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.

Fluorescence in situ hybridization analysis of 110 hematopoietic disorders with chromosome 5 abnormalities: do de novo and therapy-related myelodysplastic syndrome-acute myeloid leukemia actually differ?

A retrospective cytogenetic study of acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) was conducted by the Groupe Francophone de Cytogénétique Hématologique (GFCH) to evaluate the structural abnormalities of chromosome 5 associated with other chromosomal abnormalities, in particular of chromosome 7, in these pathologies. In all, 110 cases of AML/MDS were recruited based on the presence of chromosome 5 abnormalities under conventional cytogenetics and supplemented by a systematic fluorescence in situ hybridization study of chromosomes 5 and 7. The abnormalities of the long arm of chromosome 5 (5q) were deletions of various sizes and sometimes cryptic. The 5q abnormalities were associated with translocations in 54% of cases and were simple deletions in 46%. In 68% of cases, 5q deletions were associated with chromosome 7 abnormalities, and 90% of these presented a complex karyotype. Of the 110 patients, 28 had a hematopoietic disorder secondary to chemotherapy, radiotherapy, or both. Among 82 patients with de novo AML/MDS, 63 were older than 60 years. Chromosomal abnormalities often associated hypodiploidy and chromosome 5 and 7 abnormalities in complex karyotypes, features resembling those of secondary hemopathies. Systematic investigation of the exposure to mutagens and oncogenes is thus essential to specify the factors potentially involved in MDS/AML with 5q abnormalities.

[A cytological, immunophenotypical and cytogenetical study of 136 consecutive cases of B-cell chronic lymphoid hemopathies]. / Etude cytologique, immunophénotypique et cytogénétique d'une série de 136 cas consécutifs d'hémopathies lymphoïdes chroniques à cellules B matures.

A cytological, immunophenotypical and cytogenetical study of 136 chronic B-cell proliferations (93 CLL, 43 B-cell lymphomas) was led in order to precise diagnosis and to characterize and appreciate chromosomal rearrangements. In this series, mainly selected on blood lymphocytosis criteria, B-CLL were twice more frequent than small B-cell lymphomas. Probes used revealed cryptic abnormalities, which remained unknown by conventional cytogenetics (CC). The frequency of clonal abnormalities (CC and FISH) was 74.8% for this series, with 74.4% for lymphomas and 75.3% for CLL, mainly of Binet stage A (69 A, 13 B, 1 C, 10 unspecified). Proportion was 88.4% in A stages and 84.6% in B stages. In CLL, 13q14 cryptic deletions and translocations were widely majority, 14q32 translocations and trisomy 12 being predominant in lymphoma series. Interphase FISH study of non-clonal metaphasic abnormalities with locus-specific probes often revealed unrecognised clones.

Loss of the NPM1 gene in myeloid disorders with chromosome 5 rearrangements.

The assignment with chromosome banding techniques of the breakpoints of the recurrent translocation t(3;5) which leads to NPM1/MLF1 gene fusion in myeloid malignancies has not been unequivocal. In order to assess whether this is due to uncertainty in interpretation of the observed banding pattern or whether it reflects true genomic heterogeneity, we decided to analyze the breakpoint positions using fluorescence in situ (FISH) techniques in eight patients with myeloid malignancies and rearrangements of chromosomes 3 and 5. In three patients, colocalization of the NPM1 and MLF1 spanning BACs was demonstrated and NPM1/MLF1 fusion shown by PCR in one while in the remaining cases breakpoints were located outside the NPM1 and MLF1 loci. Interestingly, loss of a copy of the NPM1 gene was found in three of these latter patients. This findings suggest that haploinsufficiency of NPM1 may play a role in subtypes of myelodysplasias and leukemias.

Cytogenetic study of 75 erythroleukemias.

