Cytogenetics in pre-B and B-cell acute lymphoblastic leukemia: a study of 208 patients diagnosed between 1981 and 2008.

The detection of chromosome abnormalities by conventional cytogenetics, now combined with analyses using fluorescence in situ hybridization (FISH), is an important component in assessing the risk stratification of acute lymphoblastic leukemia (ALL). Identification of specific chromosome abnormalities led to the recognition of genetic subgroups based on modal chromosomal number, reciprocal translocations in B-cell ALL, or both. We report here the cytogenetic analysis of 208 patients with pre-B and B-cell ALL referred to a single laboratory between 1981 and 2008. Chromosome abnormalities were observed in 82.9% of L1/L2 ALL patients and in 83.3% of L3 patients with successful analysis at diagnosis. The proportion of diploid karyotypes tended to decrease during the period of study, from 26% to 13%, in association with technical progress and the introduction of FISH techniques. As previously reported, the incidence of high hyperdiploidy (51-67 chromosomes) was higher among children, whereas pseudodiploidy and hypodiploidy were higher among those >15 years of age. Structural chromosome abnormalities were more frequently observed among patients older than 15 years than in children (75.9% vs. 68.5%, respectively). As previously reported, t(9;22)(q34;q11) and t(12;21)(p13;q21) were the most frequent structural rearrangements among adults (26.9%) and children (19.7%), respectively. Almost 17% of the patients studied at diagnosis had further cytogenetic analyses at relapse, the majority showing clonal evolution toward a more complex karyotype. Although the detection of chromosome abnormalities by conventional cytogenetics and FISH techniques is an important tool in assessing risk stratification of ALL, some patients lack abnormalities with clinical relevance. The use of array comparative genomic hybridization (aCGH) offers an alternative for identifying copy number alterations, but cannot detect balanced chromosomal rearrangements.

Philadelphia chromosome-positive acute lymphoblastic leukemia: a cytogenetic study of 33 patients diagnosed between 1981 and 2008.

BACKGROUND: The Philadelphia (Ph) chromosome, resulting from a t(9;22)(q34;q11), is one of the most frequent chromosomal abnormalities observed among patients with acute lymphoblastic leukemia (ALL). Main of study: To analyze the distribution of Ph chromosome-positive ALL patients. PATIENTS AND METHODS: Conventional cytogenetic analysis was performed on bone marrow cells at the time of diagnosis and/or relapse of 208 patients shown to have B-cell ALL. Fluorescent in situ hybridization studies using probe LSI BCR-ABL dual ES color (Abbott, Rungis, France) and other types of probes were performed on the available cytogenetic pellets. RESULTS: Thirty-three Ph chromosome-positive ALL patients were identified between 1981 and 2008. The Ph chromosome was present in 39.7% of the patients older than 25 years, but in only 4.3% of the patients younger than 15 years. A pseudodiploid karyotype was found in 68.75% of the patients and hypodiploidy in a further 15.6% of the patients. FISH studies revealed the breakpoint to be located in the major breakpoint cluster region in 20% of the patients and in the minor in the remaining 80% of the patients. Complex rearrangements of the derivative chromosomes 9 or 22 were identified in four patients, including a complex Ph translocation, t(9;22;X). CONCLUSION: Ph chromosome-positive ALL patients show cytogenetic characteristics that differ from those of other ALL patients.

Chromosome 20 deletions in myelodysplastic syndromes and Philadelphia-chromosome-negative myeloproliferative disorders: characterization by molecular cytogenetics of commonly deleted and retained regions.

