The costimulatory molecule CD70 is regulated by distinct molecular mechanisms and is associated with overall survival in diffuse large B-cell lymphoma.

In diffuse large B-cell lymphomas (DLBCL), a recurrent deletion of the 19p13 region has recently been described. CD70 and TNFSF9 genes are suspected tumor suppressor genes, but previous studies suggest an oncogenic role for CD70. Therefore, we studied the consequences of variation in CD70 copy number and epigenetic modifications on CD70 expression. Copy-number variation was investigated in 144 de novo DLBCL tissues by comparative genomic hybridization array and quantitative multiplex PCR. Gene expression was assessed by quantitative RT-PCR, and CD70 promoter methylation was determined by pyrosequencing. The 19p13.3.2 region was deleted in 21 (14.6%) cases, which allowed the minimal commonly deleted region of 57 Kb that exclusively includes the CD70 gene to be defined. Homozygous deletions were observed in four (2.7%) cases, and acquired single-nucleotide variations of CD70 were detected in nine (6.3%) cases. CD70 was highly expressed in both germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL compared to normal tissue, with distinct molecular mechanisms of mRNA expression regulation. A gene dosage effect was observed in the GCB subtype, whereas promoter methylation was the predominant mechanism of down regulation in the ABC subtype. However, high CD70 expression levels correlated to shorter overall survival in both the GCB (P = 0.0021) and the ABC (P =0.0158) subtypes. In conclusion, CD70 is targeted by recurrent deletions, somatic mutations and promoter hypermethylation, but its high level of expression is related to an unfavorable outcome, indicating that this molecule may constitute a potential therapeutic target in selected DLBCL.

Cytogenetics in the management of mature B-cell non-Hodgkin lymphomas: Guidelines from the Groupe Francophone de Cytogénétique Hematologique (GFCH).

Non-Hodgkin lymphomas (NHL) consist of a wide range of clinically, phenotypically and genetically distinct neoplasms. The accurate diagnosis of mature B-cell non-Hodgkin lymphoma relies on a multidisciplinary approach that integrates morphological, phenotypical and genetic characteristics together with clinical features. Cytogenetic analyses remain an essential part of the diagnostic workup for mature B-cell lymphomas. Karyotyping is particularly useful to identify hallmark translocations, typical cytogenetic signatures as well as complex karyotypes, all bringing valuable diagnostic and/or prognostic information. Besides the well-known recurrent chromosomal abnormalities such as, for example, t(14;18)(q32;q21)/IGH::BCL2 in follicular lymphoma, recent evidences support a prognostic significance of complex karyotype in mantle cell lymphoma and Waldenström macroglobulinemia. Fluorescence In Situ Hybridization is also a key analysis playing a central role in disease identification, especially in genetically-defined entities, but also in predicting transformation risk or prognostication. This can be exemplified by the pivotal role of MYC, BCL2 and/or BCL6 rearrangements in the diagnostic of aggressive or large B-cell lymphomas. This work relies on the World Health Organization and the International Consensus Classification of hematolymphoid tumors together with the recent cytogenetic advances. Here, we review the various chromosomal abnormalities that delineate well-established mature B-cell non-Hodgkin lymphoma entities as well as newly recognized genetic subtypes and provide cytogenetic guidelines for the diagnostic management of mature B-cell lymphomas.

The complex karyotype in hematological malignancies: a comprehensive overview by the Francophone Group of Hematological Cytogenetics (GFCH).

Karyotype complexity has major prognostic value in many malignancies. There is no consensus on the definition of a complex karyotype, and the prognostic impact of karyotype complexity differs from one disease to another. Due to the importance of the complex karyotype in the prognosis and treatment of several hematological diseases, the Francophone Group of Hematological Cytogenetics (Groupe Francophone de Cytogénétique Hématologique, GFCH) has developed an up-to-date, practical document for helping cytogeneticists to assess complex karyotypes in these hematological disorders. The evaluation of karyotype complexity is challenging, and it would be useful to have a consensus method for counting the number of chromosomal abnormalities (CAs). Although it is not possible to establish a single prognostic threshold for the number of CAs in all malignancies, a specific consensus prognostic cut-off must be defined for each individual disease. In order to standardize current cytogenetic practices and apply a single denomination, we suggest defining a low complex karyotype as having 3 CAs, an intermediate complex karyotype as having 4 CAs, and a highly complex karyotype as having 5 or more CAs.

Comparison of a quantitative PCR method with FISH for the assessment of the four aneuploidies commonly evaluated in CLL patients.