Chromosomal abnormalities of erythroleukemia (EL) are often described as complex and unspecific. A retrospective study of 75 EL defined following the WHO classification was performed by the Groupe Francophone de Cytogénétique Hématologique (GFCH) in order to reexamine the cytogenetics of this infrequent leukemia subtype. Clonal chromosomal abnormalities were found in 57 patients (76%), distributed in 4 subgroups according to their ploidy status: pseudodiploid (16%), hypodiploid (47%), hyperdiploid (19%), and 18% mixed cases associating 2 different clones (hypodiploid+hyperdiploid) or (pseudodiploid+hyperdiploid). Complex rearrangements and hypodiploid chromosome number were widely dominant (50%). Partial or entire monosomies represented 56% of abnormalities. Chromosomes 5 and 7 were the most frequently involved (41 and 33 times, respectively), followed by chromosomes 8, 16, and 21 (19 times each). Unbalanced abnormalities were more frequent than balanced. All these kinds of abnormalities were observed in de novo as well as in secondary EL. Four out of 7 cases of "pure erythroid" leukemia were associated with a BCR-ABL fusion. Lastly, no chromosome abnormality specific to EL could be established. However, the large overlap of chromosomal abnormality patterns of EL (pure erythroid form excepted) and refractory anemia with excess of blasts in transformation (RAEB-t) favors the hypothesis of similarities between these 2 hematologic disorders.

t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukemia. A collaborative study of the Groupe Français de Cytogénétique Hématologique (GFCH).

To accurately estimate the incidence of HOX11L2 expression, and determine the associated cytogenetic features, in T-cell acute lymphoblastic leukemia (T-ALL), the Groupe Français de Cytogénétique Hématologique (GFCH) carried out a retrospective study of both childhood and adult patients. In total, 364 patients were included (211 children

High incidence of Hox11L2 expression in children with T-ALL.

The orphan homeobox gene HOX11L2 was previously found to be transcriptionally activated as a result of the t(5;14)(q35;q32) translocation in three T-ALL cases. We now tested by RT-PCR Hox11L2 expression in 23 consecutive cases of T-ALL (15 children aged 0.8-14 years, eight adults aged 17-55 years) and as control 13 B-ALL patients from a single institution. Hox11L2 expression was undetectable in all patients with B-ALL, nor in adults with T-ALL. Nine children (60% of the cases), all boys, expressed Hox11L2. Blast cells from most of the latter patients carried surface CD1a, CD10 and not CD34 antigens, in contrast to the other children. FISH, M-FISH and IPM-FISH analysis failed to detect a t(5;14)(q35;q32) in one of them, which suggests a possible distinct genetic mechanism in Hox11L2 expression induction. Hence, Hox11L2 expression seems to be the most frequent abnormality in childhood T-ALL to date, comparable to the t(12;21) in child B-ALL.

Translocation t(5;14)(q35;q32) in three cases of childhood T cell acute lymphoblastic leukemia: a new recurring and cryptic abnormality.

We report three cases of T-ALL in which conventional cytogenetic analysis yielded normal karyotypes, but for which a new M-FISH technique (IPM-FISH) was able to detect a translocation. For these patients this technique highlighted a new, recurring and cryptic translocation t(5;14)(q35;q32) in childhood T-ALL which might be phenotypically restricted. The most innovative part of this technique is the use of interspersed polymerase chain reaction (IRS-PCR) painting probes that show an R-band pattern simultaneous with the combinatorial labeling. Contrary to the DOP-PCR, IRS-PCR-derived probes provide stronger hybridization signals at the telomeric ends that potentially increase the possibility of detecting cryptic translocations. All the IPM-FISH findings were validated by FISH with whole chromosome painting and unique sequence probes. These results demonstrate the efficient use of IPM-FISH as an improved, single-step method for the identification of cryptic chromosomal abnormalities. This new IPM-FISH technique is a good tool to display cryptic chromosomal abnormalities.

Spiroplasma turonicum sp. nov. from Haematopota horse flies (Diptera: Tabanidae) in France.