Deletion of the long arm of chromosome 20 is a recurrent abnormality observed in myelodysplastic syndromes (MDS) and in Philadelphia-chromosome-negative myeloproliferative disorders (MPD). Our objective was to characterize the deletion size among 38 MDS and MPD patients using fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) probes and to define commonly deleted and retained regions on chromosome 20. Patients were distributed in three groups according to the World Health Organization classification: MDS (22 patients), MPD (12 patients) and myelodysplastic/myeloproliferative diseases (four patients). FISH with centromeric, subtelomeric, and unique sequence probes was performed to characterize the deletion whereas its size was delineated using BAC clones. All 38 deletions were found to be interstitial. A commonly deleted region was identified for each of the three groups; it varied from 6.62 to 10.4 Mb and showed considerable overlapping. Two commonly retained regions (CRR), also showing overlapping, were identified in all three groups, one in the centromeric region, the other in the telomeric region. The deletion size is highly variable, with no apparent recurrent breakpoint. The deletion may result in the loss of one or several tumor suppressor genes but the target genes remain unknown. Loss of genes plays an important part in the myeloid leukemic process associated with del(20q). However, genes located in the retained chromosomal regions may also play a role in the oncogenetic mechanisms.

Inhibitory effect of the substance P and its derivative on erythropoietin-independent growth of erythroid progenitors in polycythemia vera.

Regulation of normal hematopoiesis by neuropeptide substance P (SP) and its amino terminal fragment, SP(1-4), has been reported. Endogenous erythroid colony (EEC) formation without erythropoietin is characteristic of polycythemia vera (PV), a chronic myeloproliferative disorder. We investigated the effect(s) of SP and SP(1-4) on EEC formation from PV BM mononuclear cells (BMMCs) and purified CD36+ erythroid progenitors. We found a potent in vitro inhibitory effect of SP and SP(1-4) on PV EEC formation for both BMMCs and CD36+ erythroid progenitors. The influence of SP and SP(1-4) on PV progenitor erythroid differentiation and cell viability was also investigated, and the impact of neurokinin receptors and G proteins in the inhibition were analyzed by quantitative PCR and with specific inhibitors. This progenitor inhibition was: (1) not mediated by accessory cells; (2) characterized by an increase in cell death and inhibition of the EPOindependent terminal erythroid differentiation; and (3) not mediated by the same neurokinin receptor. NK-1R and NK-2R antagonists completely abrogated the SP inhibitory effect but not SP(1-4)-induced inhibition. Furthermore, the truncated form of the NK-1R was predominant over the full-length mRNA and could mediated the SP inhibitory effect on PV CD36+ erythroid progenitors. Different G proteins were also implicated according to the erythroid differentiation stage of the PV cells. The observation of an inhibitory effect of SP and its related peptide, SP(1-4), on PV EEC formation at physiological concentrations (10-8M) suggests that neuropeptides represent a way to downregulate pathologic expansion of PV progenitors.

Evaluation of chromosome 5 aberrations in complex karyotypes of patients with myeloid disorders reveals their contribution to dicentric and tricentric chromosomes, resulting in the loss of critical 5q regions.

Dicentric chromosomes have often been observed in complex karyotypes in previously reported studies of therapy-related myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Fluorescence in situ hybridization (FISH) has now made the characterization of these rearrangements much easier. Dicentric and tricentric chromosomes were identified in 21 patients (9 MDS and 12 AML) among the 133 consecutive MDS/AML patients (17%) who had a structural or numerical aberration of chromosome 5 using conventional cytogenetic analysis. One third (7/21) of the patients had received alkylating drugs for a previously diagnosed cancer or chronic myeloproliferative disease. Loss of 5q material was identified in all 21 patients. One copy of the EGR1 (5q31) or the CSF1R (5q33 approximately q34) genes was lost in 20 of the 21 patients. Dicentric and tricentric chromosomes involving chromosome 5 are frequently observed in complex karyotypes among patients with de novo or therapy-related MDS/AML. They lead to deletions of various parts of the long arm of chromosome 5.

Deletion size characterization of der(9) deletions in Philadelphia-positive chronic myeloid leukemia.

