BASELINE AND TREATMENT-INDUCED CHROMOSOMAL ABNORMALITIES IN PERIPHERAL BLOOD LYMPHOCYTES OF HODGKIN S LYMPHOMA PATIENTS

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1 doi: /s (03) Int. J. Radiation Oncology Biol. Phys., Vol. 57, No. 2, pp , 2003 Copyright 2003 Elsevier Inc. Printed in the USA. All rights reserved /03/$ see front matter CLINICAL INVESTIGATION Lymphoma BASELINE AND TREATMENT-INDUCED CHROMOSOMAL ABNORMALITIES IN PERIPHERAL BLOOD LYMPHOCYTES OF HODGKIN S LYMPHOMA PATIENTS R. M KACHER, PH.D.,* T. GIRINSKY, M.D., S. KOSCIELNY, PH.D., J. DOSSOU, PH.D., D. VIOLOT, M.S., N. BÉRON-GAILLARD, PH.D., V. RIBRAG, M.D.,* J. BOURHIS, M.D., PH.D., A. BERNHEIM, M.D., C. PARMENTIER, M.D., AND P. CARDE, M.D.* Departments of *Medicine, Radiation Oncology, and Biostatistics; Laboratory of Radiosensitivity and Radiocarcinogenesis; and Cytogenetic and Oncologic Genetic Laboratory Institut Gustave Roussy, Villejuif, France Purpose: To study chromosomal abnormalities in 49 patients with Hodgkin s lymphoma (HL), before and after treatment and at several times during a 2-year period. Methods and Materials: Simple chromosomal aberrations (CAs) and complex chromosomal rearrangements (CCRs) were counted in peripheral lymphocytes by painting of chromosomes 1, 3, and 4 (fluorescence in situ hybridization). A control population was composed of 20 healthy donors and 69 untreated cancer patients who had undergone various radiologic scans. Results: A greater frequency (p < 10 4 ) of spontaneous cytogenetic abnormalities was observed in untreated HL patients compared with the control populations. CCRs were observed exclusively in the HL population (p < 10 4 ). Chemotherapy was associated with a significant increase in the frequency of CAs (p < 10 4 ), according to the chemotherapy regimen (p 0.002). Immediately after radiotherapy, a significant increase (p < 10 4 ) was observed in CAs according to the size of the irradiation field. Conversely, the significant increases in the frequency of CCRs observed after treatment did not correlate with the chemotherapy regimens, radiotherapy dose, or size of the irradiation field. The evolution of CAs vs. CCRs over time was also dissociated: during the follow-up of these patients, a significant decrease was observed in the frequency of CAs at 6 months and 1 and 2 years. In contrast, after an initial decrease for up to 6 months after treatment, the frequency of CCRs remained constant for up to 2 years. Conclusion: Increased cytogenetic abnormalities were observed in untreated HL patients compared with the control populations. The greater frequency of cytogenetic abnormalities persisted in some patients. The presence of CCRs supports the concept of a unique genetic environment in HL patients that persists in response to potentially noxious treatments Elsevier Inc. Hodgkin s lymphoma, Chromosomal abnormalities, Complex chromosomal rearrangements, FISH. INTRODUCTION More than 75% of patients diagnosed with Hodgkin s lymphoma (HL) are cured with radiotherapy (RT) and/or chemotherapy given either initially or as salvage therapy (1). Unfortunately, HL patients have been shown to be at increased risk of developing second malignancies (2). Chromosomal abnormalities in peripheral blood lymphocytes have been shown to be sensitive cytologic indicators of the DNA damage induced by various agents. Biologic monitoring of chromosomal abnormalities in HL patients is important, because the results of such investigations may Reprint requests to: Radhia M kacher, Ph.D., Institut Gustave Roussy, 39 Rue Camille Desmolulins, Villejuif France. Tel: ; Fax: ; mkacher@igr.fr Supported by grants from Electricité de France (EDF) 98-04, Institut Gustave Roussy CCR 98-25, CCR , Suzanne Axel Gran, and Comite de l Essonne de la Ligue Française contre le Cancer. correlate with the long-term effects of therapy and thus influence the choice of subsequent treatment modalities. Estimating the cytogenetic effects should also help to identify treatment-induced risk factors. Multiple studies have demonstrated abnormalities in peripheral blood lymphocytes from untreated HL patients using conventional staining (3 8). Fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries is a technique used to score translocations and other chromosomal changes rapidly (9). The introduction of chromosome painting into radiation cytogenetics has not only widened the spectrum of aberra- Acknowledgments We are grateful to the nurses, technologists, and secretaries of the Departments of Medicine and Radiation Oncology (Institut Gustave Roussy) for their support and participation in this study. We are indebted to Lorna Saint Ange for editing, to Mrs. Kuchenthal for preparing the manuscript, and to Dr. A. G. Turhan for his suggestions. Received Mar 4, Accepted for publication Apr 11,

