal karyotype in elderly patients with acute myeloid leukemia: a GOELAMS study of 186 patients with unfavorable cytogenetic abnormalities

The prognosis of acute myeloid leukemia (AML) is very poor in elderly patients, especially in those classically defined as having unfavorable cytogenetics. The recent monosomal karyotype (MK) entity, defined as 2 or more autosomal monosomies or combination of 1 monosomy with structural abnormalities, has been reported to be associated with a worse outcome than the traditional complex karyotype (CK). In this retrospective study of 186 AML patients older than 60 years, the prognostic influence of MK was used to further stratify elderly patients with unfavorable cytogenetics. CK was observed in 129 patients (69%), and 110 exhibited abnormalities according to the definition of MK (59%). MK(+) patients had a complete response rate significantly lower than MK(-) patients: 37% vs 64% (P = .0008), and their 2-year overall survival was also decreased at 7% vs 22% (P < .0001). In multivariate analysis, MK appeared as the major independent prognostic factor related to complete remission achievement (odds ratio = 2.3; 95% confidence interval, 1-5.4, P = .05) and survival (hazard ratio = 1.7; 95% confidence interval, 1.1-2.5, P = .008). In the subgroup of 129 CK(+) patients, survival was dramatically decreased for MK(+) patients (8% vs 28% at P = .03). These results demonstrate that MK is a major independent factor of very poor prognosis in elderly AML.

Chromosomal aberrations and their prognostic value in a series of 174 untreated patients with Waldenström's macroglobulinemia

Waldenströms macroglobulinemia is a disease of mature B cells, the genetic basis of which is poorly understood. Few recurrent chromosomal abnormalities have been reported, and their prognostic value is not known. We conducted a prospective cytogenetic study of Waldenströms macroglobulinemia and examined the prognostic value of chromosomal aberrations in an international randomized trial. The main aberrations were 6q deletions (30%), trisomy 18 (15%), 13q deletions (13%), 17p (TP53) deletions (8%), trisomy 4 (8%), and 11q (ATM) deletions (7%). There was a significant association between trisomy of chromosome 4 and trisomy of chromosome 18. Translocations involving the IGH genes were rare (<5%). Deletion of 6q and 11q, and trisomy 4, were significantly associated with adverse clinical and biological parameters. Patients with TP53 deletion had short progression-free survival and short disease-free survival. Although rare (<5%), trisomy 12 was associated with short progression-free survival. In conclusion, the cytogenetic profile of Waldenströms macroglobulinemia appears to differ from that of other B-cell lymphomas. Chromosomal abnormalities may help with diagnosis and prognostication, in conjunction with other clinical and biological characteristics. This trial is registered with Clinicaltrials.gov, numbers NCT00566332 and NCT00608374.

Acute myeloid leukaemia with 8p11 (MYST3) rearrangement : an integrated cytologic, cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique

Thirty cases of acute myeloid leukaemia (AML) with MYST histone acetyltransferase 3 (MYST3) rearrangement were collected in a retrospective study from 14 centres in France and Belgium. The mean age at diagnosis was 59.4 years and 67% of the patients were females. Most cases (77%) were secondary to solid cancer (57%), haematological malignancy (35%) or both (8%), and appeared 25 months after the primary disease. Clinically, cutaneous localization and disseminated intravascular coagulation were present in 30 and 40% of the cases, respectively. AMLs were myelomonocytic (7%) or monocytic (93%), with erythrophagocytosis (75%) and cytoplasmic vacuoles (75%). Immunophenotype showed no particularity compared with monocytic leukaemia without MYST3 abnormality. Twenty-eight cases carried t(8;16)(p11;p13) with MYST3-CREBBP fusion, one case carried a variant t(8;22)(p11;q13) and one case carried a t(8;19)(p11;q13). Type I (MYST3 exon 16-CREBBP exon 3) was the most frequent MYST3-CREBBP fusion transcript (65%). MYST3 rearrangement was associated with a poor prognosis, as 50% of patients deceased during the first 10 months. All those particular clinical, cytologic, cytogenetic, molecular and prognostic characteristics of AML with MYST3 rearrangement may have allowed an individualization into the World Health Organization classification.

Chromosomal translocations involving the IGH@ locus in B-cell precursor acute lymphoblastic leukemia: 29 new cases and a review of the literature

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are recurrent but rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and various partner genes have been described. Here, we report a new series of 29 cases of BCP-ALL with IGH@ translocations. The partner gene was identified by fluorescence in situ hybridization and/or molecular cloning in 20 patients. Members of the CEBP gene family (n = 11), BCL2 (n = 3), ID4 (n = 3), EPOR (n = 2), and TRA/D@ (n = 1) were identified and demonstrated by quantitative real-time reverse transcriptase-polymerase chain reaction to be markedly up-regulated. The present cases, added to those already reported, confirm the diversity of the partner genes, which, apart from BCL2, are specific to BCP-ALL. Collectively, patients with IGH@ translocations may represent a novel sub-group of BCP-ALL occurring in adolescents and young adults.