About 95% of the CML patients with chronic myeloid leukemia (CML) have a Philadelphia chromosome resulting from a reciprocal translocation between bands 9q34 and 22q11.2 that juxtaposes the 3' region of the ABL gene to the 5' region of BCR. Over the past few years, submicroscopic deletions due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint have been found using fluorescence in situ hybridization (FISH). Among 150 CML bone marrow samples analyzed by molecular cytogenetics in our laboratory, 11 had a der(9) deletion detectable by FISH (deletion of the 5'ABL region and 3'BCR region in 10 samples and deletion of the 5'ABL region solely in 1 sample). To delineate the size of the deletions, FISH mapping was performed using 22 bacterial artificial chromosomes (BACs), 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. The deletion size of the 5'ABL region on the der(9) extended from 2 to 5 Mb, the minimal deletion size being localized between BACs RP11-101E3 and RP11-83J21. In two patients, the deletion size of the 3'BCR region was about 500 kb (between RP11-80O7 and RP11-681C06). The poor prognosis associated with these deletions was postulated by several workers to be explained by haploinsufficiency of a tumor suppressor gene. However, in our cases, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment. We think that there could be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions.

Chromosome 1 abnormalities in multiple myeloma.

Multiple myeloma (MM) is a malignancy of the terminally-differentiated B cells and accounts for 10% of all hematological malignancies. Chromosome 1 aberrations are frequently described, the short arm being preferentially involved in deletions and the long arm in gains. The abnormalities were identified in the bone marrow of 37 MM patients by conventional cytogenetics. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the abnormalities and to better characterize them. Chromosome 1 abnormalities were grouped into 4 categories: balanced translocations, deletions, amplifications and jumping translocations (JT). Breakpoints involved in balanced translocations were randomly distributed. The smallest region of overlap for deletions was 1p11 --> 1p21 (present in 27% of the patients) and for gains 1q31 --> 1qter (present in 54% of the patients). The whole long arm was found to be the donor segment for the majority of patients with JT, the most frequent recipients being chromosomes 16 and 19. Our results share some similarities with those obtained for 143 published patients studied by FISH. Band 1p21 was found to be frequently deleted, leading to the assumption that a 1p deletion could lead to hemizygosity of at least 1 tumor suppressor gene. Two regions of 1q showed preferential gains: q12 to q22 and q31 to q42; these amplifications could induce the overexpression of 1 or more oncogenes. In conclusion, our results confirm that chromosome 1 abnormalities play an important role in the pathogenesis of multiple myeloma.

Screening by fluorescence in situ hybridization for MLL status at diagnosis in 239 unselected patients with acute myeloblastic leukemia.

A large number of abnormalities involving the MLL gene have been associated with hematological malignancies, including acute myeloblastic leukemias (AML). Given the overall unfavorable prognosis of AML with an MLL abnormality, its reliable and accurate detection is needed for informed treatment decision. We therefore investigated the occurrence of MLL abnormalities in 239 unselected consecutive AML patients, using conventional cytogenetic and fluorescent in situ hybridization (FISH) analyses. FISH analysis for MLL was performed using a commercial dual-color probe. Of the 239 patients, 30 (12.6%) showed MLL abnormalities under FISH analysis, 10 (4.2%) showed a split signal indicating the disruption of the MLL gene by translocation or insertion, and 20 (8.4%) showed overrepresentation of the MLL gene without evidence of rearrangement. MLL abnormalities were more frequently found in AML-M5 and M4, mainly as rearrangements, and in AML-M2, mainly as overrepresentation. Our results are in agreement with those reported in other studies. All M2, M4, and M5 AML patients without known recurrent translocations, such as t(8;21) and inv(16), should be investigated by FISH with an MLL probe because it allows the detection of MLL rearrangement that would go undetected by conventional cytogenetics and because it has the ability of detecting multiple copies of the MLL gene in, for example, marker chromosomes and double minutes.

Jumping translocations in multiple myeloma.

