Diabetes insipidus revealing acute myelogenous leukaemia with a high platelet count, monosomy 7 and abnormalities of chromosome 3: a new entity?

We describe three cases of acute myeloid leukaemia revealed by diabetes insipidus. The patients were 42, 38 and 39 yr old and they had marked hyperleukocytosis, circulating immature granular cells and a normal or elevated platelet count. The leukaemia was type AML-M2 according to the FAB classification. Cytogenetic studies showed inversion of chromosome 3 (q21;q26) in 2 cases and a translocation (3;3)(q21;q29?) in the remaining case, both associated with monosomy 7. All the cerebral CT scans were normal. Complete remission was never achieved, and all three patients survived less than 14 months. Desmopressin therapy was active but treatment could not be reduced. The association of dysmegakaryopoiesis with a chromosome 3 abnormality and diabetes insipidus is probably not fortuitous and could represent a new entity.

Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Successive transformation of chronic myelomonocytic leukaemia into acute myeloblastic then lymphoblastic leukaemia, both with minor-bcr rearrangement.

We report a case of chronic myelomonocytic leukaemia (CMML), which transformed first into acute myeloblastic leukaemia (AML) and then into acute lymphoblastic leukaemia (ALL). In the AML and ALL phases, chromosome analysis showed a classic Philadelphia chromosome (Ph) t(9:22)(q34:q11). Molecular studies showed breakpoint cluster region rearrangement between exons e1 and a2 compatible with a p190(bcr/abl) breakpoint as observed in Ph-positive lymphoblastic acute leukaemia. The minor (m-bcr) rearrangement was also detected during complete remission. This observation supports a multistep pathogenesis of leukaemias, and that the p190(bcr/abl) breakpoint may influence the course of the disease.

Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL).

We sequentially performed cytogenetic analysis and RT-PCR analysis of BCR-ABL transcripts in 17 cases of Ph1-positive ALL who had achieved hematological complete remission (CR) with intensive chemotherapy (CT). Sixteen cases were studied prospectively. All but one of the patients had reached cytogenetic CR, but cytogenetic has low sensitivity in predicting relapse. Twelve patients relapsed, three died in first CR and two were alive in first CR. Two of five, two of four, and five of nine patients who were allografted (in first or second CR), autografted and received consolidation CT, respectively, achieved negative two-round PCR in the bone marrow (BM): three died in CR, three remained in CR with negative two-step PCR in the BM and three relapsed after 22 to 28 months. In all cases, relapse was preceded by switch to PCR positivity in the BM by 4 to 6 months. The remaining nine patients remained PCR-positive in the BM and relapsed after 2 to 16 months. In the four autografted cases, PCR was positive at the time of bone marrow harvest. The two patients who received a purged transplant achieved negative PCR and prolonged CR, whereas the two patients who received an unpurged transplant remained PCR positive and relapsed. In 34% of the samples where analysis was concomitant, sensitivity of PCR proved lower in the blood than in the BM. These findings show that RT-PCR is a useful tool in the monitoring of MRD in Ph1 positive ALL.

Automated counting of in situ hybridization dots in interphase cells of leukemia samples.

Twenty-seven samples (cell cultures prepared for routine cytogenetics) of leukemia patients with known cytogenetic abnormalities were stained by in situ hybridization for interphase cytogenetics with centromere specific probes for chromosome Nos 4, 6, 7, 8, 9, 12, 17, 18, X and Y. The number of hybridization domains per nucleus was quantified using a semi-automated system developed in our laboratory. Results of this automated counting procedure (with and without verification of the counting results by the operator) were compared with conventional cytogenetic data and with visual scoring of the number of hybridization dots. The findings show that the system is capable of analysing 1000 cell nuclei in less than 30 min, including the necessary verification of the results by the operator. Automated counting and visual scoring were in good agreement. Conventional cytogenetics and interphase cytogenetics agreed in only 50% of the cases, confirming other studies showing that conventional cytogenetic results are not always representative for the majority of the cell population.