2 322 I. J. Radiation Oncology Biology Physics Volume 57, Number 2, 2003 tions amenable to detection (reciprocal translocations, insertions, and deletions), but has made it possible to refine the detection and definition of complex chromosomal rearrangements (CCRs) that were hitherto only possible with conventional staining (10). In this study, we assessed spontaneous chromosomal abnormalities, the cytogenetic effects of treatment, and the kinetic profile reflecting the disappearance of chromosomal abnormalities in circulating lymphocytes in 49 HL patients. METHODS AND MATERIALS Patients Forty-nine patients (24 men and 25 women, mean age 34 years, range 18 80) with HL consecutively treated in the Department of Hematology and Radiation Therapy at the Institut Gustave Roussy between 1998 and 2001 were included in the present study. Among these patients, disease was classified as Stage I in 9, Stage II in 28, Stage III in 8, and Stage IV in 4. Before RT, all patients received four to six cycles of chemotherapy consisting of adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) in 22 patients, epirubicin, bleomycin, vinblastine, and prednisone (EBVP) in 8 patients, mustargen, vincristine, procarbazine, and prednisone/adriamycin, bleomycin, and vinblastine (MOPP/ ABV) in 10 patients, and bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, and prednisone (BEACOPP) in 2 patients. Involved- or extendedfield RT (20 40 Gy) was delivered to 40 patients. Blood samples were obtained before treatment, after chemotherapy, immediately after RT, and at regular intervals during the mandatory follow-up period (6 months, 1 year, and 2 years). The present study compared HL patients with two control populations. The first control group consisted of 20 healthy donors, with a mean age 40 years (range 19 66). Of the 20 controls, 11 were women and 9 were men, none of whom had recently been exposed to ionizing radiation for diagnostic or therapeutic purposes. The second control group consisted of 69 cancer patients (thyroid, urologic, head and neck), with a mean age of 41 years (range 18 69). Of the 69 control cancer patients, 45 were women and 24 were men. All had undergone a radiologic workup, including CT before treatment. This study was performed in accordance with local ethical rules. All patients and healthy donors signed an informed consent form consistent with institutional review board guidelines. METHODS AND MATERIALS Chromosome painting (FISH) was used to score chromosomal aberrations (CAs; translocations, dicentrics, and breaks) and CCRs (abnormal exchanges involving three or more breaks in two or more chromosomes) in chromosomes 1, 3 and 4, chromosomes not known to be involved in HL. One to four hundred metaphases were counted per blood sample. Lymphocyte culture and chromosome preparation Five milliliters of medium were added to 0.5 ml of the blood sample and incubated at 37 C for 48 h. The medium consisted of 5 ml RPMI 1640 supplemented with 10% fetal calf serum, 0.1 ml phytohemagglutinin M (Gibco/BRL, Grand Island, NY), 1% glutamine, 1 mm sodium pyruvate, 1% bromodeoxyuridine, and penicillin and streptomycin. Colcemid (0.1 g/ml) was added 2 h before harvesting, and slides were prepared with metaphase chromosomes following the standard methanol/acetic (3/1 vol/vol) procedure. The slides were stored at 20 C until use (11). Fluorescence in situ hybridization Slide pretreatment. Slides were rinsed for 30 min at 37 C in 2 saline sodium citrate buffer (SSC)/0.5% nonylphenylpolyethylene glycol (NP-40) and dehydrated for 2 min in three alcohol solutions (70%, 85%, and 100%). They were then denatured for 2 min in 70% formamide/2 SSC at 72 C and dehydrated. Probe preparation. The probe was prewarmed at 37 C for 5 min. An aliquot of 3 L of the red total