NUP98 rearrangements in hematopoietic malignancies: a study of the Groupe Francophone de Cytogénétique Hématologique.

The NUP98 gene is fused with 19 different partner genes in various human hematopoietic malignancies. In order to gain additional clinico-hematological data and to identify new partners of NUP98, the Groupe Francophone de Cytogénétique Hématologique (GFCH) collected cases of hematological malignancies where a 11p15 rearrangement was detected. Fluorescence in situ hybridization (FISH) analysis showed that 35% of these patients (23/66) carried a rearrangement of the NUP98 locus. Genes of the HOXA cluster and the nuclear-receptor set domain (NSD) genes were frequently fused to NUP98, mainly in de novo myeloid malignancies whereas the DDX10 and TOP1 genes were equally rearranged in de novo and in therapy-related myeloid proliferations. Involvement of ADD3 and C6ORF80 genes were detected, respectively, in myeloid disorders and in T-cell acute lymphoblastic leukemia (T-ALL), whereas the RAP1GDS1 gene was fused to NUP98 in T-ALL. Three new chromosomal breakpoints: 3q22.1, 7p15 (in a localization distinct from the HOXA locus) and Xq28 were detected in rearrangements with the NUP98 gene locus. The present study as well as a review of the 73 cases previously reported in the literature allowed us to delineate some chromosomal, clinical and molecular features of patients carrying a NUP98 gene rearrangements.

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.

Alternative origin of p13MTCP1-encoding transcripts in mature T-cell proliferations with t(X;14) translocations.

The MTCP1 gene is involved in the t(X;14)(q28;q11) translocation associated with T-cell prolymphocytic leukemia and related conditions. This gene is unusual in that it codes for two distinct proteins: a small mitochondrial protein, p8MTCP1, and a putative oncogenic protein, p13MTCP1. Scarcity of material from t(X;14)-associated proliferations and very low levels of mRNA expression have so far prevented a thorough description of p13MTCP1-encoding transcripts. Here, we characterize two additional t(X;14) bearing leukemias allowing this analysis. In one case, with a breakpoint located 5' to the MTCP1 gene, the level of transcription of previously described p13MTCP1-encoding transcripts is enhanced. In the second case, with a breakpoint within the MTCP1 intron I, an alternative transcription initiation site is demonstrated in the tumor cells at 229 bp upstream to exon II. The identification of this internal promoter, together with the similarity between TCL1 and MTCP1 genomic structures, allow us to propose a model in which the duplication of an ancestral gene was followed by the insertion of one copy within the intron of a p8-encoding gene, accounting for the unusual feature of the MTCP1 gene.

Prognostic impact of p27KIP1 expression in cyclin D1 positive lymphoproliferative disorders.

Nodal mantle cell lymphoma (MCL) is a well-defined entity, but non-nodal leukemic cyclin D1 positive lymphoproliferative disorders have been reported and their relationship with MCL remains controversial and their prognosis heterogeneous. We prospectively studied the expression of cyclin D1 in CD5 positive leukemic B lymphoproliferative disorders at diagnosis and identified 65 cases overexpressing cyclin D1. We did not distinguish any clinical or biological criteria allowing one to identify a non-MCL group. Multivariate analysis identified age, anemia and p27kip1 expression as independent prognostic factors of survival. By univariate analysis, p27kip1 high expression proved to be the strongest predictor of prolonged survival. The median survival of p27 low expressors was 30 months, while it was not reached for p27 high expressors. A high level of p27 expression was often found associated with the absence of nodal involvement and the presence of somatic mutations, but neither of them was restricted to the p27 high expression group. In conclusion, we hypothesize that MCL and these cyclin D1 positive leukemic lymphoproliferative disorders represent a continuous spectrum of diseases. Determination of p27 expression level appears as a routine applicable test allowing identification of a subset of patients who could be considered for different therapeutic approaches.

Quantitative RNA slot-blot analysis of CCND1/cyclin D1 expression in suspected mantle cell lymphoma.

