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The replication status of malignant cells is usually asynchronous. However, to date the pattern of replication has not been studied in myeloproliferative disorders nor has the effect of chemotherapy been systematically evaluated. Therefore, we used fluorescence in situ hybridization to interphase nuclei in PHA-stimulated peripheral blood lymphocytes to examine replication timing of three alleles associated with the malignant process. The study group comprised hydroxyurea treated and untreated patients with essential thrombocytosis (ET) or polycythemia vera (PV). A significantly higher rate of the asynchronous pattern of replication in both treated and untreated patients was found as compared to healthy controls. The highest rate of asynchronous replication was observed in untreated patients. Also, the frequency of the two doublets pattern was significantly higher in the untreated group compared to the treated patients and to the control groups. In conclusion, patients with PV and ET have a higher rate of asynchronous pattern of replication. A possible correlation between disease activity and the pattern of replication is suggested. The effect of hydroxyurea on the pattern of replication is variable.

Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal chronic myeloproliferative disorders originating from a multipotent stem cell. Bone marrow examinations reveal chromosomal abnormalities in 15-43% of PV patients and 5% of ET patients, but no specific recurring abnormality has been found to date. We aimed to find cytogenetic aberrations in PV and ET by comparative genomic hybridization (CGH), a relatively new molecular cytogenetic technique. In this study, CGH analysis was performed on peripheral blood leukocytes of 12 PV patients and 8 ET patients. One patient (8.3%) with PV had an abnormal karyotype with a deletion in 7q11.2 and one patient with ET (12.5%) had a gain in 18p. Peripheral blood analysis by CGH revealed a low frequency of cytogenetic abnormalities in PV and ET patients. However, using CGH we were able to detect two cytogenetic aberrations that were not reported previously in these disorders.

Asynchronous replication of homologous loci was reported in lymphocytes of patients with lymphoma, ovarian and renal cancer as well as in lymphocytes of patients with premalignant conditions, for example, essential mixed cryoglobulinemia associated with hepatitis C virus and in monoclonal gammopathy of unknown significance. In the present study we evaluated the replication pattern in lymphocytes of four groups of patients with intermediate grade of non-Hodgkin lymphoma at various stages of their disease: 1) at diagnosis; 2) during cytotoxic treatment; 3) in remission; and 4) in relapse. A significantly higher proportion of the asynchronous pattern of replication at diagnosis, during cytotoxic treatment, and in relapse was noted as compared to healthy controls and to patients who achieved remission of their lymphoma. Also, the frequency of the two doublets (DD) pattern in every group studied was significantly lower than in the controls. If our findings can be confirmed in larger, long-term prospective studies, it may allow the use of a simple and inexpensive tool to closely observe patients with lymphoma who are at high risk for relapse.

Recently, a working-model of a stepwise malignant transformation in the molecular pathogenesis of multiple myeloma (MM) was proposed, involving the tumor suppressor gene TP53 and retinoblastoma gene (RB1) as prominent components of cell cycle control. To further define the role of TP53 and RB1 in disease progression, we retrospectively analyzed by fluorescence in situ hybridization (FISH) cytological material from 16 patients who underwent sequential bone marrow biopsies during the course of their disease. For TP53, no deletions were detected at presentation or during follow-up. It is possible that the patients reported here represent a subset with relatively long survival, and therefore did not demonstrate the TP53 deletions that had been reported in patients with a very poor prognosis. For RB1, monoallelic deletion was demonstrated in nine patients. In each case, the deletion appeared already in the first biopsy analyzed. The presence of a deletion did not affect the rate of tumor progression or the length of follow-up, and thus prognosis. Monoallelic deletions of RB1 appear to be a frequent and early event in the pathogenesis of MM, without obvious relevance for disease progression.

In this study we evaluated the replication pattern and cell-cycle dynamics of cells from patients considered to have a premalignant condition (monoclonal gammopathy, or MGUS) and patients with multiple myeloma (MM), as well as healthy controls. We applied the fluorescence in situ hybridization (FISH) technique with the TP53, RB-1 and 21q22 loci on the patient's cells. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. The rate of asynchronic replication was significantly higher in the cells from MM patients, with intermediate value in the cells from MGUS, while the lowest rate was in cells from controls. We suggest that these results may reflect the changes in gene replication and cell-cycle progression that occur in premalignant and malignant cells.

Chromosomal aberrations can be detected in 50% of patients with chronic lymphocytic leukemia (CLL). A role for tumor suppressor genes in the genesis of lymphoid tumors has been reported. In B-CLL, p53 gene mutations were found in 10-15% of the patients. We used fluorescence in situ hybridization (FISH) to detect p53 deletion in B-CLL. We also correlated the cytogenetic findings with the clinical course. In situ hybridization to interphase nuclei showed monallelic p53 deletion in 6 of 23 patients (26%). The percentage of cells with one p53 signal ranged from 12 to 100. A statistically significant correlation between p53 deletion and progression of CLL was demonstrated. We conclude that FISH is a sensitive and reliable method to detect deletion of specific genes (i.e., p53) in CLL. The finding of p53 deletion is associated with disease progression.

