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.

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.

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.

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

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.

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.

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.

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.

[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.

[Juvenile hormone in diapausing Pieris brassicae and mutations. Tetrahydrofolic acid and pterins incubated in chrysalids, provoking ontogenic and mutagenic genetic information alterations in Drosophila melanogaster]. / Hormone juvénile de Pieris brassicae diapausante et mutations. Acide tétrahydrofolique et ptérines incubés dans ces chrysalides provoquant chez Drosophila melanogaster des altérations de l'information génétique ontogènes et mutagènes

Study of AMPc phosphodiesterase shows presence of JH in diapausing chrysalids and antogonistic action of FH and pterines. Study of farnesoldeshydrogenase and farnesal deshydrogenase in Dm shows that FH4 and pterines inhibite FDH, active ADH. Conclusion is JH in diapause chrysalides is active factor with FH4 provoking genesis of pigmentary mutation, cellular proliferation or growth deficiencies. Comparison with JH+FH4+ teromes incubated in Bar (Muller 5) mutants of Dm in place of diapausing chrysalids reproduce larval deficiences, mosaics and mutations observed in precedent experiments.

[Study on deshydrogenases implicated in control of farnesol and juvenile hormone metabolisms in Pieris brassicae (author's transl)]. / Etude de deux déshydrogénases intervenant dans le métabolisme du farnésol et dans le contrôle des hormones allatiques (JH).

Activity of ODH is high during larval development on pars intercerebralis which is releasing its allatotropic neurosecretion towards 4 th day of 5 th instar under photophase 16 h. ADH is inhibited during the same period and then activated at the time of spinning when the level of JH I and II is falling; by oxidization of JH I and JH II, possibly JH III is produced.

[Influence of reduced pterine and juvenile hormone on the pigmentation of the phasms Carausius morosus (author's transl)]. / Influence des ptérines réduites et de l'hormone juvénile sur la pigmentation du phasme Carausius morosus

Tetrahydrofolic acid and dimethyltetrahydropterine, FH4 and DMH4P, have an antagonist action. FH4 darkens the phasms. DMH4P and the tryptophane have an inhibitrice influence on the melanisation, probably by the increase of the synthesis or the release of serotonin, in the hypoderm. For the adult phasm remaining ivory, xanthopterin must be added in the treatment. The addition of diapausing chrysalids pieridae extract (containing juvenile hormone JH) and treated by FH4, in the treatment precedent, transmits the pgimentary effect at the first generation, which presents a strong percentage of dark or ivory phasms.