Comparison between interphase and metaphase cytogenetics in detecting chromosome 7 defects in hematological neoplasias

Monosomy 7 (–7) is one of the most common chromosomal abnormalities found in the leukemic cells of patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Because patients with –7 have a poor prognosis, their identification is important for treatment planning. Conventionally, –7 is detected by the G-banding technique. This study examines the use of fluorescent in situ hybridization (FISH) methodology to detect –7 cells in interphase nuclei and metaphase chromosomes. Fifteen AML or MDS patients whose leukemic cells were found to have –7 by G-banding at disease presentation were studied. In 13 of these patients, –7 could be detected in interphase by FISH using a chromosome 7-specific centromeric DNA probe. The two patients whose leukemic cells were not detectable by interphase FISH had –7 and t(1q;7p), which were detectable by FISH in metaphase using a chromosome 7-specific painting probe. Metaphase FISH was particularly useful in further defining chromosome 7 defects in cells that contained aberrant or marker chromosomes. For example, in 6 patients, chromosome 7 sequences were detectable in aberrant or marker chromosomes by metaphase FISH, but not by G-banding. These results suggest that metaphase FISH is an important adjunct to conventional cytogenetic methods for defining chromosome 7 abnormalities in AML and MDS patients. Furthermore, interphase FISH is useful for follow-up studies in patients who are found informative for the FISH study at presentation.     

Detection of monosomy 7 and trisomy 8 in myeloid neoplasia: a comparison of banding and fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful tool for detection of numerical and structural chromosomal aberrations. We have compared conventional banding techniques and FISH for the detection of monosomy 7 (-7) and trisomy 8 (+8) in 89 patients with myeloid malignancies. Of these patients, 21 had -7, 30 had +8, four had both, and 34 had no aberrations or aberrations other than -7 or +8 as assessed by banding techniques. Sequential samples were available in 23 patients. Alphoid DNA probes specific for chromosomes no. 7 and 8 were used for FISH. As controls, 10 normal bone marrow (BM) samples were hybridized with the chromosomes no. 7 and 8 probes, and in addition all tumor samples were hybridized with a chromosome no. 1 specific probe. The cut-off value for -7 was 18% one-spot cells, and for +8 was 3% three-spot cells. FISH analysis of 44 samples with -7 or +8, and at least 10 metaphases evaluated, showed that the proportions of aberrant metaphase cells mirrored the interphase clone sizes. Most samples with nonclonal metaphase aberrations, including those with only a few metaphases, had increased numbers of aberrant interphase cells: 20% to 80% for -7, and 3% to 43% for +8. Interphase cytogenetics of the 34 samples without -7 or +8 did not show significant cell populations with -7 or +8. In four patients, -7 or +8 could not be confirmed by FISH due to additional structural aberrations, marker chromosomes, or wrongly interpreted banding results. As FISH will be used more and more in cytogenetic diagnosis, clinical follow-up, and therapy monitoring, it will be necessary to standardize FISH procedures and supplement the Standing Committee on Human Cytogenetic Nomenclature (ISCN) definitions of a clone with criteria specifically for in situ hybridization.     