chromosome 1 DNA probe, 3 L of the green total chromosome 4 DNA probe, 1.5 L of the red and 1.5 L of the green total chromosome 3 DNA probe (Appligene Oncor) was denatured at 70 C for 5 min. The slides were analyzed under a fluorescence microscope and translocations, insertions, deletions, and breaks were visually scored. The reproducibility of scoring of abnormalities in painted chromosomes 1, 3, and 4 was assessed by counting the chromosomal abnormalities in circulating lymphocytes obtained from 5 healthy donors and irradiated at known doses: the coefficient of variation was 14.8% for 0.2 Gy, 12.2% for 0.5 Gy, 4.3% for 1 Gy, and 4% for 2 Gy. The reproducibility of the assay system was considered acceptable even for low doses. Chromosomes 1, 3, and 4 represent 20.4% of the whole genome. The results were not extended to cover the whole genome using Lucas formula, because the same chromosomes were painted in the patient and control populations. In this case, using Lucas formula (12) would have been equivalent to multiplying all the results by the same coefficient. The scoring of chromosomal abnormalities was determined according to the Savage and Simpson scheme (10): simple abnormalities included reciprocal translocations (2B), dicentrics (2A), and centric rings with a single fragment (CR1), and CCRs involved a minimum of three or more breaks in two or more chromosomes. Statistical analysis Statistical analysis was performed with Statistical Analysis System software. The comparison between the frequency of CAs and CCRs was performed with the nonparametric Wilcoxon rank order test (two classes) or the

3 Chromosomal abnormalities in HL patients R. M KACHER et al. 323 Table 1. Frequency of chromosomal abnormalities in HL patients before treatment, untreated cancer patients, and healthy donors using chromosome 1, 3, and 4 painting Population n Counted cells (n) CAs/cell Cells with CCRs (%) CAs* CCRs* Control populations Healthy donors ( ) (0 0.01) 0 Cancer patients (32 400) (0 0.02) ( ) HL vs. controls Control populations (32 500) ( ) ( ) HL before treatment (50 400) (0 0.12) 0.23 (0 1.15) Abbreviations: HL Hodgkin s lymphoma; CAs chromosomal aberrations; CCRs complex chromosomal rearrangements. Data presented as the mean, with the range in parentheses, unless otherwise noted. *Given by nonparametric Wilcoxon rank test (two classes) or the Kruskal-Wallis test (three classes). Kruskal-Wallis test (more than two classes). To provide quantitative information on the relevance of statistically significant results, the minimal and maximal intervals are reported. All statistical tests were two-sided, and p 0.05 was considered statistically significant. RESULTS Frequency of spontaneous cytogenetic abnormalities Before treatment, a greater frequency of spontaneous cytogenetic abnormalities was found in the peripheral lymphocytes from HL patients. The frequencies of CAs and CCRs detected by chromosome 1, 3, and 4 painting in control populations (healthy donors and untreated cancer patients) and in HL patients before treatment are shown in Table 1. The frequency of CAs in other untreated cancer patients was greater than in the healthy donors but the difference was not statistically significant. Conversely, HL patients had a statistically significant (p 10 4 ) greater frequency of CAs than did the control populations. There was a sevenfold greater frequency of CAs in the HL patients than in the control populations. CCRs are rare events in the absence of RT. No CCR was evident in healthy donors and only 1 cancer patient exhibited one CCR before treatment. The frequency of CCRs was significantly greater in HL patients (6 HL patients exhibited CCRs vs. 1 patient in the control populations; p 10 3 ). Histologic type, disease stage, gender, and age were not significantly associated with the frequency of spontaneous cytogenetic abnormalities. Increased cytogenetic abnormalities after chemotherapy The frequency of CAs and CCRs increased significantly after chemotherapy (p 10 4 ; Table 2). Statistically significant differences (p 0.005) in the frequency of CAs were found among the various chemotherapy regimens. The highest frequency of CAs was induced by BEACCOP. Although the number of patients was very small, the BEA- COPP regimen gave rise to a significantly greater number