Four chromosomal defects associated with outcome are commonly evaluated by fluorescent in situ hybridization (FISH) in chronic lymphocytic leukemia (CLL), namely deletions of the 13q13-q14, 11q22 and 17p13 regions and trisomy 12. In this study, we compared a quantitative PCR method--quantitative multiplex PCR of short fluorescent fragment (QMPSF)--with FISH for the detection of these acquired aneuploidies in a series of 110 patients with Binet stage A CLL. Genes located in the deleted or gained regions were selected as target genes and amplified using a method based on the simultaneous amplification of short fluorescent genomic fragments under quantitative conditions. A chromosomal imbalance involving one or several of the four loci was detected by either method in 72 patients (65%). A chromosome 13 deletion was present in 61 patients (54%), a 11q22 deletion in nine (8%), a trisomy 12 in nine and a 17p deletion in one. FISH and QMPSF results were identical for 103 out of 110 patients and discrepancies could be explained in most cases. This study demonstrates that a quantitative multiplex PCR represents a cost-effective method that could replace FISH in CLL patients. However, although QMPSF is perfectly adapted to the detection of primary defects, care should be taken when searching for clonal evolutions present in a small proportion of tumor cells.

NUP98 is rearranged in 3.8% of pediatric AML forming a clinical and molecular homogenous group with a poor prognosis.

Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.

Amplification of AML1 gene is present in childhood acute lymphoblastic leukemia but not in adult, and is not associated with AML1 gene mutation.

The AML1/CBFA2/RUNX1 gene is the target of many recurrent translocations seen in different leukemia subtypes. The t(12;21)(p13;q22) is the most frequent translocation observed in childhood B acute lymphoblastic leukemia (ALL), occurring in 20% to 25% of cases. In adult ALL this rearrangement is scarce. Another route of AML1deregulation could be point mutations in the runt domain. We now report on AML1amplification in two cases of childhood ALL, found in a series of 107 consecutive children with B-lineage ALL analyzed by fluorescence in situ hybridization (FISH). A parallel analysis of 42 adult B-ALL failed to detect any AML1 rearrangement by FISH. The two patients with AML1 amplification were further analyzed using molecular techniques. SSCP analysis did not detect any mutation. Furthermore, direct sequencing of the cDNA did not reveal any mutation. In conclusion, AML1amplification seems to be observed only in childhood ALL and is not associated with AML1 gene mutation. Other mechanisms, such as gene dosage effects could be hypothesized.

Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11).

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most reported JAK2 fusions cases reveal an aggressive clinical course and long-term remissions have only been achieved after allogeneic stem cell transplantation (ASCT). To the best of our knowledge, this is the thirteenth case reported worldwide to describe a BCR-JAK2 fusion transcript in MPN-U. The present report revealed a sustained complete clinical, hematologic, and cytogenetic remission 35 months after diagnosis and ~24 months after ASCT. Regarding BCR-ABL1 negative MPN patients this case report provides strong support for a role of JAK2 activation in the oncogenesis and suggests a possible diagnostic and therapeutic target that should be investigated.

[Hypercalcemia a sign of medullar transformation of low grade malignant lymphoma. Apropos of a case]. / Hypercalcémie: signe de transformation médullaire d'un lymphome de faible grade de malignité. A propos d'une observation.

The authors report a case of transformation of a low grade non-Hodgkin's lymphoma (LGL) to an agressive lymphoma in a 55 year-old woman who was treated by fludarabine phosphate. The only sign of transformation was the supervention of an hypercalcemia. This complication is rare in the evolution of the LGL and the mechanism is original.

CD66c expression in B-cell acute lymphoblastic leukemia: strength and weakness.

INTRODUCTION: In B-cell acute lymphoblastic leukemia (B-ALL), testing at diagnosis for BCR/ABL1 gene rearrangements is mandatory for prognostic stratification and treatment decisions. Several diagnostic methods have been proposed using flow cytometry to identify BCR/ABL1(+) B-ALL. METHODS: We evaluated expression of the myeloid antigen CD66c by flow cytometry in B-ALL. We studied 94 patients with B-ALL. The t(9;22)(q34;q11) or BCR/ABL1 rearrangement was detected by cytogenetic analysis or RT/PCR. Myeloid antigens CD66c, CD13, CD33, CD117, Myeloperoxidase, CD15 and CD65 were determined by flow cytometry. RESULTS: Of these 94 cases, 17 (18%) cases displayed BCR/ABL1 gene rearrangements and 38 (40%) cases were CD66c positive. CD66c was the most common myeloid antigen expressed on malignant lymphoblasts. Its expression was correlated with BCR/ABL1 rearrangements (P = 0.0001): sensitivity 82%, specificity 69%, positive predictive value 37% and negative predictive value 95%. Co-expression of CD66c(+) CD13(+) was more frequent in BCR/ABL1(+) B-ALL (29%) than BCR/ABL1(-) cases (4%) (P = 0.0044). Some BCR/ABL1(-) B-ALL cases (including hyperdiploid or cases with normal karyotype) were CD66c positive (31%). CONCLUSION: CD66c expression is correlated, but not specifically, with BCR/ABL1 rearrangement. It would seem better to interpret the absence of CD66c expression with a lack of BCR/ABL1 rearrangement. This myeloid antigen could be interesting in the detection of minimal residual disease.

Recurrent genomic aberrations combined with deletions of various tumour suppressor genes may deregulate the G1/S transition in CD4+CD56+ haematodermic neoplasms and contribute to the aggressiveness of the disease.