Cytogenetic analysis in 941 consecutive patients with haematologic disorders

Clinical and cytogenetic findings were reevaluated in 941 consecutive patients with suspected neoplastic haematological conditions studied during 1973-1984. A total of 1652 attempts at cytogenetic analysis with banding technique were performed in 240 patients with acute nonlymphocytic leukaemia (ANLL), 177 with chronic myeloid leukaemia (CML), 157 with myelodysplasia (MDS), 82 with myeloproliferative disorders (MPD), 114 with acute lymphoblastic leukaemia (ALL), 42 with non-Hodgkin lymphoma or other lymphoproliferative disorders (NHL + LPD), and 120 patients with benign disorders. Only 1 patient with a benign disorder had an acquired clonal chromosomal abnormality (diagnostic specificity 0.99), whereas abnormalities were detected in 50.0% of patients with malignant haematologic disorders (diagnostic sensitivity 0.50). Success rate was 73-74.4% in ALL, MPD, and NHL + LPD, versus 87-94% in ANLL, MDS, CML, and benign disorders. The frequencies of detected abnormalities in diagnostic subgroups were within the limits of previous reports. Striking differences in cytogenetic pattern in relation to age were found in MDS and ANLL. Results from 1973-80 were compared to 1981-84. In spite of a marked reduction in failure rate of bone marrow (BM) analyses in the second time period, the fraction of patients with only inadequate cytogenetic analyses and the frequencies of detected chromosome abnormalities remained essentially unchanged. Peripheral blood samples had a high failure rate, and seldom provided additional information to BM analyses. Delay in transportation time of samples did not in general affect the outcome of cytogenetic analysis, with possible exceptions for a higher failure rate in ALL and lower frequency of detected abnormalities in ANLL.     

Relationship of patient survival and chromosome anomalies detected in metaphase and/or interphase cells at diagnosis of myeloma

The clinical efficacy of evaluating genetic anomalies in metaphase cells versus interphase nuclei for multiple myeloma (MM) is poorly understood. Therefore, survival for 154 patients with newly diagnosed untreated MM was compared with results from analysis of metaphase and interphase cells. Metaphases were studied by conventional cytogenetics and fluorescent-labeled DNA probes (fluorescence in situ hybridization [FISH]), whereas inter-phase nuclei were evaluated only by FISH. All FISH studies were done using DNA probes to detect t(4;14)(p16;q32), t(11;14)(q13;q32), t(14;16)(q32;q23), del(17) (p13.1), and chromosome 13 anomalies. Metaphases were abnormal by cytogenetics and/or metaphase FISH in 61 (40%) patients. Abnormal interphase nuclei were observed in 133 (86%) patients, including each patient with abnormal metaphases. FISH was a necessary adjunct to cytogenetics to detect t(4;14) and t(14;16) in metaphase cells. Patient survival was especially poor for patients with greater than 50% abnormal interphase nuclei, although this result was more likely due to level of plasma cells than specific chromosome anomalies. For metaphase data, patients with t(4;14), t(14;16), del(17) (p13.1), and/or chromosome 13 anomalies (primarily monosomy 13) had poor survival. A different outcome was observed for interphase data as patients with t(4;14) or t(14;16) had poor survival, whereas patients with chromosome 13 anomalies had intermediate survival: interphase FISH did not substitute for metaphase analysis.     

Trisomy 3 in marginal zone B-cell lymphoma: a study based on cytogenetic analysis and fluorescence in situ hybridization

Trisomy 3 represents the most frequent and consistent chromosomal abnormality characterizing the recently defined entity marginal zone B-cell lymphoma (MZBCL). By cytogenetic analysis and/or fluorescence in situ hybridization (FISH) on interphase nuclei we found an increased copy number of chromosome 3 in 22/36 (61%) successfully analysed cases, including 8/12 cases with extranodal MZBCL, 8/13 cases with nodal MZBCL, and 6/11 patients with splenic MZBCL. Sensitivity of interphase cytogenetics was somewhat higher than that of conventional cytogenetic investigation. Structural chromosomal changes involving at least one chromosome 3 were seen in 11/20 cases with an increased copy number of chromosome 3: +del(3)(p13) was demonstrated in three cases, and was the sole chromosomal abnormality in one of them; +i(3)(q10) was seen in two other patients; and rearrangements involving various breakpoints on the long arm of chromosome 3 were found in the remaining cases. FISH on metaphase spreads confirmed these structural abnormalities and additionally showed two unexpected translocations involving chromosome 3. We conclude that: (1) trisomy 3 occurs in a high proportion of extranodal, nodal and splenic MZBCL; (2) FISH on interphase nuclei is an additional and sensitive tool in detecting an increased copy number of chromosome 3 in MZBCL; (3) additional structural abnormalities involving the long arm of chromosome 3 are frequent but non-recurrent and are perhaps secondary changes; and (4) abnormalities such as +del(3)(p13) and +i(3)(q10) suggest that genes located on the long arm of chromosome 3 are of particular importance in the pathogenesis of MZBCL.     