Jumping translocations (JT) have been defined as nonreciprocal translocations involving a same donor chromosome arm or chromosome segment onto two or more recipient chromosomes in different cell lines in the same patient, leading to a mosaic karyotype. This definition has been expanded to also include extra copies of a same donor segment on different recipient chromosomes in a single clone. Six patients with multiple myeloma and JT involving chromosome arm 1q were identified among 37 patients presenting with chromosome 1 abnormalities. All six patients had an advanced disease and a short survival. The literature review allowed us to identify 24 additional patients with JT. Chromosomes 16 and 19 were the recipients in 11 (45.8%) and 6 (25%) of these 24 patients, respectively. Breakpoints on the recipient chromosomes were pericentromeric in 46.2% and telomeric in 40.4% of the breakpoints recorded. Since telomeres are made of (TTAGGG)n tandem DNA repeats that are also found in the pericentromeric heterochromatic regions (interstital telomeric sequences), it is presumed that jumping translocations arise through illegimate recombination between telomere repeat sequences and interstitial telomeric sequences.

Characterization of IGH rearrangements in non-Hodgkin's B-cell lymphomas by fluorescence in situ hybridization.

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin's B-cell lymphomas (NHL) and correlated to clinical relevant subgroups. However, the detection rate varied greatly with the technique used. The incidence of IGH rearrangements was analyzed using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 57 patients with nodal NHL. An IGH rearrangement was identified in 42 cases (73.7%). A t(14;18)(q32;q21) was found in 17 of the 20 follicular lymphomas (85%) studied and a t(11;14)(q13;q32) in 10 of the 11 mantle cell lymphomas (91%). IGH rearrangements were identified in 12 of the 26 diffuse large B-cell lymphomas (46%), including 5 t(14;18)(q32;q21) and 2 t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using Multi-FISH and/or chromosomal whole paint enabled the characterization of complex IGH translocations in follicular lymphomas and mantle cell lymphomas and the identification of all the chromosomal partners involved in the IGH rearrangement in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe could be the first approach in NHL, after which chromosome painting and M-FISH could be used to identify the chromosomal partner involved in the IGH rearrangement.

Rearrangement of the MLL gene in acute myeloblastic leukemia: report of two rare translocations.

Band 11q23 is known to be involved in translocations and insertions with a variety of partner chromosomes. They lead to MLL rearrangement, resulting in a fusion with numerous genes. We report here 2 male adults in whom a diagnosis of acute myelomonoblastic leukemia (FAB M4) and acute monoblastic leukemia (FAB M5) was made. Conventional cytogenetic techniques showed a 45,XY,t(1;11)(p32;q23),-7 karyotype in the first case and a 46,XY, t(11;17)(q23;q21) in the second case. Fluorescent in situ hybridization (FISH) with a specific MLL probe showed the gene to be disrupted, the 3' region being translocated on the derivative chromosomes 1 and 17, respectively. Fourteen and 24 patients, including ours, with acute myeloblastic leukemia associated with a t(1;11)(p32;q23) and a t(11;17)(q23;q21), respectively have been reported in the literature. Several patients with the latter translocation have also been identified to have acute lymphoblastic leukemia (ALL). Although both translocations are preferentially associated with monocytic differentiation, the t(11;17)(q23;q21) is more common in adults and has been reported in many patients with ALL, compared to the t(1;11)(p32;q23).

Interphase FISH for follow-up of Philadelphia chromosome-positive chronic myeloid leukemia treatment.

BACKGROUND: Several attempts have been made to determine whether interphase fluorescence in situ hybridization (I-FISH) on bone marrow or peripheral blood specimens is a good alternative to conventional cytogenetics (CC) in calculating the residual proportion of Philadelphia (Ph) chromosome-positive cells during treatment follow-up of patients with chronic myeloid leukemia. MATERIALS AND METHODS: Nineteen patients were selected for I-FISH follow-up compared to CC. All samples were also classified into 4 groups according to the percentage of residual Ph chromosome-positive metaphases analyzed in CC. I-FISH was performed using the LSI bcr/abl dual ES color probe (Vysis). RESULTS: A high correlation was observed between the frequency of Ph chromosome-positive cells, assessed by CC and I-FISH (p<0.001). A high correlation was found between CC and I-FISH for 12 patients, but not for the remaining 7. Applying the same classification for I-FISH did not show a good relationship between the two techniques (p<0.001). CONCLUSION: Dual color I-FISH is a reliable method to monitor the size of the Ph chromosome-positive clone in bone marrow of treated CML patients. However, it has to be complementary to conventional cytogenetics because it cannot detect the emergence of other chromosomal abnormalities in Ph chromosome-positive or -negative cells.