CD4+CD56+ haematodermic neoplasms (HDN) constitute a rare disease characterized by aggressive clinical behaviour and a poor prognosis. Tumour cells from HDN are leukaemic counterparts of plasmacytoid dendritic cells (pDCs). Despite increased knowledge of the ontogenetic origin of these tumours, the genetic causes and oncogenic signalling events involved in malignant transformation are still unknown. To delineate novel candidate regions and disease-related genes, we studied nine typical CD4+CD56+ HDN cases using genome-wide high-resolution array comparative genomic hybridization (CGH). Genomic imbalances, which were predominantly losses, were frequently detected. Gross genomic losses or gains involving an entire chromosome were observed in eight cases. The most frequent imbalances were deletions of chromosome 9, chromosome 13 and partial losses affecting 17p or 12p. Combinations of deletions of tumour suppressor genes (TSG), namely RB1, CDKN1B (p27), CDKN2A, (p16(ink4a), p14(arf)) or TP53 (p53), were observed in all cases. These results indicate that deletion events altering G1/S regulation are crucial for HDN oncogenesis. Furthermore, in addition to frequent sporadic gene losses, in one case we observed a 8q24 interstitial deletion that brought MYC closer to miR-30b/miR-30d, which may be related to their deregulation. Taken together, these results indicate that in addition to frequent G1/S checkpoint alterations, various genetic events could contribute to the chemoresistance of the tumour.

Genome profiling of acute myelomonocytic leukemia: alteration of the MYB locus in MYST3-linked cases.

The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia. It fuses two genes encoding histone acetyltransferases (HATs), MYST3 located at 8p11 to CREBBP located at 16p13. Variant translocations involve other HAT-encoding genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked acute myeloid leukemias (AMLs) share specific clinical and biological features and a poor prognosis. Because of its rarity, the molecular biology of MYST3-linked AMLs remains poorly understood. We have established the genome and gene expression profiles of a multicentric series of 61 M4/M5 AMLs including 18 MYST3-linked AMLs by using array comparative genome hybridization (aCGH) (n=52) and DNA microarrays (n=44), respectively. We show that M4/5 AMLs have a variety of rare genomic alterations. One alteration, a gain of the MYB locus, was found recurrently and only in the MYST3-linked AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor.

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.

[Comparison of chemiluminescence and polynuclear neutrophil count after aplasia in hematologic malignancies]. / Comparaison de la chémoluminescence et de la numération des polynucléaires neutrophiles après aplasie dans les hémopathies malignes.

Polymorphonuclear neutrophils (PNN) chemiluminescence results from the luminescence emission in oxidative reactions during phagocytosis. This activity is easily detectable and measurable using a luminometer. PNN luminescence allows precociously the evaluation of the end of the aplasia. We have analysed PNN luminescence emission stimulated by latex beads in 69 patients with hemopathies after 72 aplasias following autologous bone marrow transplantation (9 pts), allogeneic bone marrow transplantation (26 pts) and acute myeloblastic leukemia (AML) induction chemotherapies (37 pts). Luminescence emission was measured in whole blood using a luminometer before, during and after aplasia and was compared to PNN manual count. Chemiluminescence measurement is a simple and reproductable method. Its allows more earlier than the PNN blood count the detection of PNN recovery: chemiluminescence nadir is reached on average on the fourth day of aplasia and correspond to the value procured by sample tubes without PNN. The onset of the chemiluminescence increasing is definite by the doubling of the nadir value. It is reached on average on the fourteenth day of aplasia. It precedes 3.5 days and 10.5 days a total PNN count > or = 0.1 x 10 9/1 and > or = 0.5 x 10 9/1 respectively, in the 72 aplasias. In autologous and allogeneic bone marrow transplantation, chemiluminescence increasing precedes at least PNN > or = 0.1 x 10 9/1 of 4.6 days whereas in AML induction chemotherapies, the advantage is only 1.5 day (p = 0.0078). The chemiluminescence could be considered as an additional tool in daily management of sustained aplasia.

Molecular analysis of chromosome arm 17q gain in neuroblastoma.

Complete or partial gain of the long arm of chromosome 17 (17q) has been shown recently by molecular cytogenetic techniques to be the most frequent chromosomal change in neuroblastoma and to be associated with adverse prognosis. Few reports, however, have focused on the precise mapping of the commonly overrepresented region. We have investigated 17q gain by the analysis of allelic imbalances at microsatellite loci dispersed along chromosome 17 in a series of 69 neuroblastomas. Allelic imbalances for at least two consecutive loci were observed in 39/59 informative cases, that is in agreement with previously reported frequencies of 17q gain. In a subset of the cases, comparative genomic hybridization analysis established the relationship between these allelic imbalances and the gain of 17q material. A partial 17q gain was observed in 9 cases, delineating a common region of 17q gain between the marker D17S787 (75 cM, 360 cR) and the telomere. In most cases, molecular results were suggestive of partial tri- or tetrasomy, whereas in 4 cases a higher copy number was documented. Our results also confirm that the presence of additional 17q material is closely associated with 1p36 deletion, MYCN amplification, and diploid or tetraploid chromosomal content. Genes Chromosomes Cancer 28:276-284, 2000.