Interphase cytogenetic analysis of in vivo differentiation in the myelodysplasia of Down syndrome

In Down syndrome, acute megakaryoblastic leukemia (AMKL) occurs frequently during the first 4 years of life and is usually preceded by a period of myelodysplasia (MDS), often associated with chromosomal abnormalities. Archival peripheral blood and/or bone marrow films of six patients with Down syndrome and MDS whose leukemic cells contained monosomy 7 or trisomy 8 were studied to determine whether the abnormal precursors produce mature cells in vivo. Using fluorescence in situ hybridization (FISH) of interphase nuclei with chromosome-specific centromere probes for either chromosome 7 or 8, we were able to determine which cells had one, two, or three signals indicative of one, two, or three no. 7 or 8 chromosomes. In five patients with trisomy 8, 80% to 100% (94.5% +/- 6.2%) of the megakaryoblasts had three signals using a chromosome 8 probe; in one patient with monosomy 7, 96.5% of the megakaryoblasts had one signal using a chromosome 7 probe. In all six patients, the myeloid and lymphoid series did not have evidence of the chromosomal abnormality present in the blasts. In three of five patients with trisomy 8, three signals were observed in 27%, 33%, and 41% of normoblasts, respectively. These data are evidence that the abnormal cell in MDS is a progenitor cell with the potential of forming cells of megakaryocyte and erythroid lineages.     

rHuEpo administration in patients with low-risk myelodysplastic syndromes: evaluation of erythroid precursors' response by fluorescence in situ hybridization on May-Grunwald-Giemsa-stained bone marrow samples

The issue of whether, in patients affected by myelodysplastic syndromes (MDS), haematological response to cytokines, particularly to recombinant human erythropoietin (rHuEpo), is a phenomenon related to the stimulation of normal haemopoietic cells or to the differentiation of cells belonging to the abnormal clone remains an open question. To assess the pattern of response to rHuEpo treatment of bone marrow (BM) cells, we evaluated in 13 low-risk MDS patients with known cytogenetic abnormalities the number of cytogenetically normal and abnormal cells by conventional cytogenetic analysis (CCA) and by a fluorescence in situ hybridization (FISH) technique, enabling the simultaneous visualization of FISH chromosomal abnormalities in morphologically and immunophenotypically identifiable BM elements. Patients responding to rHuEpo presented a lower number of abnormal metaphases at diagnosis in comparison with patients who did not respond (22.74% vs 76.23%, P = < 0.001). This was confirmed by the combined morphological FISH analysis, showing that, before treatment, BM samples from patients responding to rHuEpo had a lower proportion of both FISH abnormal erythroid (36.48% vs 66.93%, P = 0.002) and myeloid (40.76% vs 67.70%, P = 0.014) elements than unresponsive patients. After rHuEpo treatment, responding patients presented a significantly lower proportion of FISH abnormal erythroid precursors than observed before treatment (16.93%vs 36.48%, P = 0.017). Likewise, in responding patients, a significantly lower proportion of FISH abnormal erythroid elements (16.93% vs 66.30%, P < 0.001) was detected in comparison with unresponsive patients. These findings provide evidence that, in low-risk MDS patients with known cytogenetic abnormalities, response to rHuEpo may be due to the proliferation of karyotypically normal erythroid precursors, possibly representing residual normal erythroid elements.     