Strain Tab4cT, a helical prokaryote that was isolated from the body of a Haematopota sp. fly collected in Champchevrier, Indre-et-Loire, Touraine, France, was found to be a member of the class Mollicutes. The cells of strain Tab4cT were small, motile helices that were devoid of a cell wall. The organism passed through filters with mean pore diameters as small as 0.20 mm. Strain Tab4cT grew rapidly in liquid SP-4 medium at both 30 and 37 degrees C. The organism fermented glucose but did not hydrolyse arginine or urea, and did not require serum for growth. In preliminary electrophoretic analyses, the cell protein patterns of strain Tab4cT were distinct from those of 14 other spiroplasmas found in mosquitoes, deer flies and horse flies from Europe and the Far-East. In reciprocal metabolism inhibition and deformation serological tests, employing antigens and antisera representative of spiroplasma groups I-XXXIII (including all sub-groups), plus ungrouped strains BARC 1901 and BARC 2649, no serological relationship with Tab4cT was found. The G + C content of the DNA of strain Tab4cT was about 25 +/- 1 mol% and its genome size was 1.305 kbp. It is proposed that spiroplasma strain Tab4cT be assigned to group XVII (presently vacant) and that strain (ATCC 700271T) is the type strain of a new species, Spiroplasma turonicum.

Three spiroplasmas isolated from Haematopota sp. (Diptera:Tabanidae) in France.

On August 1991, three spiroplasmas (Tab 2A, Tab 2B and Tab 4C) were isolated from Haematopota sp. flies collected from Indre-et-Loire, France. Isolations were made at 28 degrees C in MD1 medium from ground individual flies, but not from external washings. All isolates adapted well to SP4 medium at 30 and 37 degrees C and were triple cloned before serological identification. Using the cross deformation test, the 3 isolates were compared with 42 known spiroplasmas isolated from diseased plants, flowers, various insects and ticks belonging to 25 serological groups. Each isolate cross-reacted with others and exhibited weak 1-way reactions with TABS-2 or PLHS-1, two spiroplasmas isolated in the United States from respectively Tabanus abactor Philip and Panorpa helena L, a scorpion-fly. These results suggest that Tab 2A, Tab 2B and Tab 4C together with TABS-2 and PLHS-1 may represent, members of a new serological supergroup.

Serological and molecular characterization of Mesoplasma seiffertii strains isolated from hematophagous dipterans in France.

Three strains of nonhelical mollicutes previously isolated in France from two different mosquitoes and one tabanid fly were designated strains Ar 2328 (isolated from Aedes detritus), Ar 2392 (isolated from Aedes caspius), and CP 13 (isolated from Chrysops pictus). All of these strains exhibited properties of the genus Mesoplasma, a recently described genus of non-sterol-requiring mollicutes isolated from plants and insects. The results of metabolism inhibition and growth inhibition tests revealed that these strains and Mesoplasma entomophilum TAC or Mesoplasma florum L1 were not serologically related, but all three dipteran strains reacted strongly with Mesoplasma seiffertii F7T (T = type strain) antibodies. Using metabolism inhibition and growth inhibition tests, we found that the dipteran strains were related to each other and to strain F7T but were not identical. We also found that they were able to multiply and persist in the central nervous systems of suckling mice inoculated intracerebrally, a property that makes their use as biological control agents for pest dipterans inadvisable. Scanning electron microscopy revealed marked differences in the morphologies of the colonies of the different strains on SP4 solid medium. The levels of DNA-DNA homology for strains Ar 2328, Ar 2392, CP 13, and F7T were more than 70%, indicating that these strains are closely related members of the same species, M. seiffertii. In addition, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that each strain produced about 40 protein bands. This technique also revealed differences between strains. Using the coefficient of Smeath-Jacquart, we constructed a dendrogram that allowed us to estimate of the levels of relatedness of these four strains. The results which we obtained were confirmed by two-dimensional protein electrophoresis results.

Genetic recombination and DNA transpositions induced by pteridines and extracts of pteridine-treated diapausing chrysalids and mutants injected in Drosophila melanogaster.

