Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Effect of conditioned media, nutritive elements, and mitotic synchronization on the accuracy of the cytogenetic analysis in acute nonlymphocytic leukemia patients presenting with inv(16)/t(16;16) or t(15;17).

To improve the yield of the cytogenetic analysis in patients with acute nonlymphocytic leukemia (ANLL), six culture conditions for bone marrow or peripheral blood cells were tested in parallel. Two conditioned media (CM), phytohemagglutinin leukocyte PHA-LCM and 5637 CM, nutritive elements (NE), and methotrexate (MTX) cell synchronization were investigated in 14 patients presenting with either inv(16)/ t(16;16) (group 1, n = 9 patients) or t(15;17) (group 2, n = 5). The criteria used to identify the most favorable culture conditions were the mitotic index (MI), the morphological index (MorI), and the percentage of abnormal metaphases. In the presence of PHA-LCM and 5637 CM, the MI were significantly increased in group 2, whereas in the MTX conditions, MI remained very low in both groups. The values of the MorI did not reveal any significant changes in chromosome resolution between the conditions in either group. The addition of NE did not have a positive effect in quantity or quality of metaphases. Because of the variability of the response of leukemic cells to different stimulations in vitro, several culture conditions in parallel are required to ensure a satisfactory yield of the chromosome analysis in ANLL.

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

[Fluorescent in-situ hybridization technique (FISH) in the diagnosis of Philadelphia translocation in chronic myeloid leukemia]. / Application de la technique d'hybridation in situ fluorescente (FISH) au diagnostic de la translocation de Philadelphie dans la leucémie myéloïde chronique.

The Philadelphia chromosome (Ph) resulting from translocation t(9;22)(q34;q11) is observed in more than 90% of patients with chronic myeloid leukemia (CML). Its molecular consequence is the genesis of a fusion gene BCR-ABL between the 5' sequences of the BCR gene (chromosome 22) and the 3' end of the ABL gene (chromosome 9). Fluorescence in situ hybridization (FISH) using specific DNA probes provides a useful tool for the detection of t(9;22) and BCR-ABL rearrangement. We report our results using the FISH technique for t(9;22) assessment in the hematopoietic cells of patients with Ph-positive CML. The DNA libraries pBS 9 and pBS 22 containing multiple sequences derived from chromosomes 9 and 22 have been used to identify t(9;22) in metaphase cells. The cos bcr-51 and cos abl-18 probes that hybridize to unique sequences specific to the BCR and ABL genes have the ability to detect the BCR-ABL rearrangement in metaphase cells as well as in interphase nuclei. FISH is a sensitive and specific technique that represents a valuable complement to conventional cytogenetics. The BCR-ABL rearrangement can be detected in metaphase spreads of insufficient quality or from interphase nuclei in the case of terminally differentiated cells or of cells which do not divide in vitro. When the efficiency of hybridization and detection is good, a large number of cells can be analyzed. This is of major significance in assessment of response to treatment and definition of a cytogenetic remission. However, interphase cytogenetics may be difficult due to variations in signal resolution and background level. The FISH technique can also be used to detect the BCR-ABL rearrangement in cases of Ph negative BCR-ABL positive CML.

[Significance, diagnosis and prognosis in the cytogenetic analysis in acute leukemias and myelodysplastic syndromes]. / Signification diagnostique et pronostique de l'analyse cytogénétique dans les leucémies aiguës et les syndromes myélodysplasiques.

Cytogenetic analysis of leukemic cells has been shown to be a mandatory part of the diagnosis of malignant hemopathies. Recurring abnormalities may be divided into those exclusively found in myeloid disorders, those associated with lymphoid diseases and those detected in both types of hemopathy. Several of the common defects are characteristic of specific FAB types or subtypes and associated with clinico-pathologic manifestations. Cytogenetic abnormalities have served to identify relatively homogenous subsets of malignant hemopathies. In view of the significant progress realized in the treatment of malignant hemopathies, the assessment of prognostic factors is particularly important for therapeutic decision making. The chromosome status at diagnosis has proven to be a major prognostic indicator for survival and outcome in individual patients. This article will focus on the diagnostic and prognostic significance of the karyotype in de novo acute leukemia and primary myelodysplastic syndromes.

Etoposide and secondary haematological malignancies: coincidence or causality?