Clonal CD5-positive B lymphocytes in myelodysplastic syndrome with systemic vasculitis and trisomy 8

 Bone marrow and peripheral blood from a myelodysplastic syndrome (MDS) patient with trisomy 8 and associated systemic vasculitis was investigated for clonal lymphoid lineage involvement using simultaneous metaphase and interphase fluorescence in situ hybridization (FISH) and immunocytochemistry with antibodies against CD13 (granulocytic), glycophorin A (GPA, erythroid), and the lymphocytic antigens CD3, CD5, CD20, and CD22. Trisomy 8 was detected in 55% of CD13+, 40% of GPA+, 6% of CD5+, and 5% of CD20/22+, but not in CD3+ cells. In a complementary experiment using interphase FISH on bone marrow cells sorted by flow cytometry, 13% of CD5/CD19 double-positive cells (76% purity) were found to be trisomic. The results indicate the existence of a small CD5-positive B-lymphoid clone as part of the MDS process in this patient. Since CD5/19-positive cells have been proposed to be autoantibody producing, this finding might be a clue to the pathogenesis underlying the propensity for MDS patients to develop immune-mediated complications. Received: 2 September 1996 / Accepted: 17 October 1996     

Interphase cytogenetic analysis detects minimal residual disease in a case of acute lymphoblastic leukemia and resolves the question of origin of relapse after allogeneic bone marrow transplantation

We used in situ hybridization with a probe for the X chromosome to study interphase cells of bone marrow and peripheral blood specimens from a male patient with acute lymphoblastic leukemia characterized by hyperdiploidy, including trisomy X. In a posttreatment bone marrow specimen, which was interpreted as a regenerating bone marrow morphologically and which demonstrated a normal karyotype cytogenetically, trisomy X was found in 16 of 1,000 interphase cells. This finding indicated the presence of leukemic cells that were undetected by conventional morphologic and cytogenetic techniques (ie, minimal residual disease). Cytogenetic studies of a relapse specimen obtained after a sex-mismatched bone marrow transplant showed only a normal female karyotype in each of 40 metaphase cells, suggesting that the relapse occurred in donor cells. However, interphase analysis demonstrated trisomy X in more than 80% of interphase cells and indicated that the relapse was of the original clone and was not a transformation of donor cells. This case illustrates that interphase analysis can be useful as an adjunct to conventional cytogenetic analysis in the detection of minimal residual disease and in the analysis of interphase cells that are not accessible to routine cytogenetic methods. It also illustrates that previously reported instances of relapse of leukemia in donor cells could have been incorrect if supported by cytogenetic data alone.     

Association between trisomy 8 and the immunophenotype of blast cells from acute leukemias secondary to a myelodysplastic syndrome or chronic myeloproliferative disorders

 In the present study we have used FISH to analyze the incidence of trisomy 8 in acute leukemias following either a primary myeloproliferative disorder (MPD) or a myelodysplastic syndrome (MDS) and correlated it with both the immunophenotype and the cell-cycle distribution of the leukemic blast cells. Six of the 21 (28%) acute leukemias studied displayed trisomy 8 by FISH. The number of trisomic cells in these cases ranged from 20 to 84%, with a mean of 46±24%. Trisomy 8 was associated with a homogeneous population of leukemic cells, phenotypically characterized by CD34+ / HLADR+ / CD13+ / CD33+ / CD11b– / CD15– / CD14–. No significant differences were observed on the proliferative rate of cases with trisomy 8, as compared with blast cells from the remaining patients. Overall, our findings suggest that in acute leukemias secondary to MPD or MDS, trisomy 8 is associated with a blockade of myeloid maturation at an early step of the differentiation process. Received: 2 December 1996 / Accepted: 12 February 1997     

Combined immunophenotyping and DNA in situ hybridization to study lineage involvement in patients with myelodysplastic syndromes.