In this study, we used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) as complementary techniques for the analysis of two therapy-related secondary myelodysplastic syndrome (t-MDS) cases with complex karyotypes, previously analyzed by G-banding. Different types of SKY's cytogenetic contributions include confirmation of G-banding results, identification of partially characterized rearrangements, identification of marker chromosomes unidentified by G-banding, and detection of cryptic reciprocal translocations. In particular, the ability of SKY to clarify a number of markers led to the comprehension of clonal evolution. The common aberration found in these two t-MDS cases was the fragility of chromosome 5 and monosomy of chromosome 18. We clearly present that the use of SKY combined with conventional G-banding analysis and FISH has assisted in the identification of important chromosomal events that may play a key role in the development of t-MDS.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in humans, with the major cytogenetic aberrations of trisomy 12 and deletion of 13q14. This study examined the influence of these aberrations on general gene replication. The study group included three subgroups: (1) 15 CLL patients, (2) 4 CLL patients with trisomy 12, (3) 3 CLL patients with deletions in 13q14. Five healthy individuals served as a control group. Monocolor fluorescence in situ hybridization (FISH) with probes for c-myc, HER-2/neu, and p53 was applied to lymphocyte nuclei for the evaluation of replication timing. Asynchronous replication (SD) rate was significantly higher in all CLL patients (P0.01) when compared to the control group and was even higher in the group of CLL patients with trisomy 12 and 13q14 deletion (P0.01). The asynchrony rate was significantly higher in cells with trisomy 12 for all three probes analyzed, compared to "healthy" cells in the same patients (P0.001). To conclude, in CLL patients with a chromosomal aberration such as trisomy 12 and 13q14 deletion we were able to demonstrate a high rate of asynchrony of replication. The high correlation between cells with trisomy 12 and SD pattern could reflect direct influence of the aberration on gene replication and cell cycle control.

The risk of myelodysplastic syndrome (MDS) or acute myeloblastic leukemia (AML) in patients with multiple myeloma has been estimated to be 10-20% after 10 years. Most myeloma patients develop MDS/AML after 3-4 years of treatment with alkylating agents, mainly melphalan; chromosomes 5 and 7 are most frequently involved. We studied 14 patients with myeloma by fluorescence in situ hybridization (FISH) with a probe to 5q31 (the critical area of deletion on chromosome 5) to verify whether deletion of 5q31 occurs during the course of stable, uncomplicated myeloma, and to assess the clinical importance of this abnormality. We found 2 patients (14%) with deletion of 5q31 in 30-40% of their peripheral white blood cells. One patient with this deletion received a high cumulative amount of melphalan, and the other patient was treated with multiple alkylating agents, including melphalan. In these patients, no clinical or laboratory evidence of transformation occurred 14 and 12 months after the finding of the aberration. These findings suggest that 5q-may occur months prior to the overt development of (t)-MDS/AML, and raise important concerns regarding the management of patients with this and similar aberrations, including modification of treatment and performance of cytogenetic evaluation prior to autologous or PSC transplantation. The clinical and biological implications of these findings should be evaluated in larger clinical and laboratory studies.

Nonrandom deletions of the long arm of chromosome 6 (6q) are associated with various lymphoid malignancies. It has been suggested that deletions of 6q25∼27, 6q21, and 6q23 typically occur in intermediate-grade, high-grade, and low-grade lymphomas, respectively. We used fluorescence in situ hybridization (FISH) to evaluate the occurrence of 6q27 deletion in chronic lymphatic leukemia (CLL) and multiple myeloma (MM). 6q27 deletion was detected in 21% of patients with CLL and in 28% of patients with MM. The percentage of cells containing deletions ranged between 25∼49. Two patients with MM had progressive disease and the aberration was detected in both. We conclude that FISH is a sensitive method to detect 6q27 deletion in lymphoproliferative disorders. Also, this deletion is not specific to intermediate-grade lymphomas, but occurs also in CLL and MM.

Data concerning oncogene activation in CLL are very limited. When studied by Southern blot, rearrangements of bcl -1, bcl -2, and bcl -3 have been only infrequently reported. We evaluated the role of fluorescence in situ hybridization (FISH) in the detection of gene rearrangements in two CLL patients. We used multiple DNA probes, including those of chromosome 12, immunoglobulin heavy and light chains, and the oncogenes bcl -1, bcl -2, and bcl -3. Additionally, routine cytogenetic study was performed. In one patient, trisomy 12 and bcl -2 translocation were demonstrated by both methods, while trisomy 12 and bcl -1 translocation were seen in the second patient, who had a normal karyotype. Larger studies should evaluate the role of FISH in the detection of oncogene involvement in CLL and compare it with other molecular methods.