of CAs than the other regimens. This could have been due to the presence of etoposide. A statistically significant (p 0.025) greater frequency of CAs was observed in patients who received an etoposide-containing regimen than among those treated with the other regimens. Chemotherapy regimens with alkylating agents (MOPP/ABV and BEACOPP) led to a statistically significant difference in the frequency of CAs compared with the regimens without alkylating agents (ABVD and EBVP; p 0.05). Conversely, the significant increases in the frequency of CCRs observed after chemotherapy did not correlate with the chemotherapy regimens. Cytogenetic effects immediately after RT A statistically significant (p 10 4 ) greater frequency of CAs and CCRs was found in the peripheral blood lymphocytes immediately after RT (Table 2) compared with that found in the samples obtained before RT. The relationship between the frequency of CAs and CCRs and the radiation dose and size of the irradiated field was assessed. The relationship between the frequency of CAs and the radiation dose was not statistically significant (p 0.57). However, the frequency of CAs correlated significantly with the size of the irradiation field (p 0.005). The frequency of CAs after RT delivered to an involved field and a mantle field was not significantly different statistically (p 0.56), but it was significantly greater after subtotal RT than after mantle field RT (p 0.001). The greater frequency of CCRs observed after RT did not correlate with the radiation dose or size of the irradiated field. Evolution of frequency of cytogenetic effects after treatment The evolution over time of simple vs. complex abnormalities was dissociated. During the follow-up of these patients, a statistically significant decrease in the frequency of CAs was observed at 6 months (p 10 4 ), 1 year (p 0.008), and 2 years (p 0.01) after treatment. The greatest decrease (47%) was observed during the first 6 months after RT. The frequency of reciprocal translocations was greater than that of dicentrics. Conversely, after 6 months, the frequency of CCRs remained constant for up to 2 years (Fig. 1B).

4 324 I. J. Radiation Oncology Biology Physics Volume 57, Number 2, 2003 Table 2. Frequency of chromosomal abnormalities in HL patients before treatment, at different periods and according to treatment characteristics using chromosomes 1, 3, and 4 painting Period/treatment characteristics n Counted cells (n) CAs/cell (n) Cells with CCRs (%) CAs* CCRs* Before treatment (50 400) (0 0.12) 0.23 (0 1.15) After chemotherapy (34 200) 0.08 ( ) 0.9 (0 1.15) MOPP/ABV (50 200) ( ) 0.5 (0 2) ABVD (34 200) ( ) 1.07 (0 4) EBVP (56 200) ( ) 1.39 (0 2.5) BEACCOP ( ) 0.17 ( ) 1 (1 2) Presence of etoposide Yes ( ) 0.16 ( ) 1.66 (1 2) No (34 200) 0.08 ( ) 0.98 (0 4) Alkaline/alkylating regimen Yes (50 200) 0.07 ( ) 0.70 (0 2) No (34 200) 0.1 ( ) 1.15 (0 4) After RT (34 165) 0.87 ( ) 6.82 (1 2.94) Dose (Gy) (34 100) 1.00 ( ) 8.87 (1 2.94) (50 165) 0.81 ( ) 5.89 (1 2.20) Field Involved 3 65 (49 85) 0.68 ( ) 0.68 ( ) Mantle (45 165) 0.79 ( ) 5.99 (1 22) Subtotal 4 82 (34 100) 1.51 ( ) 1.51 (3 29.4) 6 mo after RT (31 100) 0.47 ( ) 3.7 ( ) mo after RT (28 200) 0.33 ( ) 2.6 (0 9) mo after RT (24 200) 0.19 ( ) 1.83 (0 7) Abbreviations: MOPP/ABV mustargen, vincristine, procarbazine, and prednisone/adriamycin, bleomycin, and vinblastine; ABVD adriamycin, bleomycin, vinblastine, dacarbazine; EBVP epirubicin, bleomycin, vinblastine, prednisone; BEACCOP bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbarbazine, prednisone; RT radiotherapy; other abbreviations as in Table 1. *Given by the nonparametric Wilcoxon rank test (two classes) or the Kruskal-Wallis test (three classes). Corresponds to comparison with previous period. After 2 years, the frequency of simple and complex cytogenetic abnormalities was comparable to that observed before treatment in some patients (62%) and remained greater in others (38%; 25-fold between the minimum and maximum). As shown in Fig. 1A, the frequency of chromosomal abnormalities at 2 years ranged from 0.02 to Throughout the study period, a significant correlation was always observed between CAs and CCRs (r 0.80; p 10 3 ). DISCUSSION The most important and striking observation was the greater frequency of spontaneous cytogenetic