Deletion of the 5'ABL region in Philadelphia chromosome positive chronic myeloid leukemia: frequency, origin and prognosis.

The use of new nuclei probes in fluorescent in situ hybridization (FISH) at diagnosis and during follow up has recently allowed the detection of a deletion of the 5'abl region on the derivative chromosome 9 among some CML patients. This deletion seems to be a powerful and independent prognostic factor. The aim of our study was not only to estimate the frequency of the deletion of the 5'abl region among chronic myeloid leukemia (CML) patients with bcr-abl fusion gene, but also, to assess whether this deletion is concomitant with the formation of the Philadelphia (Ph) chromosome or represents a sign for progression of the disease, and finally to evaluate the prognostic implications of this abnormality. One hundred and twelve patients were analysed using FISH with LSI bcr-abl dual ES color probes, at the moment of the diagnosis when possible or, if not, on a sample with a strong rate of Ph+ metaphases evaluated by conventional cytogenetics. When the deletion was highlighted in a patient, we performed an hybridization on all the samples available during the follow-up. The deletion of the 5' region of the gene abl was detected among 9 patients. When the deletion was found in a patient, it was present in all the Ph+ metaphases and nuclei and in all the samples studied at diagnosis and during follow up. In these patients, we never identified cells carrying the Ph chromosome translocation without the deletion. None of the patients with the deletion had a major cytogenetic response to treatment with interferon. The deletion of the 5'abl region on der(9), present in approximately 9% of the CML, takes place at the same time as the formation of the Ph chromosome translocation and seems of worse prognosis. The detection of this deletion could thus constitute an argument to start STI treatment in first intent for these patients.

Trisomy 15 as the sole abnormality in myelodysplastic syndromes: case report and review of the literature.

Trisomy 15 as the sole autosomal anomaly is uncommon in hematological malignancies but could be preferentially associated with myelodysplasia. We report a 61-year-old man who developed pancytopenia following two courses of chemotherapy for chronic lymphoid leukemia. Cytogenetic studies at diagnosis of pancytopenia with R banding showed a 47,XY, + 15[3]/45,X[3]/46,XY[14] karyotype. A review of the 53 cases of myelodysplastic syndromes (MDS) and myeloid related disorders associated with trisomy 15 reported in the literature showed that 18 of the 31 men also lost the Y chromosome in the trisomic 15 cell line. Their mean age was significantly higher than that of males who had not lost the Y chromosome (p < 0.05). The main feature of the patient reported here is the presence of two abnormal cell lines, one having lost the Y chromosome, the other having gained a chromosome 15. Therefore, the two events occurred independently, the loss of the Y chromosome being possibly due to aging and the trisomy 15 to the hematologic disorder.

Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights.

Aceruloplasminemia is an autosomal recessive disease of iron overload associated with mutation(s) in the ceruloplasmin gene. We report here a new case of aceruloplasminemia in a woman who is a compound heterozygote for two new mutations. Besides this novel genotypic profile, this observation provides new insights on: (i) iron metabolism with normal erythroid repartition, in the absence of serum non-transferrin-bound iron and with an increase of 59Fe plasma clearance; (ii) hepatic abnormalities associated with the presence of iron-free foci; (iii) the therapeutic management of the disease, chronic subcutaneous infusion of deferrioxamine being remarkably effective at reducing hepatic iron overload.