Elena Gaber

Temporal differences in replication timing of homologous loci in malignant cells derived from CML and lymphoma patients

A close association usually exists between replication timing of a given locus and its transcriptional activity: expressed loci replicate early whereas silent ones replicate late. Accordingly, alleles that show concomitant expression replicate synchronously, while those displaying an allele‐specific mode of expression show temporal differences in their replication timing, i.e., they replicate asynchronously. We aimed in our study to see whether the cancer phenotype is accompanied by a relaxation in the temporal control of allelic replication. Fluorescence in situ hybridization (FISH) was used to determine the level of synchronization in replication timing of four pairs of homologous loci in samples of malignant cells derived from patients with chronic myeloid leukemia (CML) and lymphoma and in samples from healthy individuals. Four loci, HER2 mapped to 17q11.2–q12, a locus at 21q22, TP53 mapped to 17p13.1, and MYC mapped to 8q24 were studied. In each sample we analyzed two chromosomal regions, either 17q11.2–q12 and 21q22 or 17p13.1 and 8q24. The results showed distinct differences between healthy individuals and CML/lymphoma patients: all samples derived from noncancerous subjects showed high levels of synchrony in replication timing of alleles, whereas those of cancer patients displayed a large temporal difference in replication timing, indicating early and late replicating alleles. Thus, as judged by four unrelated loci, malignancy is associated with changes in the replication pattern of homologous loci. Genes Chromosomes Cancer 22:225–231, 1998.

Asynchronous replication of allelic loci in Down syndrome.

We have used FISH to determine the level of sychronisation in replication timing of four pairs of alleles, unrelated to chromosome 21 (p53, HER2, RB1, and c-myc), in foetal (amniotic fluid) cell samples of Down syndrome and in normal foetuses. All samples derived from the Down syndrome subjects showed large temporal differences in replication timing, in contrast to the high level of synchrony shown in all samples of normal individuals. Thus, as judged by four independent loci which are not associated with chromosome 21, the additional chromosome in the Down syndrome genome induces changes in the replication pattern of an allelic pair: from a synchronous pattern characteristic to concomitantly expressed alleles to an unsychronised one shown by alleles displaying an allele-specific mode of expression.

Asynchronous replication of alleles in genomes carrying an extra autosome.

Transcriptional activity of genes appears to be highly related to their replication timing; alleles showing the common biallelic mode of expression replicate highly synchronously, whereas those with a monoallelic mode of expression replicate asynchronously. Here we used FISH to determine the level of synchronisation in replication timing of alleles in amniotic fluid cells derived from normal foetuses and from those with either of the trisomies for autosomes 21, 18 or 13, or for sex chromosomes (47,XXX and 47,XXY). Two pairs of alleles, not associated with the extra chromosome, were studied in subjects with each trisomy and three in normal subjects. In cells derived from normal foetuses and from foetuses with sex chromosome trisomies, each pair of alleles replicated synchronously; yet these very same alleles replicated asynchronously in cells derived from foetuses with trisomy for any of the three autosomes studied. The results suggest that the gross phenotypic abnormalities associated with an extra autosome are brought about not only by over-expression of genes present in three doses, but also by modifications in the expression of genes present in the normal two doses.

Replication pattern of the p53 and 21q22 loci in the premalignant and malignant stages of carcinoma of the cervix

Loss of replication synchrony during the S‐phase of the cell cycle has been shown to be associated with aneuploidy in malignant cells.

Asynchronous replication of p53 and 21q22 loci in chronic lymphocytic leukemia.

Abstract In this study, in order to evaluate the replication pattern and the cell cycle dynamics of normal and malignant cells from patients with chronic lymphocytic leukemia, we applied the FISH technique with the p53 gene. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. The rate of asynchronous replication was significantly higher in malignant cells than in normal cells (a mean of 28 vs 13, respectively, P = 0.023). There were proportionately more cells with two single dots among the normal cells (P = 0.0047). These results probably reflect the changes in gene replication and cell cycle progression that occur in malignant cells.

Clinical detection of BCR-ABL fusion by in situ hybridization in chronic myelogenous leukemia

We describe the use of the fluorescence in situ hybridization (FISH) technique to detect residual Philadelphia chromosome-positive (Ph+) cells in a patient with blastic phase chronic myelogenous leukemia (CML) after aggressive cytoreductive treatment. The analysis was made in interphase nuclei because of the very small number of recognizable metaphases in leukemic patients. FISH was a reliable tool for the detection of chromosome translocations in interphase nuclei as compared with conventional cytogenetic and polymerase chain reaction (PCR) techniques.

In situ hybridization: A simple and sensitive method for detection of trisomy 12 in chronic lymphocytic leukemia

Chromosome aberrations are detected in only 50% of patients with chronic lymphocytic leukemia (CLL), owing usually to the low mitotic rate exhibited by the neoplastic lymphocytes. Fluorescence in situ hybridization (FISH) is a simple method for identifying numerical abnormalities of the target chromosome in interphase nuclei. Therefore, we used the FISH procedure with chromosome 12-specific a-satellite probe to evaluate 19 patients with CLL. Trisomy 12 was detected in interphase cells of 12 patients (63%). Cytogenetic analysis, performed in nine patients, yielded trisomy 12 in four (44%). FISH detected three patients with trisomy 12 in whom conventional cytogenetic method yielded a normal karyotype. FISH is a simple, reliable, and sensitive method for detection of trisomy 12 in patients with CLL.

Deletion of 6q27 in Chronic Lymphocytic Leukemia and Multiple Myeloma Detected by Fluorescence In Situ Hybridization

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.

Replication Pattern in Cancer: Asynchronous Replication in Multiple Myeloma and in Monoclonal Gammopathy

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 patients 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.

Monoallelic p53 deletion in chronic lymphocytic leukemia detected by interphase cytogenetics

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.