BACKGROUND: Secondary haematological malignancies (SHM) induced by alkylating agents show cytogenetic abnormalities involving mainly chromosomes 5 and 7. Etoposide and other topoisomerase II inhibitors including anthracyclines induce secondary leukaemias with a short latency period, briefly preceded by a myelodysplastic phase, and frequently associated with balanced translocation characteristically involving the long arms of chromosomes 11 and 21. PATIENTS AND METHODS: Etoposide with doxorubicin containing chemotherapy was administered to 26 patients with Hodgkin's disease (HD), eight of whom received alkylating agents at relapse or progression, and to 59 patients with small-cell lung carcinoma (SCLC), thirty-three of whom received alkylating agents in the same combination. RESULTS: Four of 85 patients developed SHM of the myelodysplastic or acute myeloid type at, respectively, 20, 30, 35 and 38 months after therapy. In 3 of them, chromosome abnormalities were observed: two balanced translocations, t(6;9)(p23;34) and t(11;19)(q23;p13), and one monosomy 7q due to t(1;7)(cen;cen) with trisomy 8. The cumulative risk estimate of SHM is 12% (95% confidence interval (CI): 3%-46%) at 5 years for patients with HD and 18% (95% CI: 5%-49%) at 3 years for patients with SCLC. CONCLUSIONS: Our observations lend further support to the existence of SHM induced by topoisomerase II inhibitors that present early after initial treatment with balanced translocations to 11q23 and 21q22. However, other defects such as unbalanced abnormalities of chromosomes 5 and 7 can occur and may be related to the combination of topoisomerase II inhibitors with alkylating agents. Balanced t(6;9) was observed for the first time in SHM. A synergism between epipodophyllotoxins, anthracyclines, alkylating agents, cisplatin and radiotherapy is suggested, given the observed high risk of SHM.

Trisomy 8 detection in granulomonocytic, erythrocytic and megakaryocytic lineages by chromosomal in situ suppression hybridization in a case of refractory anaemia with ringed sideroblasts complicating the course of paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) was diagnosed in a 20-year-old male patient who suffered from anaemia since the age of 11. Eighteen years after diagnosis, PNH transformed into refractory anaemia with ringed sideroblasts (RARS). Trisomy 8 was observed in 27%, 45% and 53% of the bone marrow metaphase cells analysed in 1987, 1988 and 1990 respectively. In order to determine which bone marrow cell lineages were affected by trisomy 8 and at which stage of stem cell differentiation, MAC (Morphology, Antibody, Chromosomes) and CISS (Chromosomal In Situ Suppression) hybridization techniques were combined. The MAC technique enables karyotypic analysis of morphologically and immunologically classified mitotic cells. CISS hybridization makes it possible to detect individual chromosomes and chromosome aberrations using recombinant DNA libraries from sorted human chromosomes. Trisomy 8 was detected in granulomonocytic (50.6%), erythrocytic (67.2%) and megakaryocytic (one megakaryocyte with trisomy 8, one normal) lineages, providing evidence for the occurrence of trisomy 8 in early haematopoietic cell precursors, at the GEMM or pluripotent level. Cytogenetic and clinical data suggest that the sideroblastic clone originated from a mutation affecting a cell of the PNH clone, progressively replaced by the PNH/RARS clone, due to proliferative advantage.

Three new cases of chromosome 3 rearrangement in bands q21 and q26 with abnormal thrombopoiesis bring further evidence to the existence of a 3q21q26 syndrome.

Defects of 3q in bands q21 and q26 have been reported in more than 70 cases of acute nonlymphocytic leukemia (ANLL), myelodysplastic syndrome (MDS), and myeloproliferative disorder (MPD) in blast crisis. In this paper three additional patients are described: patient 1 with refractory anemia with excess of blasts in transformation (RAEB-T) and inv(3)(q21q26), patient 2 with RAEB-T and t(3;3)(q21;q26), and patient 3 with myelofibrosis with myeloid metaplasia (MMM) in blast crisis and inv(3)(q21q26). In addition to 3q rearrangements, monosomy 7 and del(7)(q22q36) were observed in patients 1 and 2, respectively. In the three patients, the most characteristic clinical features were elevated platelet counts, marked hyperplasia with dysplasia of the megakaryocytes, and poor prognosis. Although disturbance of thrombopoiesis was not systematically observed in all patients with t(3;3)(q21;q26), inv(3)(q21q26), and ins or dup(3)(q21----q26), study of the 77 cases reported and of the three cases presented here brings further evidence to the existence of a cytogenetic syndrome involving bands q21 and q26 simultaneously, which represents a subtype of ANLL, MDS, and MPD, characterized by normal or elevated platelet counts, hyperplasia with dysplasia of megakaryocytes, multilineage involvement, young median age of patients with MDS, preferential involvement of women in t(3;3), high incidence of chromosome 7 defects in MDS and ANLL, short duration of the MDS phase, no response to chemotherapy, short survival, and por prognosis.