Abnormal CCND1 expression is found in the majority of mantle cell lymphomas (MCL) and in a minority of other mature B cell malignancies. Its evaluation can therefore aid diagnostic classification, in conjunction with clinical, morphological, immunophenotypic and cytogenetic analysis. We describe a rapid slot-blot hybridization technique allowing quantitative assessment of CCND1 expression relative to beta-actin, with a sensitivity cut-off of approximately 10%. This allowed clear separation (P < 0.01) of CCND1 MCL (0.89 +/- 0.4; range 0.23-1.81; n = 25) from control samples (0.02 +/- 0.04; range 0-0.09; n = 22) on limited quantities of RNA (1-3.5 microg). Of nine samples in which a potential diagnosis of MCL lymphoma was based on morphological analysis of paraffin-embedded material, without adequate immunophenotype analysis, all were CCND1 negative and subsequent immunophenotype demonstrated features compatible with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) (CD5+, CD23+, FMC7-) in all cases tested. This study demonstrates the feasibility of slot-blot CCND1 quantification and the importance of the availability of cryopreserved material.

Fluorescence in situ hybridization analysis of masked (8;21)(q22;q22) translocations.

The translocation (8;21)(q22;q22) is associated with acute myeloblastic leukemia (AML M2). The accurate detection of this chromosomal rearrangement is vital due to its association with a favorable prognosis. Variant translocations exist; these may be hidden within an unusual or complex karyotype. In such cases, it is often difficult to confirm the presence of t(8;21)(q22;q22) by conventional cytogenetic analysis alone. The molecular detection of the AML1/ETO fusion gene is possible by reverse transcriptase polymerase chain reaction (RT-PCR) or dual-color fluorescence in situ hybridization (FISH) using probes specific for AML1 and ETO. Four cases of AML M2, with unusual or complex structural chromosomal abnormalities, without cytogenetic evidence of the classical t(8;21)(q22;q22), were studied by FISH. Two were AML1/ETO positive by RT-PCR, one showed a rearrangement by AML1 by Southern analysis, and the fourth had morphological features characteristic of t(8;21). The FISH results showed a co-localization of one AML1 and one ETO signal in interphase and metaphase nuclei in all four cases, demonstrating the presence of variant t(8;21)(q22;q22) rearrangements. Therefore, FISH analysis with the AML1 and ETO probes is extremely valuable, in cases of AML M2, because of its ability to reveal masked t(8;21)(q22;q22) translocations and thus quickly confirm the diagnosis, allowing patients to be assigned to the correct risk group in terms of treatment.

[Spectral karyotyping (SKY) principle, avantages and limitations]. / Le caryotype spectral (SKY): principe, avantages et limites en cytogénétique constitutionnelle et tumorale.

Banding karyotype is a routine technique, which allows the identification of numerous aneusomy and/or aneuploïdy in congenital diseases and cancers. However, this analysis fails to detect small or complex chromosome rearrangements. Molecular cytogenetic techniques like fluorescence in situ hybridization (FISH) analysis can overlap these limitations. Particularly, multicolor karyotyping by spectral karyotyping (SKY) may rectify or precise the conventional karyotype results. With two examples, we present here, the principle, the indications and the limits of this technique for constitutional and cancer chromosomal abnormalities characterization. Moreover, we present an easy way to build efficient sky probes with a best sensitivity than the probes classically used.

Flow cytometric detection of dyserythropoiesis: a sensitive and powerful diagnostic tool for myelodysplastic syndromes.

Several groups have published flow cytometry scores useful for the diagnosis or prognosis of myelodysplastic syndromes (MDS), mainly based on the detection of immunophenotypic abnormalities in the maturation of granulocytic/monocytic and lymphoid lineages. As anemia is the most frequent symptom of early MDS, the aim of this study was to identify markers of dyserythropoiesis relevant for the diagnosis of MDS analyzed by selecting erythroblasts in a whole no-lysis bone marrow strategy by using a nuclear dye. This prospective study included 163 patients, including 126 with cytopenias leading to MDS suspicion and 46 controls without MDS. In a learning cohort of 53 unequivocal MDS with specific markers, there was a significant difference between the coefficients of variation of mean fluorescence intensities of CD71 and CD36 in MDS patients compared with controls. These two parameters and the hemoglobin level were used to build a RED-score strongly suggestive of MDS if ≥ 3. Using the RED-score in the whole cohort, 80% of MDS or non-MDS patients were correctly classified. When combined with the flow score described by Ogata et al., this strategy allowed to reach a very high sensitivity of 88% of patients correctly classified.

