We found that patients who had been treated up to 17 years earlier with MOPP, either with or without radiotherapy, presented GIN as demonstrated by the occurrence of structural chromosomal damage or GCR. However, we found no evidence of increased chromosomal number aberrations or CIN with respect to controls. The types of aberrations observed were random and complex, consistent with persistent genomic instability events that were induced by chemotherapy and radiotherapy treatment in HL patients. These results suggest that anticancer treatment may affect genes with a role in DNA surveillance or repair, which, in turn, allows the accumulation of nontargeted structural chromosomal damage in future generations of cells.
The Anticancer Treatment MOPP Does Not Alter the Frequency of Aneuploidy
To our knowledge, there are currently no published reports on the effects of MOPP chemotherapy (with or without radiotherapy) on the frequency of aneuploidy in human lymphocytes. In this study, the total aneuploidy frequencies were 2.0% for healthy individuals, 1.1% for pretreatment HL patients and 2.2% for posttreatment HL patients; no significant differences were found in CIN among the three groups studied (Tables II and III). This result is consistent with the lack of persistence of aneuploidy in germ cells after anticancer treatment [Robbins et al.,1997; Frias et al.,2003].
Hyperdiploidy was the least frequent aneuploidy found among the three groups studied (Table II), and its frequency in healthy individuals was up to eight times lower than those reported previously (0.1–4.17%) [Juberg et al.,1985; Leopardi et al.,2002]. The frequency of hypodiploidy in the lymphocytes of healthy individuals observed in this study (Table II), was also four times lower than those found in other studies [Cimino et al.,1986; Eastmond and Pinkel,1990]. These differences are most likely the result of the very strict criteria employed during analysis to avoid cells to be mistakenly identified as aneuploid due to technical artifacts.
Although some components of the chemotherapy studied here are able to induce aneuploidy, we did not find CIN in Group III patients. Even though aneuploidy cells may have been generated immediately (or during) treatment, they may have disappeared from the population over the time, presumably because the genetic imbalances created by aneuploidy imposed a selective disadvantage [Yuen and Desai,2008].
Complex Structural Chromosome Damage is Increased in HL Survivors
Cells with one chromatid or chromosome break, considered as unstable damage, were observed in all three study groups (Tables III and IV). We found a higher frequency of chromosome breaks in PBLs from HL patients up to 17 years after treatment. Complex GCR was the most frequently type of aberrations found in HL survivors and it was significantly higher in Group III with respect to the other groups (Tables III and IV). All GCR found in this study were stable aberrations [Shaffer et al.,2009] such as translocations, deletions, inversions, marker chromosomes and complex rearrangements, while asymmetrical exchanges (dicentrics, acentrics, and ring chromosomes) were not found in any of the studied groups.
A high frequency of chromosomal aberrations in pretreatment HL patients has been reported [M'Kacher et al.,2003], suggesting that these patients harbor basal chromosome instability. In our study, we detected cells with GCR in two of five pretreatment patients that consisted of clones of 3 and 15 cells with del(7)(q36) and del(21)(q22), respectively (Table IV). These chromosomal abnormalities have been related to cytogenetic changes in cancerous Reed-Sternberg Hodgkin cells [Barrios et al.,1988; M'Kacher et al.,2003]. Interestingly, these cells may be related to viral infections which even 2 years after treatment, may generate chromosomal instability in the peripheral blood of patients [M'Kacher et al.,2010]. In this study, these rearrangements were considered to be aberrations related to the disease and were discarded as a possible GIN in noncancerous lymphocytes [Nagai et al.,1986; Dennis et al.,1989].
GCRs can be transmitted through mitosis of hematopoietic stem cells (HSCs); these types of events, therefore, may be detected many years after exposure as clonal events. In our study, only one clone with the same gross chromosomal aberration was found, (1 of 13 individuals with GCR in Group III), whereas nonclonal GCRs were found in 12 of 20 individuals within the same group. All of these 12 individuals displayed chromosomal structural aberrations 2–17 years after treatment, consistent with persistent nontargeted effects of anticancer treatment. Because of the time that elapsed between treatment and the initiation of the study, it is unlikely that the high incidence of breaks and their rejoined product GCR was due to direct chromosomal damage produced by the treatment.
These results suggest that the origin of the observed GIN is due to mutations in long-term self-renewal HSCs, that affects one or more of the main mechanisms that contribute to maintain genomic stability: (a) high-fidelity of DNA replication in S-phase, (b) accurate distribution of chromosomes among daughter cells during mitosis, (c) error-free repair of DNA damage throughout the cell cycle, and (d) cell cycle progression and checkpoint control [Wright,1999; Shen,2011]. If the anticancer treatment induced mutations affecting any of these mechanisms, HSCs with a mutator phenotype could arise in bone marrow and be maintained for long periods of time; these abnormal HSCs may constantly generate blood cells with a heterogeneous spectrum of chromosomal abnormalities. In this study, only one patient showed a clone with the same chromosomal aberration del(17)(p). This clone may be originated by additional genomic changes that conferred a selective advantage that permitted the mutated HSCs to evolve as a lymphocyte clone.