Expression of BCL-2 proto-oncogene in adult acute lymphoblastic leukemia.

The products of the BCL-2 gene prolong survival of lymphohematopoietic cells by inhibition of programmed cell death. We studied bcl-2 protein expression in a series of 43 adult acute lymphoblastic leukemia (ALL) at diagnosis, using a specific monoclonal antibody and flow cytometry. All samples expressed bcl-2 with a mean percentage of positive cells of 77.9. The level of bcl-2 in positive cells expressed as mean equivalent of soluble fluorescence (MESF) was highly variable ranging from 5 x 10(3) to 552 x 10(3) (mean +/- s.d.: 96.5 +/- 109 x 10(3)). Neither the percentage of positive cells nor bcl-2 MESF levels were correlated with initial characteristics including blood counts, immunological phenotype, or cytogenetics. The survival of leukemic cells maintained in cytokine-free liquid culture was not correlated with bcl-2 expression. However, cells from ALL with higher white blood cell (WBC) counts, with t(9;22) translocation, or expressing myeloid surface antigens exhibited significantly longer survival in this culture system. The outcome after intensive chemotherapy did not differ according to bcl-2 expression. Factors associated with poor outcome included WBC counts, presence of t(9;22) translocation, presence of myeloid antigens and prolonged survival of cultured cells. These results indicate that high levels of bcl-2 are not associated with distinct clinical or biological characteristics in ALL.

Detection of 11q13 rearrangements in hematologic neoplasias by double-color fluorescence in situ hybridization.

Rearrangements within the chromosome 11q13 region are frequent in hematologic malignancies. 50% of 75% of mantle cell lymphomas (MCLs) carry a translocation t(11;14) (q13;q32). Using Southern blot analysis, a BCL1 breakpoint can be detected in approximately 50% of MCLs. It is not known whether other MCLs harbor also breakpoints at 11q13. Breakpoints in this region not involved in t(11;14), are detected in chronic lymphocytic leukemia and acute myeloid leukemia. To detect and localize breakpoints at 11q13 more accurately, we have developed fluorescence in situ hybridization using two probe sets of differently labeled cosmids, symmetrically localized at either side of the major translocation cluster of BCL1. These probes span a region of 450 to 750 kb. We applied this assay to a series of hematologic malignancies with 11q13 abnormalities identified by classical cytogenetics. All four samples with a t(11;14) (q13;q32) showed dissociation of the differently colored signals in metaphase and interphase cells, thereby indicating a chromosomal break in the region defined by the probe sets. The frequency of abnormal metaphase and interphase cells was comparable with that observed in any of the 13 malignancies with other chromosomal 11q13 abnormalities, indicating that these chromosomal breaks occurred outside the 450- to 750-kb region covered by the probes. One patient showed triplication and one patient showed monoallelic loss of this region. The current data show that double-color fluorescence in situ hybridization is a simple and reliable method for detection of the t(11;14)(q13;q32) in interphase cell nuclei and that is can be used to distinguish this translocation from other 11q13 rearrangements in hematologic malignancies.

[Follow-up of allogenic bone marrow graft with DNA polymorphisms. Use of two minisatellite MS31 and MS43 probes. Comparison with data of chromosomal polymorphisms]. / Suivi de greffe allogénique de moelle osseuse par les polymorphismes de l'ADN. Utilisation de deux sondes minisatellites MS 31 et MS 43. Comparaison avec les données des polymorphismes chromosomiques.

OBJECTIVES: Allogenic bone marrow transplantation is widely used to treat many diseases of the haemopoietic system as well as metabolic disorders. Follow-up is essential to assess acceptance, rejection or post-graft relapse. This study was undertaken to evaluate the usefulness of the minisatellite probes MS31 and MS43 used as a routine follow-up test after bone marrow transplantation. METHODS: Twenty receivers of allogenic bone marrow transplants were followed-up. Two monoclonal minisatellite probes, MS31 and MS43, were used for comparison with the classical polymorphism methods. RESULTS: Fourteen cases of total chimeras, 3 cases of rejections and 3 cases of mixed chimeras were observed with the molecular probe techniques. In 19 of the 20 cases, this technique gave results compatible with classical polymorphism results. CONCLUSIONS: The minisatellite probes MS31 and MS43 were found to be sensitive, effective tests for bone marrow transplants which can be used in routine follow-up.