Transient familial haemophagocytic lymphohistiocytosis reactivation post-CD34 haematopoietic stem cell transplantation.

Familial haemophagocytic lymphohistiocytosis (FHLH) is a genetic disorder caused by defective lymphocyte cytotoxicity, resulting in impaired lymphocyte homeostasis and macrophage infiltration of solid tissues and bone marrow, with extensive haemophagocytosis. It is invariably fatal unless treated by allogeneic haematopoietic stem cell transplantation (HSCT). In a retrospective analysis of 11 cases of FHLH, transplanted in one centre between January 1999 and December 2003, it was found that host T cell expansion occurred early after HSCT in a setting of a viral infection (cytomegalovirus and Epstein-Barr virus respectively) in two cases who received T cell-depleted HSCT. Transient recurrence of clinical and biological manifestations of FHLH was observed, despite evidence for donor cell engraftment. Secondary development of donor T cells led to stable mixed chimaerism and sustained remission of FHLH. Detection of host-derived T cells soon after HSCT in a patient with FHLH should thus not mistakenly be taken as a manifestation of graft rejection.

T-cell prolymphocytic leukemia with autoimmune manifestations in Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is characterized by growth retardation, microcephaly, mental retardation, immunodeficiency, and predisposition to malignancies, especially B-cell lymphomas. In contrast, leukemia is rare. A 23-year-old NBS patient presented with anemia, thrombocytopenia, and hyperlymphocytosis. The diagnosis of T-cell prolymphocytic leukemia (T-PLL) was confirmed by cytological and immunological assays (TdT(-), CD2(+), CD5(+), CD3m, and CD7(+)). Biological assays also showed a hemolytic anemia and a clotting factor V decrease. The patient was first treated by methylprednisone for 3 weeks. During this period the lymphocyte count decreased. The simultaneous normalization of the hemolysis and of factor V suggested that both could be related to T-PLL. Since T-PLL is refractory to conventional therapies with a poor prognosis, an intensive chemotherapy such as 2'-deoxycoformycin with anti-CDw52 monoclonal antibodies is usually favored. In the present case, however, because of the specific context (i.e., NBS-induced immunodepression, severe hemolytic anemia, and acquired factor V deficiency), he received pentostatin weekly during 1 month and in maintenance during 6 months. At last follow-up (7 months) he showed a persistent control of the lymphocytosis with no side effect.

A spontaneous remission of lymphoid blast crisis in chronic myelogenous leukaemia following blood transfusion and infection.

We report a case of spontaneous remission of lymphoid blast crisis in chronic myelogenous leukaemia (CML) which returned to chronic phase, without the use of cytostatic chemotherapy, following an episode of viral infection and blood transfusion. Although complete remissions of acute leukaemia have been described, this evolution is extremely rare and has never been reported in CML blast crisis. The role of hypothetical factors leading to such a rare event are briefly discussed.

Simultaneous detection of MYC, BVR1, and PVT1 translocations in lymphoid malignancies by fluorescence in situ hybridization.

The rapid detection of chromosome band 8q24 rearrangements, including classical translocations involving MYC and variant 3' translocations, is important for the accurate diagnosis and appropriate treatment of lymphoid malignancies. We have identified and characterized a CEPH YAC, 934e1, which extends from at least 190 kbp upstream to over 280 kbp downstream to MYC, allowing detection of classical t(8; 14)(q24;q32) and variant t(8;22)(q24;q11) and t(8;14)(q24;q11), extending distal to PVT1 and therefore, by extrapolation, to BVR1. This YAC also allowed clarification of complex chromosome 8 abnormalities and the identification of translocations in interphase nuclei. A second CEPH YAC, 904c3, previously shown to contain the PVT1 locus but not MYC, allowed distinction between translocations occurring centromeric and telomeric to MYC. Use of the 934e1 YAC will aid classification of a variety of lymphoid proliferations and further characterization of rearranged cases with the 904c3 YAC will simplify mapping of their diverse breakpoints.

NG2 expression in MLL rearranged acute myeloid leukaemia is restricted to monoblastic cases.