Clonality of myeloid and lymphoid cell fractions obtained from peripheral blood (PB) or bone marrow (BM) of five patients with a myelodysplastic syndrome (MDS), was studied by combined immunophenotypic analysis and DNA in situ hybridization. This novel technique enables quantitative and direct analysis of cytogenetic alterations in nondividing cells of distinct cell lineages. Four patients with a trisomy 8 and one patient with a translocation (1;7) were studied. For cell lineage determination, antibodies specific for progenitor cells (CD34), myeloid cells (CD15), monocytes (63D3), T cells (CD3), and B cells (CD19,20,22) were used. In one patient with a trisomy 8, BM cells were available and the erythroid lineage could be studied. For detection of cytogenetic aberrations, we used chromosome-specific repetitive DNA probes. In three patients, all nonlymphoid cells carried the cytogenetic abnormality; in two patients, mosaicism within these lineages was suggested by the relative low numbers (35% to 55%) of aberrant cells. None of the T or B cells of the five patients carried the chromosomal aberrations. We conclude that combined immunophenotyping and in situ hybridization is a feasible technique to study lineage involvement. Our data suggest that the chromosomal aberrations studied in MDS are restricted to the myeloid lineages.     

Detection of monosomy 7 in interphase cells of patients with myeloid disorders

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated.     

Trisomy 12 in chronic lymphocytic leukemia detected by fluorescence in situ hybridization: analysis by stage, immunophenotype, and morphology

Fluorescence in situ hybridization (FISH) with a chromosome 12 specific alpha-centromeric probe was performed on interphase cells from 183 patients with B-cell chronic lymphocytic leukemia (CLL). Twenty one cases with trisomy 12 (11.5%) were detected. The number of trisomic cells ranged from 5.5% to 76% (mean 38.5%). No correlation was found between the presence of trisomy 12 and white blood cell count, hemoglobin level, platelet count, a specific immunophenotype, clinical stage, sex, splenomegaly, or lymphadenopathy. Morphologic review of all cases with trisomy 12 showed seven (33%) with more than 10% prolymphocytes and three (14%) with CLL of mixed cell type. While trisomy 12 is the most common chromosomal abnormality in CLL, it is more frequent in morphologically atypical cases, some of which may be undergoing transformation. There was a statistically significant difference in the incidence of atypical cases between those with (47%) and without (7.6%) trisomy 12 (P < .001). It remains to be determined whether this abnormality is associated with a worse prognosis; this is currently being investigated in the context of a national therapeutic trial. The technique used is more sensitive than conventional cytogenetic analysis, which in this series failed to detect trisomy 12 in six cases. FISH allows the systematic study on a large number of patients without the need of metaphase preparations.     

Cytogenetic and FISH studies in myelodysplasia,acute myeloid leukemia,chronic lymphocytic leukemia and lymphoma

Conventional cytogenetic studies are widely used today to diagnose and manage patients with hematological malignancies. The application of fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes helps to further define molecular subclasses and cytogenetic risk categories for patients with these disorders. Moreover, FISH permits analysis of proliferating (metaphase cells) and non-proliferating (interphase nuclei) cells, and is useful in establishing the percentage of neoplastic cells before and after therapy (minimal residual disease). For patients with myelodysplasia or acute myeloid leukemia, these chromosome techniques are important for accurate diagnosis and classification of disease and to help decide treatment and monitor response to therapy. Conventional cytogenetic studies have been problematic in chronic lymphocytic leukemia because the neoplastic cells divide infrequently. However, interphase FISH studies now permit detection of chromosome anomalies with prognostic significance in chronic lymphocytic leukemia. The World Health Organization recognizes that genetic anomalies are one of the most reliable criteria for classification of malignant lymphomas. New methods to extract individual nuclei from paraffin-embedded tissue are now available which permit the use of interphase FISH to detect important chromosome anomalies in lymphoma.     