We found a large variation in the number of events (total number of chromosome breaks per cell) that lead to GCR among patients. Specifically, 7 of 20 patients did not have gross chromosomal damage, whereas 13 of 20 displayed GCR and there was no correlation with personal habits, age, medical records or time after treatment among these patients. Consistent with our findings, M'Kacher et al.  analyzing eight patients treated with MOPP/ABV found no correlation between the frequency of GCR and the patient's age, time after treatment, disease stage, or chemotherapy dose, indicating that there are clear individual variability in the response to anticancer treatment [M'Kacher et al.,2003]. Individual variance is most likely caused by differences in DNA-metabolizing enzymes or DNA repair capabilities [M'Kacher et al.,2003; Bonassi et al.,2004; Hagmar et al.,2004; Allan and Travis,2005].
We found a significantly higher frequency of GCRs in posttreatment patients than the healthy and pretreatment group (Table IV) suggesting that radiation and chemotherapy caused permanent damage to HSCs in these patients. Studies have shown that cells exposed to radiation may develop mutations and chromosomal aberrations that persist for several cell generations (over a year) [Wright,1999]. In this study, GCRs were found in 10 of 14 patients treated with both chemotherapy and radiotherapy, and in 3 of 6 treated only with chemotherapy, without significant difference in the frequency of GCR between the two groups. The immediate damage to cells may be greater due to the combined treatment; however, in survivors treated a long time before analysis (12–24 years), both radiotherapy and chemotherapy might affect genes related to DNA surveillance or repair, and once these genes are mutated, new cells with nontargeted chromosomal damage are continuously produced in sensitive patients [Wright,1999].
The development of secondary neoplasms as a result of the treatment of primary malignant tumors is frequent; the highest excess risk is reported for HL patients among survivors of childhood cancer [Friedman et al.,2010]. Cancer risk is associated with many factors, one of the most important being chromosomal damage. Several studies have indicated a clear association between cancer risk and the frequency of GCRs in PBLs, suggesting that GCRs in PBLs can be used as a biomarker of cancer risk [Blanco et al.,2001; Heng et al.,2006; Norppa et al.,2006; Boffetta et al.,2007]. In our patients, the majority of GCRs were observed in only one cell (nonclonal); however, GCRs are the primary source of genomic variation that can lead to unstable cell populations and, ultimately, initiate the conversion of a healthy cell into a cancer cell [Heng et al.,2006]. In our study, we found 6 GCRs per 1,000 lymphocytes (Table IV) or 6,000 GCRs in 106 cells. If the chromosomal damage found in PBLs is similar to the damage that occurs in other tissues, the frequency of GCRs may be an indication of cancer risk in any tissue [Norppa et al.,2006; Boffetta et al.,2007].
In the post treatment group, we found three chromosomal alterations that have been identified in patients with hematological neoplasias, or in patients with diseases associated with a high risk of developing cancer like myelodysplastic syndromes (MDS). The del(7)(q35) is present in MDS and acute myeloid leukemia (AML). The inv(14)(q11q32.1) is present in patients with T-cell leukemia/lymphoma, and the del(17)(p11), which was the only clonal alteration detected in this study, has been detected in MDS, blastic crisis of chronic granulocytic leukemia, de novo AML, or t-AML associated with alkylating agents, as well as in patients with polycythemia vera or essential thrombocytopenia treated with hydroxyurea [Mitelman,1991]. As a consequence of GCR, it is likely that oncogenes and tumor suppressor genes may be misregulated and facilitate the development of second malignancies [Smith et al.,1992; Bilban-Jakopin and Bilban,2001].
Previous studies that analyzed chromosomal abnormalities using nonbanded chromosomes or FISH, have found a significantly increased frequency of structural chromosomal aberrations in HL patients treated with MOPP chemotherapy in combination with radiotherapy [Papa et al.,1984; Smith et al.,1992; Bilban-Jakopin and Bilban,2001; M'Kacher et al.,2003; Ryabchenko et al.,2003]. Smith et al.  analyzed five HL patients 12–21 years after treatment with chemotherapy/radiotherapy, using chromosome 4 painting probe and found a high frequency of chromosomal translocations (mean = 0.016). Similarly, M'Kacher et al.  studied 50–200 cells from eight patients treated with chemotherapy including MOPP/ABV, either with or without radiotherapy using FISH painting probes for chromosomes 1, 3, and 4, and found a twofold higher frequency of chromosomal aberrations per cell in HL patients 2 years after treatment, as compared with pretreatment group. In our study, we analyzed all 46 chromosomes in 1,000 cells per patient, and found a 30-fold higher frequency in posttreatment patients as compared with healthy individuals and pretreatment group. These results show that the use of GTG banding by allowing the identification of each of the 46 chromosomes permits the detection of not only more cytogenetic alterations, but also the specific chromosomes involved in structural aberrations and the detection of balanced rearrangements.
In conclusion, we found genomic instability in lymphocytes of HL patients treated with MOPP many years after the end of chemotherapy. As these patients are very susceptible to developing secondary cancers [Van der Velden et al.,2003], our results suggest that genomic instability may affect the function of genes important for preventing cancer development. In particular, balanced exchanges that may modify the expression of oncogenes, or tumor suppressor genes but allow the survival of carrier cells without lethal chromosomal damage (i.e., unbalanced exchanges such as dicentrics) may represent a mechanism by which secondary cancers arise in these patients. Real-time PCR studies to detect populations of cells with chimeric proteins that may confer an increased risk of developing secondary cancers after HL treatment is an avenue for future research to confirm this hypothesis.