abnormalities (CAs and CCRs) before any treatment in patients with HL compared with that found in the control populations. It is noteworthy that we did not find any difference in the frequency of cytogenetic abnormalities between untreated cancer patients and healthy donors. CCRs were only found in patients with HL. We also demonstrated that the decrease in CAs and CCRs after treatment was very different, with no further decline in the number of CCRs 6 months after treatment. The frequency of CAs was influenced by the various treatment modalities, but this was not the case for CCRs. The results of this study confirm previous conventional cytogenetic studies performed on untreated HL patients (3 7) that suggested a complex karyotype and a possible relationship between these abnormalities and genetic instability using chromosome painting (FISH). We also confirmed that healthy donors and untreated cancer patients had a low frequency of CAs, which has already been reported by other authors (13 15). The low frequency of CAs could stem from poorly defined environmental aggressions (e.g., low-dose X-ray exposure due to radiologic examinations). Because HL patients had undergone previous radiologic examinations, which could have induced CAs, we decided to use untreated cancer patients who had undergone the same radiologic examinations (especially CT scans) as controls. The HL patients had a significantly greater frequency of CAs than the untreated cancer patients. Moreover, the presence of CCRs before treatment was almost exclusively confined to HL patients (1 case among the cancer controls) and may imply a DNA repair defect in these patients. Chemotherapy induced a statistically significant increase in the frequency of CAs, and certain regimens gave rise to different CA frequencies. In addition to its hematologic toxicity (16), the MOPP regimen has been shown to cause CAs in the sperm of HL patients (17). In our study, we found that the frequency of CAs in the peripheral blood lymphocytes after the MOPP/ABV regimen was lower than after other regimens. The ABVD regimen has a favorable toxicity profile and causes less myelotoxicity, acute leukemia, or sterility than other treatments (18). Nevertheless, we scored a greater fre-

5 Chromosomal abnormalities in HL patients R. M KACHER et al. 325 Fig. 1. Frequency of (A) CAs and (B) CCRs in chromosomes 1, 3, and 4 before and after treatment in peripheral lymphocytes from HL patients. Peripheral lymphocytes were obtained from patients before treatment (TRT), after chemotherapy (CT), immediately after RT, and at several intervals. Cells were cultured and stained with whole chromosome probes against chromosomes 1, 3, and 4 (FISH). Chromosomal abnormalities were scored under the fluorescence microscope. The minimum, maximum, and confidence intervals were plotted. quency of CAs after ABVD than after MOPP/ABV. These results confirm that the MOPP regimen may induce less chromosome damage, as demonstrated in a previous study comparing the CVPP-ABDIC (cyclophosphamide, vinblastine, procarbazine, prednisone, doxorubicin (Adriamycin), bleomycin, dacarbazine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea) regimen and MOPP (19). The hematologic toxicity of the MOPP/ABV regimen, which causes necrotic cell death among cultured lymphocytes and thus fewer metaphases for analysis, could explain this finding. Although the number of patients was very small, the results of our study suggest that BEACCOP may induce a greater frequency of CAs than that observed after MOPP/ ABV or ABVD. Frequent and early cases of leukemia have been reported after BEACOPP and, notably, after escalated BEACOPP (20). Detailed cytogenetic analyses have linked exposure to etoposide to secondary acute leukemia characterized by a number of recurring chromosomal translocations involving the myeloid/lymphoid leukemia gene (21, 22). It remains possible that this greater frequency of CAs was caused by etoposide. These results need to be confirmed in a large series of patients treated with etoposide-containing regimens, and an analysis of CAs involved in secondary malignancies should also be conducted. A significant increase in CAs was found immediately after RT. The dose effect relationship could not be studied because high doses of radiation