Cytogenetic and FISH studies in myelodysplasia,acute myeloid leukemia,chronic lymphocytic leukemia and lymphoma

Conventional cytogenetic studies are widely used today to diagnose and manage patients with hematological malignancies. The application of fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes helps to further define molecular subclasses and cytogenetic risk categories for patients with these disorders. Moreover, FISH permits analysis of proliferating (metaphase cells) and non-proliferating (interphase nuclei) cells, and is useful in establishing the percentage of neoplastic cells before and after therapy (minimal residual disease). For patients with myelodysplasia or acute myeloid leukemia, these chromosome techniques are important for accurate diagnosis and classification of disease and to help decide treatment and monitor response to therapy. Conventional cytogenetic studies have been problematic in chronic lymphocytic leukemia because the neoplastic cells divide infrequently. However, interphase FISH studies now permit detection of chromosome anomalies with prognostic significance in chronic lymphocytic leukemia. The World Health Organization recognizes that genetic anomalies are one of the most reliable criteria for classification of malignant lymphomas. New methods to extract individual nuclei from paraffin-embedded tissue are now available which permit the use of interphase FISH to detect important chromosome anomalies in lymphoma.     

Cytogenetic clonality in myelodysplastic syndromes studied with fluorescence in situ hybridization: lineage, response to growth factor therapy, and clone expansion

Clonality in myelodysplastic syndromes (MDS) has been studied with various techniques including glucose-6-phosphate dehydrogenase (G6PD) isoenzyme and cytogenetic analyses, and with molecular techniques such as gene deletion studies and the analysis of restriction fragment- length polymorphisms (RFLP) of X-linked genes. In this study, we investigated the use of fluorescence in situ hybridization (FISH) with a chromosome-specific probe to examine cytogenetic clonality in peripheral blood (PB) cells from three patients with MDS. In each case, trisomy 8 was shown by conventional cytogenetic analysis at the time of the initial diagnosis. By using FISH with a probe for the centromere of chromosome 8, we identified the trisomy in individual PB cells from Wright-stained smears. With this technique, we could determine the cell lineage involved by the trisomy, and through serial analyses we could assess the response of the clonal and nonclonal cells to growth-factor therapy, and the expansion of the trisomic clone over time. In each of the three cases, various proportions of granulocytes, monocytes, eosinophils, and basophils showed trisomy 8 by FISH analysis. In none of the cases did we detect trisomy 8 in lymphocytes. By analysis of PB cells before and during therapy with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), we found that GM-CSF stimulated both trisomic and disomic cells. During a 1-year period of sequential study, we detected an abrupt increase in the percentage of trisomic cells in one patient, a stable percentage in another, and a slowly increasing percentage in the third. The abrupt increase in the first patient preceded a transformation to a more acute phase by 2 months. We conclude that FISH analysis of PB cells of patients with MDS offers an additional approach to the study of clonality in this disorder. In some cases this analysis may provide a useful and simple means of assessing response to therapy and progression of disease.     

Genomic instability in bone marrow failure syndromes

Aneuploidy is frequently seen in leukemia and myelodysplasia (MDS) but was thought to be uncommon in aplastic anemia (AA). We examined marrow cells from 96 unselected patients with bone marrow failure syndromes to assess the frequency of undetected aneuploidy for chromosomes 7 and 8 by fluorescence in situ hybridization (FISH) as compared to routine cytogenetic analysis. Twenty-eight percent (27/96) of patients had an abnormal karyotype. FISH identified an additional 27 patients with undetected monosomy 7 or trisomy 8. Those patients with undetected monosomy 7 generally had a poor clinical outcome, suffering from lack of response to medical therapy or early death. In one AA/MDS patient with normal cytogenetics, FISH identified a large population of monosomy 7 cells, which clearly heralded a clinical relapse. In another patient, FISH studies were used to delineate instability of chromosome 8, with apparent disease progression from AA to MDS. We conclude that undetected aneuploidy exists in marrow cells of a significant percentage of patients with bone marrow failure syndromes.     