This paper presents the results of two different treatments using pteridines in Drosophila melanogaster larvae: injection of pteridines alone; and injection of extracts from diapaused Pieris brassicae chrysalids treated with pteridines. Genetic analysis reveals first the induction of lethal or visible recessive mutations that give rise mostly to developmental mutants with variable phenotypes, and second the induction of genetic recombinations. Both treatments disturb genetic recombination in F1 female female issued from the treated larvae. This disturbance is evidenced by the increase in the rate of recombination particularly in the centromere region, and induces in F1 female female and male male clusters of mitotic recombinations of premeiotic origin. These two observations present an analogy with hybrid dysgenesis in the P-M system. This suggests that the treatments either promote the mobility of transposons in female and male larvae and their progeny, or affect the system controlling transposon mobility and integration at specific chromosomal sites. We used in situ hybridization to test our hypotheses, using P, I and copia-like probes. P yields a positive response both at the level of gonadal sterility (gonadal dysgenesis test) and in situ hybridization: after treatment, Oregon K and the wing-altered mutant bspw exhibit a normal number of P elements whereas the maternal strain Oregon K is totally devoid of P. This mutant bspw carries the neutral strain Q (a variant of P), which cannot produce P-M dysgenesis. The implication of these findings for understanding the mode of action of pteridines is twofold: (1) pteridines may be mutagenic agents which perturb meiotic and mitotic recombination; and (2) pteridines disturb the system regulating the mobility and insertion of P elements.

Lethal mutations induced in Drosophila melanogaster by direct or indirect action of pteridines.

The experiments described in this paper show that synthetic pteridines, especially biopterin and pterin, injected directly into Drosophila melanogaster induce recessive lethals. On the contrary, D-neopterin seems to have little effect. A mutagenic effect has previously been shown for an extract of Pieris brassicae in diapause, treated with these pteridines and tetrahydrofolic acid (FH4). It appears that chromosome II is more sensitive to these mutagenic treatments than chromosome X.

Mutations induced by a hormonal imbalance in Drosophila melanogaster.

Pterin treatment of chrysalids in diapause modified the juvenile hormone- ecdysone ratio. The treatment of Drosophila donor mutant with a mixture of reduced folic acid, pterins and extract of Pierides chrysalids in diapause induced the formation of short sequences, by the intermediary of variations in hormonal balance. The effect of this variation was seen in the germinal lines especially at the gonial stage, where recessive, visible or lethal mutations were induced in the form of clusters. Genetically active fractions were found in the 4S-8S and 18S-28S sedimentation zones after saccharose gradient ultracentrifugation. The short DNA sequences coded for tRNA and rRNA. Consequently, it is there DNA sequences from the Drosophila donor mutant that altered the genetic information of the host. 2 types of recessive visible mutation appeared: those affecting the differentiation of the imaginal discs and those affecting the pigment biosynthetic chain. Recessive lethals were induced by treatment. 3 hypothesis are proposed: the first suggests the formation of a short DNA sequence complexing at a specific locus in the acceptor. The second involves transposable factors belonging to the acceptor itself, behaving as a particular transposable factor. The third supposes the induction of alterations at loci of rRNA and tRNA synthesis at the origin of perturbations in protein synthesis. The present data do not allow us to choose between the 3 hypotheses. In conclusion, it seems that a "hormonal imbalance" can have grave consequences not only for the individual itself but also for its descendants.

[Activity of the octanol dehydrogenase, of the alcool dehydrogenase and aldehyde dehydrogenase on the farnesol metabolism. Photoperiodic and neurhormonale regulation, controlling the metabolism of the juvenile hormone, in Pieris brassicae (author's transl)]. / Activité de l'octanol déshydroénase, de l'alcool et de l'aldéhyde déshydrogénase sur le métabolisme du farnésol.

The antagonistic photoperiodic behaviour of the farnesol dehydrogenases indicates that the photonic control mechanism of the brain acts on the farnesol derivates. This cerebral control is double. The first system, linked at the allatotrope function is proportionnal at the photoperiod and acts on the octanol dehydrogenase 0,32. The second system controle the deshydrogenases ADH bands 0,50--0,58, is linked at the darkness. It is linked also at the neurocerebral activity then it stops its activity at the 4th day of the 5th stage. This last seems to be the determinating control for the establishment of the diapause since in short photoperiod, when the inhibition by this system ends, the alcool dehydrogenases 0,50-0,58 series is suractivated in rate with the lasting of the scotophase. In darkness, the 1st system functionnes cyclically and has a maximum synchron with the single maximum of the 2nd system. Inversally, in continuous light, the 2nd system is synchronisated with the 1st which has a prolongated action, maybe linked with a prolongated activity of the neurosecretory cells of the pars intercerebralis and corpora allata.