[Unusual initial manifestation in a case of refractory anemia with excess of blasts]. / Présentation initiale inhabituelle d'un cas d'anémie réfractaire avec excès de blastes.

In 1987, a 50-year-old patient presented with isolated thrombocytopenia (27,000/mm3) which proved to be refractory to steroid medication and high i.v. doses of immunoglobulin. Two years later he developed macrocytic anemia. Chromosomal analysis confirmed the diagnosis of myelodysplastic syndrome (MDS), refractory anemia type with blast excess. Cytogenetically, three cellular populations were observed: one normal (75% of metaphases) and two abnormal, clone A (2%) 46,XY, del(5q), del(11q), and clone B (23%) 46,XY, del(5q), del(11q) plus 2 other anomalies. Evolution was characterized by worsening of the bicytopenia with marked hypoplasia of the megakaryocytic and erythroid series while the percentage of blasts remained stable. Concerning the chromosomal markers, the normal population disappeared and clone A became predominant (clone A 97%, clone B 3%). This case shows that isolated thrombocytopenia can be the sole initial manifestation of MDS. We discuss the possibility that "refractory thrombocytopenia" constitutes a diagnostic category like refractory anemia or refractory anemia with ring sideroblasts. The proliferative advantage of clone A or the disadvantage of clone B may be due to the occurrence of new, cytogenetically non-detectable mutations.

Inducible and constitutive MHC class II gene expression. Distinct tissue-specific genetic controls.

B cells express MHC class II Ag in a constitutive fashion, whereas macrophages can do so only after induction by a variety of exogenous stimuli. In this study we describe interspecies somatic cell hybrids between the human B cell Raji and the murine macrophage cell P388 D1. This murine cell line does not express detectable levels of class II mRNA. Phenotypic, molecular, and karyotype analysis of a series of hybrids showed that murine macrophage class II genes can be expressed in a constitutive fashion under the control of the human B cell genome. This event is the consequence of de novo accumulation of class II specific mRNA and thus probably reflects activation of transcription. In certain cases the amount of murine class II Ag expressed on the surface of the hybrid cell was significantly higher than the one observed in the parental macrophage cells after induction with IFN-gamma and was not further modified by treatment with the murine lymphokine. Reversion from a murine class II-positive to class II-negative cell surface phenotype in the hybrids correlated with reduced expression of human markers and more important with segregation of human chromosomes. Interestingly, in this case certain hybrids still expressed detectable levels of murine class II mRNA and increased levels of murine invariant chain mRNA when compared with parental P388 D1 murine macrophage cells. These results indicate that constitutive class II gene expression behaves as a dominant trait in B cell x macrophage somatic cell hybrids. Possible mechanisms responsible of the different control of class II gene expression during cell type differentiation are discussed.

Cytogenetic analysis of 54 cases of myelodysplastic syndrome.

Fifty-four patients with myelodysplastic syndrome (MDS) (35 men and 19 women aged 34-92 years) were studied cytogenetically. Bone marrow cell culture and chromosome preparation were performed according to four different protocols used in parallel: methotrexate (MTX)-synchronized or thymidine (TdR)-unsynchronized techniques, and presence or absence of 5637 conditioned medium (CM). Some patients responded better to MTX; others had better results with TdR exposure only. Use of 5637 CM generally improved quantity and quality of metaphases. A cytogenetic result was obtained in 53 cases. 60% of the patients had a chromosome abnormality. Percentage of abnormality varied from one French-American-British (FAB) subtype to the other: 62% in refractory anemia with ringed sideroblasts (RARS, 8/13), 50% in refractory anemia (RA, 6/12), 60% in refractory anemia with excess of blasts (RAEB, 3/5), 77% in refractory anemia with excess of blasts in transformation (RAEB-T, 7/9), and 57% in chronic myelomonocytic leukemia (CMMoL, 8/14). Chromosome defects were subdivided into three categories: single, two, and complex defects. The most frequent chromosome abnormalities, either single or one of two or complex defects were del(5q) or monosomy 5 (13 cases), trisomy or rearrangement of chromosome 8 (eight cases), total or partial monosomy or rearrangement of chromosome 7 (eight cases), Y loss (seven cases), and del(20q) (two cases). With the exception of del(5q) in macrocytic RA, this study confirms the absence of chromosome defects specific to each FAB category of MDS. Recurrent defects in MDS are relatively limited, however, in terms of chromosomes involved and type of abnormality. Consequently, these defects, mostly of deleted type, are assumed to play a specific role in the genesis of myelodysplasia.