Collagen biosynthesis in cell culture from chorionic villi.

After chorionic villus biopsy of human placenta, cell cultures were propagated with Ham's F10 medium or Eagle's minimum essential medium (MEM). It was possible to study the morphology of the cells by transmission electron microscopy (TEM) after a special culture of the cells in a collagen gel. The cells embedded in a collagen gel were able to contract the gel and to organize collagen fibres, as fibroblast cultures do. TEM showed vacuolization and well-developed cisternae of the endoplasmic reticulum, especially in the case of MEM culture. The aim was to determine whether cells cultivated from early placenta were able to synthesize enough collagen for a metabolic study. A high level of collagen biosynthesis could be quantified. Types I and III collagen can be determined which is useful for studying the abnormalities of collagen synthesis in suspected cases of osteogenesis imperfecta or Ehlers-Danlos type IV syndrome. The hydroxylation of lysine can also be studied with respect to Ehlers-Danlos type VI syndrome. Moreover, these cells, in contrast to fibroblast cultures, made it possible to study the biosynthesis of type IV collagen.

[Acquired myelodysplastic syndromes after treatment of melanoma with fotemustine and dacarbazine]. / Syndromes myélodysplasiques acquis après traitement par fotémustine et dacarbazine pour mélanome.

INTRODUCTION: Secondary myelodysplasia after antimitotic therapy is a rare complication usually observed with alkylating agents. The condition usually progresses to acute leukaemia with very poor short term prognosis. CASE REPORT: We report the cases of 2 women who developed myelodysplasia 2 and 9 months after treatment associating dacarbazine and fotemustine for visceral metastases of a malignant melanoma. DISCUSSION: The frequency of these rare complications is probably underestimated because of the rapid unfavourable outcome of metastatic malignant melanoma. We were unable to determine whether dacarbazine, fotemustine or their combination was incriminated in this complication. Risk could be reduced by carefully determining the cumulative doses of these antimitotics.

[Constitutional translocation t(1;6) and Burkitt's lymphoma]. / Translocation constitutionnelle t(1;6) et lymphome de Burkitt.

BACKGROUND: The relationship between constitutional chromosome aberrations and a predisposition to malignancy has already been established. CASE REPORT: A 4 year-old boy was admitted suffering from abdominal mass. Biopsies of the tumor and bone marrow showed a stage IV Burkitt's lymphoma. Karyotype showed a constitutional t(1;6)(p36;q22) in initial bone marrow samples and peripheral lymphocytes. No classic Burkitt's type translocation was demonstrated in the malignant bone marrow cells. CONCLUSION: Our case is similar to four other reported cases. The break-points 1p36 and 6q22 seem to be involved in several malignant tumors, especially lymphomas.

Rearrangement and overexpression of the BCL-1/PRAD-1 gene in intermediate lymphocytic lymphomas and in t(11q13)-bearing leukemias.

The t(11;14)(q13;q32) translocation and its molecular counterpart, BCL-1 rearrangement, are consistent features of intermediate lymphocytic lymphoma (ILL). Rearrangement is thought to deregulate the nearby PRAD-1/BCL-1 proto-oncogene that is a newly identified member of the cyclin family. To characterize further the association between rearrangement of chromosome 11q13 and over-expression of BCL-1. Southern blot analysis was performed in 33 cases of ILL, 5 cases of t(11;14)-associated leukemias, and 1 case of leukemia carrying a variant translocation t(11;19)(q13;q13) using three separate BCL-1 locus probes. When RNA was available, BCL-1 expression was assessed by Northern blot analysis. DNA from 19 of 33 ILL (57%) showed BCL-1 rearrangement, 16 involving the major translocation cluster (MTC) region and 3 involving a new breakpoint cluster located in the 5' flanking region of the BCL-1 gene. DNA from 3 of 6 t(11q13)-associated leukemias demonstrated a rearrangement involving the MTC. Northern blot analysis showed that BCL-1 was overexpressed in 14 of 15 ILL and in all leukemias analyzed (included the t(11;19) leukemia) relative to normal and malignant lymphoid tissues. These results constitute additional elements in favor of the role of BCL-1 in lymphoid neoplasia and allow us to speculate about its mechanisms of activation.