were delivered (20 40 Gy), precluding a proper cytogenetic study. The size of the irradiation field was a significant factor, but no marked difference was found between involved-field and mantle field RT. This could be explained by too slight a difference in irradiated blood volumes (0.67 L/s for involved field and 0.81 L/s for mantle field). On the other hand, the difference in the frequency of CAs after mantle field and subtotal RT could have been due to the difference in irradiated blood volumes (2.4 L/s for the subtotal field). The frequency of CAs was huge in patients who received subtotal nodal RT (splenic and lumbar-aortic RT fields). In addition, 90% of circulating lymphocytes contained CAs. The decrease in CAs after RT was significantly greater during the first 6 months (Fig. 1), with a slower decline in frequency thereafter. This variation may be attributed to repopulation through increased activity of stem cells or the migration of lymphocytes into the bloodstream from nonirradiated areas (23, 24). More reciprocal translocations with fewer dicentrics without acentric fragments were found in the 6- and 12- month blood samples after treatment than in those collected immediately after treatment. This kind of aberration indicated that new lymphocytes had been produced by stem cells (24). CCRs were caused by treatment, but the frequency of CCRs was independent of the chemotherapy regimen or the dose and/or size of the irradiation field. This kind of CA could reflect defects in DNA repair mechanisms. The frequency of CCRs was not related to disease stage, age, gender, or the treatment modality. However, the frequency of CCRs immediately after RT had a predictive value in terms of the kinetics of variation in the frequency of CA. Our data clearly show that individual differences exist in the frequency of treatment-induced CAs (Fig. 1) and in the kinetic profile, reflecting the disappearance of CAs. This variation can exhibit three profiles. Two years after treatment, the frequency of CAs in some patients was low (0.02 aberration/cell), close to the spontaneous CA rate (0.023 aberration/cell), but it remained high (0.56 aberration/cell) in others. These differences may be attributed to several factors, including drug absorption, metabolism, and the efficiency with which damage sustained by chromosomes is repaired. Complementary studies should be undertaken to

6 326 I. J. Radiation Oncology Biology Physics Volume 57, Number 2, 2003 explain these important interindividual variations, focusing, for example, on the telomere length and its relationship with the frequency of CAs and genome integrity. The phenomenon of CCRs in lymphocytes might reflect damage sustained by other cells or tissues and play a contributory role in the origin of late complications. The presence of CCRs in other tissues could indicate a certain degree of chromosomal instability in HL patients. That HL patients tend to be at a high risk of developing secondary malignancies after treatment could signify that this genetic instability is a facilitating factor in the development of neoplasia (25). It is important to continue the cytogenetic follow-up of these patients beyond 2 years to establish a possible correlation between the frequency of CCRs and CAs and the occurrence of secondary leukemia and other malignancies. In addition, our data should lead to the study of the cytogenetic effect of treatment in other normal tissues (e.g., fibroblasts in the irradiated field) to confirm chromosomal instability in the presence of CCRs. CONCLUSION In this study, we detected a greater frequency of spontaneous CAs before treatment in HL patients than that in the control populations. The frequency of CCRs before treatment suggests the existence of a certain degree of chromosomal instability in HL patients. Treatment modalities induced a high frequency of CAs and CCRs, with high interindividual variations. Our results tentatively indicate the biologic particularity of Hodgkin s disease and its underlying genetic susceptibility to damage-causing agents (25). REFERENCES 1. Mendenhall NP, Hoppe RT, Prosnitz LR, et al. Principles of radiation therapy in Hodgkin s disease. In: Mauch PM, Armitage JO, Diehl V, et al., editors. Hodgkin s disease. 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