Patterns of genomic aberrations suggest that Burkitt lymphomas with complex karyotype are distinct from other aggressive B-cell lymphomas with MYC rearrangement.

We previously showed that complex karyotypes (CK) and chromosome 13q abnormalities have an adverse prognostic impact in childhood Burkitt lymphomas/leukemias (BL) and diffuse large B‐cell lymphomas (DLBCL). The aim of our study was to identify recurrent alterations associated with MYC rearrangements in aggressive B‐cell lymphomas with CK. Multicolor fluorescence in situ hybridization (M‐FISH) was performed in 84 patient samples (59 adults and 25 children), including 37 BL (13 lymphomas and 24 acute leukemias), 12 DLBCL, 28 B‐cell lymphomas with intermediate features (DLBCL/BL), 4 B‐cell precursor acute lymphoblastic leukemias (BCP‐ALL), and 3 unclassifiable B‐cell lymphomas. New (cytogenetically undetected) abnormalities were identified in 80% of patients. We also refined one‐third of the chromosomal aberrations detected by karyotyping. M‐FISH proved to be more useful in identifying chromosomal partners involved in unbalanced translocations and in revealing greater complexity of 13q rearrangements. Most of the newly identified or refined recurrent alterations involved 1q, 13q and 3q (gains/losses), 7q and 18q (gains), or 6q (losses), suggesting that these secondary aberrations may play a role in lymphomagenesis. Several patterns of genomic aberrations were identified: 1q gains in BL, trisomies 7 in DLBCL, and 18q‐translocations in adult non‐BL. BCP‐ALL usually displayed an 18q21 rearrangement. BL karyotypes were less complex and aneuploid than those of other MYC‐rearranged lymphomas. BCP‐ALL and DLBCL/BL were associated with a higher rate of early death than BL and DLBCL. These findings support the categorization of DLBCL/BL as a distinct entity and suggest that BL with CK are indeed different from other aggressive MYC‐rearranged lymphomas, which usually show greater genetic complexity.

Genetic differences between paediatric and adult Burkitt lymphomas.

Dysregulation of MYC is the genetic hallmark of Burkitt lymphoma (BL) but it is encountered in other aggressive mature B‐cell lymphomas. MYC dysregulation needs other cooperating events for BL development. We aimed to characterize these events and assess the differences between adult and paediatric BLs that may explain the different outcomes in these two populations. We analysed patterns of genetic aberrations in a series of 24 BLs: 11 adults and 13 children. We looked for genomic imbalances (copy number variations), copy‐neutral loss of heterozygosity (CN‐LOH) and mutations in TP53, CDKN2A, ID3 (exon 1), TCF3 (exon17) and CCND3 (exon 6). Young patients displayed more frequent 13q31.3q32.1 amplification, 7q32q36 gain and 5q23.3 CN‐LOH, while 17p13 and 18q21.3 CN‐LOH were only detected in adult BLs. ID3 mutations were present in all adult samples, but only in 42% of childhood cases. CCND3 and ID3 double‐hit mutations, as well as 18q21 CN‐LOH, seemed to be associated with poorer outcome. For the first time, we report different genetic anomalies between adult and paediatric BLs, suggesting age‐related heterogeneity in Burkitt lymphomagenesis. This may explain the poorer prognosis of adult BLs. Additional studies are needed to confirm these results in the setting of clinical trials.

14q deletions are associated with trisomy 12, NOTCH1 mutations and unmutated IGHV genes in chronic lymphocytic leukemia and small lymphocytic lymphoma

Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B‐cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1‐69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP‐array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment‐free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over‐representation of the IGHV1‐69 repertoire), NOTCH1 mutations, and a short TFS.

Detection of chromosomal abnormalities associated with chronic lymphocytic leukemia: what is the best method?

B-cell chronic lymphocytic leukemia (CLL) follows a heterogeneous clinical course, for which several biological markers may predict clinical outcome. Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of CLL patients. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. To determine the best strategy for identifying cytogenetic abnormalities, we compared results obtained by FISH analysis on peripheral blood mononuclear cells with those obtained by FISH after culture with mitogens. We studied 46 CLL patients selected from two different institutions. The most frequent structural aberrations leading to loss of genetic material were loss of the 13q14 region, in 32 cases (70%), and loss of TP53 in 11 cases (24%). Of the 46 cases patients, 10 patients (21.7%) had deletion of the ATM locus at 11q22 and 8 patients (17%) had trisomy 12. Results could be interpreted as discordant in four cases in which the abnormal clone was small and both values were around the threshold. FISH performed on stimulated cells detected the same frequency of abnormalities as FISH performed without culture. This equivalence between the two techniques allows performing both conventional cytogenetic and FISH analyses on the same sample.

“Double-hit” chronic lymphocytic leukemia: An aggressive subgroup with 17p deletion and 8q24 gain

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p‐) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p‐ and the additional aberrations influenced the prognosis in a series of 195 patients with 17p‐CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment‐free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this “double‐hit” CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p‐ CLL.