Pericentric intrachromosomal insertion responsible for recurrence of del(11)(p13p14) in a family.

The combined use of qualitative and quantitative analysis of 11p13 polymorphic markers together with chromosomal in situ suppression hybridization (CISS) with biotin labeled probes mapping to 11p allowed us to characterize a complex rearrangement segregating in a family. We detected a pericentric intrachromosomal insertion responsible for recurrence of del(11)(p13p14) in the family: an insertion of brand 11p13-p14 carrying the genes for predisposition to Wilms' tumor, WT1, and for aniridia, AN2, into the long arm of chromosome 11 in 11q13-q14. Asymptomatic balanced carriers were observed over three generations. Classical cytogenetics had failed to detect this anomaly in the balanced carriers, who were first considered to be somatic mosaics for del(11)(p13). Two of these women gave birth to children carrying a deleted chromosome 11, most likely resulting from the loss of the 11p13 band inserted in 11q. Although in both cases the deletion encompassed exactly the same maternally inherited markers, there was a wide variation in clinical expression. One child, with the karyotype 46,XY, del(11)(p13p14), presented the full-blown WAGR syndrome with aniridia, mental retardation, Wilms' tumor, and pseudohermaphroditism, but also had proteinuria and glomerular sclerosis reminiscent of Drash syndrome. In contrast, the other one, a girl with the karyotype 46,XX,del(11)(p13), only had aniridia. Although a specific set of mutational sites has been observed in Drash patients, these findings suggest that the loss of one copy of the WT1 gene can result in similar genital and kidney abnormalities.

Trisomy 12 in B-cell chronic lymphocytic leukemia: interphase study by in situ hybridization in 75 patients.

Trisomy 12 is the most common cytogenetic abnormality in chronic lymphocytic leukaemia (CLL) and may be a prognostic indicator. In the present study, fluorescence in situ hybridization (FISH) is shown to be a method of choice for detection of trisomy 12 in interphase cells. Seventy-five cases of B-cell CLL were analysed with a chromosome 12 specific alpha satellite DNA probe and results compared with those from cytogenetic analysis. FISH showed the three hybridization spots characteristic of trisomy 12 in 32/75 patients (42.6%). Sixty-three patients were also studied by conventional cytogenetics: failure in 7 cases, normal karyotype in 28, trisomy 12 in 9 (14.3%) and in 19 cases abnormalities other than trisomy 12. In these same 63 patients, trisomy 12 was detected on 29 occasions by FISH (46%): in one case of failure by cytogenetic analysis, in 9 cases thought to have a normal karyotype, in 10 cases carrying abnormalities other than trisomy 12 and in all 9 cases showing trisomy 12 by conventional cytogenetic investigation. Correlation between trisomy 12 and the three stages of the Binet classification indicated an increasing proportion of trisomy 12 from stage A to stage C. It is concluded that fluorescence in situ hybridization is a powerful and sensitive technique for detection of trisomy 12 in CLL and although more cases will be required to confirm a correlation between the incidence of trisomy 12 and the stage of the disease, this link could be important from a prognostic point of view.

Cytogenetic heterogeneity in t(11;19) acute leukemia: clinical, hematological and cytogenetic analyses of 48 patients--updated published cases and 16 new observations.

