Frequent induction of chromosomal aberrations in in vivo skin fibroblasts after allogeneic stem cell transplantation: hints to chromosomal instability after irradiation

The most worrisome long-term side effect after successful allogeneic SCT (SCT) is the increased risk of secondary malignancies.

These tumors develop several years after transplantation with rising incidence over time. Besides younger age at transplantation and chronic immunosuppression through chronic graft-versus-host disease (GvHD), intensive conditioning treatment with total body irradiation (TBI) and high-dose chemotherapy has emerged as a prominent risk factor after allogeneic SCT [1‐7].

Ionizing radiation has the capacity to induce chromosomal aberrations in a variety of human tissues. Specific chromosomal aberrations in blood cells like losses at chromosomes 5 and 7, inv(16)(p13q22) and aberrations involving 11q23, 17q21, or 21q22 have been identified in chemotherapy-related myelodysplastic syndromes or acute myeloid leukemias after chemotherapy as well as after radioiodine therapy for malignant or benign thyroid disease [8‐11]. Less cytogenetic data in secondary solid tumors after radiotherapy are available, showing a diverse spectrum from simple balanced translocations to complex aberrant karyotypes in few cases [12]. Sublethal genomic damage may induce repairing cellular mechanisms after various times of cell cycle arrest or propagation of genetic lesions, which could ultimately lead to malignant transformation [13]. Evidence has accumulated that DNA-damage not only occurs in directly irradiated cells, but also in the progeny of the irradiated cells at delayed times after radiation exposure [13, 14].

Chromosomal aberrations have been described in cultured skin fibroblasts after accidental irradiation in single patients [15, 16] and in a few patients having received radiotherapy [17‐19]. No systemic in vivo cytogenetic analysis of irradiated skin fibroblasts has been performed in patients.

We herein describe the time-dependent development of cytogenetic damage induced in skin fibroblasts after conditioning with high-dose chemotherapy and TBI and allogeneic SCT in a larger cohort of patients with a long term follow-up.

The percentage of cells with an aberrant karyotype in our study was significantly higher at all time points after high-dose conditioning compared to the biopsies taken before conditioning. Genetic damage occurred early, because most metaphases from biopsies taken 3 months after SCT already presented an aberrant karyotype.

It is unlikely, that all these aberrations developed ex vivo in culture, because most skin metaphases were normal before SCT. In the literature there is only one small series of four pediatric patients after TBI or total lymphoid irradiation, where authors found 49 to 88 % aberrant metaphases in the skin biopsies taken within radiotherapy fields [19]. A few case reports have also been published describing chromosomal aberrations in skin fibroblasts after in vivo radiation exposure [15‐18, 25], but without comparison to pre-radiation metaphases. To our knowledge, our study is the largest in vivo cytogenetic study in irradiated patients with hematologic malignancies, which follows chromosomal aberrations over time.

Patients with acute leukemias, who had received more intensive chemotherapy before TBI had a higher level of cytogenetic damage. Therefore it cannot be excluded that the extent of the observed cytogenetic damage in the present study may have been influenced in part by radiation enhancing effects of chemotherapy. However in a small series of SCT-patients, abnormal karyotypes were only observed in those with TBI, but not in those without [19].

We mainly detected clonal aberrations in our study. Most of them were only seen in single culture flasks, so that in vitro clone formation cannot be excluded. A stronger hint to in vivo clonality could be found in three of our patients with identical aberrations in cultures from independently set up pieces of the same biopsy. Hints to in vivo clone formation come from case reports by others as well [15, 17‐19]. The strongest evidence for in vivo clone formation was reported by Mouthuy and Dutrillaux, who found cells with identical aberrations in two skin biopsies, which were taken with a time interval of one year [15]. This is a unique finding neither confirmed by us nor others.

A clone is not necessarily completely homogenous since subclones may have evolved exhibiting additional aberrations [24]. This phenomenon described as karyotype evolution was observed in 13 patients of our study.

Reciprocal translocations were the most common type of aberration observed at all time points. In 16 of our patients, complex aberrations were detected. The most complex aberration was a four-break-translocation seen in a patient of the retrospective group.

The high rates of aberrations in our study, karyotype evolution and the presence of complex aberrations are in line with the concept of permanent genomic instability through the cytotoxic effects of irradiation [13, 14, 29].

Studies of in vitro irradiated fibroblasts have demonstrated that the radiation-induced breakpoints are distributed non-randomly throughout the genome [27, 30, 31]. In our study some of the clusters were detected at published breakpoints like, for example, 1p22. Both arms of chromosome 1 have been affected frequently in in vitro studies of irradiated diploid cells [27]. We are reserved in the interpretation of the results of our breakpoint analysis on the basis of this conventional cytogenetic study. We see hints for a non-random distribution of breakpoints in our study (Figs. 2 and 3, Table 3).

Interestingly, several of the observed clusters were located at bands, where genes for the chromosome instability syndromes have been mapped to. Additionally, a cluster was detected at 14q32, which is known to be a preferential site of chromosomal breakage in ataxia-telangiectasia patients [32]. The genes involved in the chromosome instability syndromes like Ataxia teleangiectasia or Fanconi anemia are critical in the early detection of induced damage and subsequent induction of cellular response to repair the damage [13, 33]. Patients with these or other related disorders have a dramatically increased malignancy risk [33]. An interesting task for future studies would be to compare known gene loci and breakpoints in these cancer-prone disorders with radiation-induced aberrations with molecular tools. We hypothesize, that a congruence of breakpoints induced by radiation with chromosomal breakage sites in chromosome instability syndromes would support the concept of radiation-induced instability through disturbances in DNA repair as suggested by others [13].

Due to the limited number of patients in our experimental study, the rates of secondary malignancies cannot be compared with large register data [1‐7], although the 20 % cumulative incidence of secondary malignancies seems rather high. As virtually all patients had cytogenetic aberrations in skin, additional cellular mechanisms must be involved in carcinogenesis in these patients. It also seems very likely, that organ-specific cellular changes are involved in organ carcinogenesis.

More advanced technologies like FISH, SKY and molecular techniques have evolved rapidly with more data being available on mechanisms of cell damage and repair due to radiation injury [34‐36]. These data, however, are either from cell culture systems and mice models or did not involve patients with TBI, while our data were from in vivo irradiated patients’ fibroblasts. Experimental approaches and organ-specific analyses might detect specific cytogenetic and molecular aberrations, which would increase our understanding of organ-specific radiation-induced cellular injury and carcinogenesis.