The aberrant asynchronous replication - characterizing lymphocytes of cancer patients - is erased following stem cell transplantation

Abstract

Background: Aberrations of allelic replication timing are epigenetic markers observed in peripheral blood cells of cancer patients. The aberrant markers are non-cancer-type-specific and are accompanied by increased levels of sporadic aneuploidy. The study aimed at following the epigenetic markers and aneuploidy levels in cells of patients with haematological malignancies from diagnosis to full remission, as achieved by allogeneic stem cell transplantation (alloSCT).

Methods: TP53 (a tumor suppressor gene assigned to chromosome 17), AML1 (a gene assigned to chromosome 21 and involved in the leukaemia-abundant 8;21 translocation) and the pericentomeric satellite sequence of chromosome 17 (CEN17) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosomes 17 and 21. Replication timing and aneuploidy were detected cytogenetically using fluorescence in situ hybridization (FISH) technology applied to phytohemagglutinin (PHA)-stimulated lymphocytes.

Results: We show that aberrant epigenetic markers are detected in patients with hematological malignancies from the time of diagnosis through to when they are scheduled to undergo alloSCT. These aberrations are unaffected by the clinical status of the disease and are displayed both during accelerated stages as well as in remission. Yet, these markers are eradicated completely following stem cell transplantation. In contrast, the increased levels of aneuploidy (irreversible genetic alterations) displayed in blood lymphocytes at various stages of disease are not eliminated following transplantation. However, they do not elevate and remain unchanged (stable state). A demethylating anti-cancer drug, 5-azacytidine, applied in vitro to lymphocytes of patients prior to transplantation mimics the effect of transplantation: the epigenetic aberrations disappear while aneuploidy stays unchanged.

Conclusions: The reversible nature of the replication aberrations may serve as potential epigenetic blood markers for evaluating the success of transplant or other treatments and for long-term follow up of the patients who have overcome a hematological malignancy.

Figure 1

Fluorescent signals in PHA-stimulated lymphocytes at interphase following FISH with the CEN17 probe. (A) Cell with two singlets (SS cell), in which neither allele has replicated; (B) cell with two doublets (DD cell), in which both alleles have replicated; and (C) cell with one singlet and one doublet (SD cell), a S-phase cell in which one allele has replicated while its partner has still to do so.

Figure 2

Mean SD values in blood samples grown with and without AZA. (A) Control samples (20 cases); (B) samples from patients with hematological malignancies obtained at diagnosis (20 cases); (C) samples from patients with malignancies taken prior to alloSCT transplantation (14 cases); and (D) samples from patients with hematological malignancies taken following alloSCT (10 cases). AZA - 5-azacytidine.

Figure 3

Mean frequency of aneuploidic cells in blood samples grown with and without AZA. (A) Control samples (20 cases); (B) samples of patients with hematological malignancies obtained at diagnosis (20 cases); (C) samples of patients with malignancies taken prior to alloSCT transplantation (14 cases); and (D) samples of patients with haematological malignancies taken following alloSCT (10 cases). Chro - Chromosome; and AZA - 5-azacytidine.

Figure 4

Distribution of blood samples according to their SD cell frequency and aneuploidy level. The SD value of each sample is expressed by the mean of the SD cell frequency values of TP53, CEN17, and AML1; and the aneuploidy is the mean of the aneuploidy values of chromosomes 17 & 21.

Figure 5

Distribution of bone marrow samples according to their AML1 and TP53 SD values.

Figure 6

Distribution of lymphocyte samples of patients with prostate cancer and cancer-free individuals, with and without inflammation of the prostate gland, according to their AML1 and CEN17 SD values. The red marked characters indicate the mean values of the corresponding subgroup; Note, that the SD values of samples derived from the cancer-free patients with chronic inflammation were similar to those of the cancer-free patients with no inflammation, and were lower than those found in the cancer patients.