We report on 16 cases of t(11;19) acute leukemia and review data of published observations: altogether updated data of 48 patients are analyzed. Four hematological groups could be distinguished: (i) 13 cases of acute lymphoblastic leukemia (ALL) of B lineage, mostly CD19+; (ii) eight cases of biphenotypic leukemia: CD19+ (most often) ALL but with simultaneous or inducible expression of differentiation marker of monocytic lineage. The B lineage and biphenotypic leukemias were predominantly found in female infants; (iii) four cases of T-ALL in children; and (iv) 23 acute non-lymphocytic leukemia (ANLL) cases generally of M4 or M5 subtype, predominantly in males. Cytogenetically, at least two subtypes were observed with possibly an identical breakpoint on 11q23 but discrete breakpoints on 19p: lymphoid, biphenotypic, and most congenital myeloid cases showed a distal breakpoint on 19p13 producing 11q- and 19p+ derivatives, while most older myeloid cases showed 11q+ and 19p- derivatives as a result of a more proximal breakpoint on 19p12 or p13.1. The latter type was clearly detected using R bands but barely visible using Q or G bands while the other translocation was easy to detect with G bands but could be missed with R bands. The white blood cell count is usually high in these t(11;19) acute leukemias and prognosis is poor, except for T-ALL cases.

FVT-1, a novel human transcription unit affected by variant translocation t(2;18)(p11;q21) of follicular lymphoma.

Variant t(2;18) and t(18;22) chromosome translocations observed in B-cell chronic lymphocytic leukemias and in follicular lymphomas have been reported to consistently involve the 5' region of the BCL-2 gene on chromosome 18 and various regions on the lg light chain loci. We show here that a variant t(2;18)(p11;q21) translocation observed in a case of follicular lymphoma leads to the juxtaposition of a J kappa segment to a chromosome 18 transcriptional unit located 10 kpb upstream of the BCL-2 locus. The cDNA of this new evolutionarily conserved gene, termed FVT-1 for follicular-variant-translocation gene, codes for a putatively secreted protein of 36 Kd that is not homologous with any described protein. The FVT-1 gene is weakly expressed in all the analyzed normal hematopoietic tissues but a very high rate of transcription is observed in some T-cell malignancies and in phytohemagglutinin-stimulated lymphocytes. The proximity of FVT-1 to the BCL-2 locus suggests that in the t(14;18) currently observed in follicular lymphomas, both genes would participate in the tumoral process.

t(2;18) and t(18;22) variant chromosomal translocations in B cell malignancies.

Variant translocations (2;18 and 18;22) are described in this review. The chromosomal and molecular findings of these translocation of BCL2 and their effect on possible BCL2 gene activation is discussed. Unanswered questions still remain and these include why this is so rare compared to the 25% incidence recorded for translocations in Burkitt's lymphoma. Further studies are obviously still needed in order to determine the true frequency of these findings and their distribution in the various B-cell disorders.

Cytogenetic experience in prenatal fra(X) detection on amniotic fluid cultures.

Since 1987, we have had experience with 13 prenatal diagnoses of 11 women at risk for the fragile X syndrome by cytogenetic studies on amniotic fluid cultures. The induction method included TC 199 medium and methotrexate. Results were obtained in all cases. Ten were males and three were prenatally diagnosed as being affected. Three were females and none of them was fra(X)-positive. Results were confirmed in 10/13 cases. In these cases, we had neither false-positive nor false-negative results.

Chromosome studies in stimulated lymphocytes of 50 patients with B-cell chronic lymphocytic leukemia.

The chromosome constitutions of stimulated lymphocytes from 50 B-cell chronic lymphocytic leukemia patients were studied using different stimulation systems, i.e., TPA alone or associated with different cytokines. Adequate metaphases were obtained in 44 subjects (88%). Among 20 patients with abnormal karyotypes (45.5%), 7 had trisomy 12. The most frequent structural abnormality was a 14q+ resulting from translocations including one t(11;14) and two t(14;17), while deletions on the long arms of chromosomes 6 and 13 constituted a second common alteration. The most important finding in this series was the recurrence of a t(18;22) observed in two cases.