Expression of NG2 has been reported in the majority of paediatric acute leukaemia (AL) cases with MLL rearrangement. We demonstrated 7. 1 positivity in 2/3 paediatric and 4/11 adult MLL rearranged acute myeloid leukaemia (AML) but in 0/28 adult AML without MLL rearrangement, thus extending the 100% specificity to adult cases. Positivity correlated with stage of maturation arrest since it was found in 0/6 immature AML but in 6/8 monoblastic cases. These data demonstrate that, if NG2 expression in AL is the (in)direct result of MLL rearrangement, such activation is restricted to a monoblastic population in AML. They also have practical implications for NG2 diagnostic screening strategies.

Remission of transformed myelodysplastic syndrome with fibrosis after danazol therapy.

Danazol has been used with success in some hematological diseases, but there is no report of this treatment in acute leukemia. We report here a case of remission of myelodysplastic syndrome with myelofibrosis in transformation after danazol therapy in a 72-yr-old man. The role of danazol in remission induction is briefly discussed.

Molecular detection of t(8;21)/AML1-ETO in AML M1/M2: correlation with cytogenetics, morphology and immunophenotype.

The t(8;21) identifies a subgroup of acute myeloid leukaemia (AML) with a relatively good prognosis which may merit different treatment. It is associated predominantly, but not exclusively, with AML M2, and corresponds to rearrangements involving the AML1 and ETO genes. AML1-ETO positive, t(8;21) negative cases are well recognized but their incidence is unknown. In order to determine optimal prospective AML1-ETO RT-PCR screening strategies, we analysed 64 unselected AML M1 and M2 cases and correlated the results with other biological parameters. Molecular screening increased the overall detection rate from 8% to 14%. AML1-ETO was found in 3% (1/32) of AML M1 and 25% (8/32) of M2, including three patients without a classic (8;21) but with chromosome 8 abnormalities. It was more common in younger patients. Correlation with morphology enabled development of a scoring system which detected all nine AML1-ETO-positive cases with a false positive rate of 7% (4/55). Although certain AML1-ETO-positive cases demonstrated characteristic immunological features (CD19 and CD34 expression, CD33 negativity), each of these markers was insufficiently specific to permit prediction in an individual case. We conclude that initial routine prospective molecular screening for AML1-ETO in all AMLs, combined with standardized morphological and immunological analysis, is desirable in order to produce improved prognostic stratification and to determine whether screening can ultimately be restricted to appropriate subgroups.

Genetic analysis of splenic lymphoma with villous lymphocytes: a Groupe Français d'Hématologie Cellulaire (GFHC) study.

In order to characterize the genetic diversity in splenic lymphoma with villous lymphocytes (SLVL), we have undertaken cytogenetic and molecular analyses of CCND1 expression and BCL1-IgH PCR rearrangement in 76 cases diagnosed predominantly on morphological criteria. Cytogenetic abnormalities were detected in 19/44 (43%) of cases, including in 16/25 (64%) of cases with an absolute lymphocytosis. Abnormalities included those involving chromosome 14q32 (9/19, 47%), predominantly t(11;14)(q13;q32) (5/19, 26%), chromosome 3 (26%), predominantly 3q, chromosome 17p (26%) and trisomy 12 (3/19, 16%) and were thus suggestive of pathogenetic diversity. CCND1 was expressed in 8/30 (27%) cases, including in all t(11;14) cases, 5/10 (50%) CD5-positive cases and also in 3/20 (15%) CD5-negative cases. Three CCND1-positive SLVL demonstrated immunophenotypic features similar to mantle cell lymphoma (MCL) but the majority differed in their CD5 negativity or CD23 positivity. BCL1-IgH rearrangement was only seen in 1/62 (2%) of cases overall and in none of the t(11;14) cases, which demonstrated FISH breakpoints both centromeric and telomeric to the BCL1/MTC, suggesting that, if genomic clustering exists in t(11;14) SLVL, it differs from MCL. Although CCND1 expressing SLVL more commonly had marked lymphocytosis, they did not demonstrate a more aggressive clinical course than their negative counterparts, demonstrating that the detection of CCND1 expression or of a t(11;14) should not suffice to alter diagnostic classification in the absence of other criteria.