A new case of myelodysplastic syndrome with 6p rearrangement.

Bone marrow cells from a patient with refractory anemia with ringed sideroblasts were studied cytogenetically. All metaphases analyzed revealed an abnormal karyotype with complex defects. The most prominent defect consisted of a rearrangement of the short arm of chromosome 6. Until now, 6p rearrangements have been preferentially observed in myelodysplastic patients with a history of previous exposure to toxic products such as alkylating agents or environmental factors of occupational origin. Although our patient was not exposed to alkylating agents, for about 20 years he has regularly consumed important quantities of analgesics, tranquilizers, and nonsteroidal antiinflammatory drugs. The eventual relationship between sideroblastic anemia and drug abuse, as well as the existence of chromosome sites preferentially rearranged in the bone marrow cells of myelodysplastic patients, are discussed.

Short-term culture of spleen lymphocytes for chromosome analysis of small rodents.

In order to improve the methods for the cytogenetic analysis of small rodents with regard to metaphase number, quality of chromosome resolution and duration of cultivation, a short-term culture technique from spleen cells, based on the capacity of Concanavalin A supernatant to stimulate the proliferation of normal T lymphocytes, was developed. Protocols of culture and chromosome preparation are described in detail. This technique not only makes it possible to obtain a high number of cells in mitosis from the spleen of very small rodents in a short time, but also insures metaphases of good quality, due to optimal control of cell cycle and processing thanks to cultivation in small aliquots. Although the method applies mainly to rodents because of species specificity of interleukin 2, it can probably be easily adapted to species from other mammalian orders.

Loss of interleukin 2 dependence in cloned interleukin 2-dependent rat T lymphocyte x BW5147 hybridomas is not associated with segregation of a specific pair of rat chromosomes.

A fusion between the mouse AKR thymoma BW5147 and a culture of homogeneously OX8+ (CD8) rat T lymphoblasts yield interleukin (IL) 2-dependent T cell hybridomas when selected in HAT medium supplemented with IL 2-containing supernatants of concanvalin A-activated cells and dexamethasone. IL 2-independent variants can be selected from cloned IL2-dependent hybrids in the absence of conditioned medium. Karyotype analysis was used to test a previously proposed hypothesis according to which IL2-independent variants arise through loss of a specific cytotoxic T lymphocyte (rat) chromosome carrying a gene responsible for IL2 dependence. Comparison of karyotypes of several independently derived hybrids with those of their IL 2-independent variants showed that the hybrids contain at least one homologue of all rat chromosomes, and that no pair of rat chromosomes is consistently absent in the IL 2-independent variants.

Constitutional karyotype in retinoblastoma. Case report and review of literature.

High resolution karyotype was performed in 13 retinoblastoma patients. A mosaic pattern for del(13)(q14.1;q14.3) was found in a girl with sporadic bilateral retinoblastoma and midface dysmorphism. In addition, 162 cases of 13q aberrations were reviewed, including 140 retinoblastoma patients and 22 non-penetrance 13q14 deletions. Some epidemiological and genetic involvements are discussed.

[Chromosomes of the Cuming rat, Phloeomys cumingi Waterhouse, 1839 (Mammalia: Rodentia)]. / Les chromosomes du rat de Cuming, Phloeomys cumingi Waterhouse, 1839 (Mammalia: Rodentia).

The chromosome formula of Phloeomys cumingi is described for the first time. The diploid number is 40 in both sexes, the fundamental number is 60. The 38 autosomes are divided into 5 pairs of submetacentrics, 4 pairs of metacentrics, 6 pairs of large acrocentrics and 4 pairs of small acrocentrics. The sex chromosomes are large and rich in C-positive heterochromatin. The comparative analysis of the chromosomes of Phloeomys with those of Mus musculus and Rattus norvegicus puts forward numerous analogies between these 3 species. According to the comparison of the chromosomes of Phloeomys with the ancestral karyotype of the Cricetidae, eleven chromosome pairs at least have conserved their original G-banding pattern. Although chromosome data reveal a close relationship between Phloeomys and Muridae, they do not exclude the possible belonging of this species to a separate family, that of Phloeomyidae.