Application of the multi-colour FISH to interphase nuclei and metaphase spreads for simultaneous examination of monosomy 7 and trisomies 8 and 11 in acute myelocytic leukaemia (AML)

We have used the multi-colour (three) fluorescence in situ hybridization (FISH) technique based on the ratio labelling for the detection of monosomy 7 and trisomies 8 and 11 in 13 cases of acute myeloid leukaemia (AML). Two out of the 13 AML cases showed monosomy 7 and two out of the remaining cases exhibited trisomy 8 in interphase nuclei. Three of these results were confirmed by metaphase-FISH study. Trisomy 11 was not found either by the interphase FISH study or in the metaphase FISH study. These results demonstrate the potential power of multi-colour FISH using ratio labelling to produce more fluorescein colour in interphase nuclei for the detection of aneuploidies in leukaemia.     

Novel translocations that disrupt the platelet-derived growth factor receptor beta (PDGFRB) gene in BCR-ABL-negative chronic myeloproliferative disorders

The BCR-ABL-negative chronic myeloproliferative disorders (CMPD) and myelodysplastic/myeloproliferative diseases (MDS/MPD) are a spectrum of related conditions for which the molecular pathogenesis is poorly understood. Translocations that disrupt and constitutively activate the platelet-derived growth factor receptor beta(PDGFRB) gene at chromosome band 5q33 have been described in some patients, the most common being the t(5;12)(q33;p13). An accurate molecular diagnosis of PDGFRB-rearranged patients has become increasingly important since recent data have indicated that they respond very well to imatinib mesylate therapy. In this study, we have tested nine patients with a CMPD or MDS/MPD and a translocation involving 5q31-33 for disruption of PDGFRB by two-colour fluorescence in situ hybridization (FISH) using differentially labelled, closely flanking probes. Normal control interphase cells gave a false positive rate of 3% (signals more than one signal width apart). Six patients showed a pattern of one fused signal (from the normal allele) and one pair of signals separated by more than one signal width in > 85% of interphase cells, indicating that PDGFRB was disrupted. These individuals had a t(1;5)(q21;q33), t(1;5)(q22;q31), t(1;3;5)(p36;p21;q33), t(2;12;5)(q37;q22;q33), t(3;5) (p21;q31) and t(5;14)(q33;q24) respectively. The remaining three patients with a t(1;5)(q21;q31), t(2;5)(p21;q33) and t(5;6)(q33;q24-25) showed a normal pattern of hybridization, with > or = 97% interphase cells with two fusion signals. We conclude that two-colour FISH is useful to determine the presence of a PDGFRB rearrangement, although, as we have shown previously, this technique may not detect subtle complex translocations at this locus. Our data indicate that several PDGFRB partner genes remain to be characterized.     

Analysis of premature centromere division (PCD) of the X chromosome in Alzheimer patients through the cell cycle

Cytogenetic analysis of the X chromosome in phytohaemagglutinin stimulated peripheral blood lymphocytes was evaluated in 12 sporadic Alzheimer disease (AD) patients and in 11 healthy subjects. For chromosome analysis two methods were used: (1) standard analysis of G-banded metaphase chromosomes and; (2) fluorescent in situ hybridization (FISH) for the detection of the X chromosome centromeric region in interphase nuclei. Cytogenetic analysis revealed that the X chromosome expresses premature centromere division (PCD) in AD females in 10.53% of metaphase cells and in 15.22% of interphase nuclei. In AD men the percentages were 3.98 and 6.06%, respectively. X chromosome PCD in the female control group showed a percentage of 7.46% in metaphase cells and 9.35% in interphase nuclei and in male controls the percentages were 2.84% in metaphases and 5.54% in interphase nuclei. The results of FISH analysis showed that PCD could occur much earlier than metaphase of mitosis, i.e. in interphase of the cell cycle, immediately after replication. The FISH method can be used for PCD verification in all phases of the cell cycle in various disorders including AD.