JC human polyomavirus is associated to chromosomal instability in peripheral blood lymphocytes of Hodgkin's lymphoma patients and poor clinical outcome.

BACKGROUND: B cells are potential sites for latency and reactivation of the human neurotropic JC polyomavirus (JCV). We investigated JCV and Epstein-Barr virus (EBV) status in peripheral blood lymphocytes (PBL) from 74 Hodgkin's lymphoma (HL) and 91 B-cell non-Hodgkin's lymphoma (B-NHL) patients. PATIENTS AND METHODS: JCV and EBV DNA were assessed by PCR, and FISH technique was used to localize viral infection and to estimate chromosomal instability (rogue cells, 'chromosomal aberrations') throughout evolution. The influence of viral infection and chromosomal instability on freedom from progression (FFP) was investigated in HL patients. RESULTS: PCR product sequencing of PBL identified JCV in 42 (57%) circulating lymphocytes of HL patients. FISH analysis revealed that the presence of cells with a high JCV genome copy number--associated to the presence of rogue cells and 'higher frequency of chromosomal aberrations'--increased from 15% before treatment to 52% (P < 10(-5)) after. The co-activation of JCV and EBV was independent of known prognostic parameters and associated with a shorter FFP (JCV and EBV co-activation P < 0.001, rogue cells P < 0.002). CONCLUSION: In HL, JCV activation and chromosomal instability have been identified in PBL and associated with a poorer prognosis, especially in EBV+.

Caspase independence of radio-induced cell death.

Colon carcinoma cells subjected to gamma-irradiation (4 Gy) manifest signs of apoptosis (caspase activation, chromatin condensation, phosphatidylserine (PS) exposure on the cell surface, sub-diploid DNA content), correlating with their radiosensitivity, which is increased in cells lacking the 14-3-3sigma protein as compared to wild-type controls. Inhibition of caspases by addition of Z-Val-Ala-DL-Asp (OMe)-fluoromethylketone, by stable transfection with the Baculovirus gene coding for p35, or by Bax knockout reduced all signs of apoptosis, yet failed to suppress radio-induced micro- and multinucleation. Moreover, pharmacological caspase inhibition, p35 expression or Bax knockout had no effect on the clonogenic survival that was reduced by gamma-irradiation and caspase inhibition failed to abolish the increased radiosensitivity of 14-3-3sigma-deficient cells. Micro- and multinucleation was detectable among non-apoptotic cells lacking PS exposure, as well as among cells undergoing apoptosis. Moreover, a fraction of micro- or multinucleated cells manifested caspase activation, and videomicroscopic analyses revealed that such cells could succumb to caspase-dependent apoptosis. Altogether, these results suggest that genomic instability induced by gamma-irradiation can trigger apoptosis, although apoptosis is dispensable for radio-induced clonogenic death.

Evidence of increased chromosomal abnormalities in French Polynesian thyroid cancer patients.

PURPOSE: The aim of this study was to evaluate the frequency of chromosomal abnormalities in thyroid cancer patients before and after radioactive iodine administration in order to assess cytogenetic particularity in Polynesian thyroid cancer patients. METHODS: Chromosomal abnormalities were studied in 30 Polynesian patients with differentiated thyroid cancer, prior to and 4 days after 131I administration. Unstable chromosomal abnormalities were counted in peripheral blood lymphocytes using a conventional cytogenetic method. Peripheral blood was irradiated in vitro at different doses (0.5, 1 and 2 Gy) in order to establish the dose-response of the lymphocytes. Control groups were composed of 50 European thyroid cancer patients before and after first administration of 131I, and of ten European healthy donors. In addition, in vitro irradiation assays were performed at different doses (0.5, 1 and 2 Gy). RESULTS: The relative risk of spontaneous dicentrics before any radiation treatment was 2.9 (95% CI 1.7-5.1) times higher among Polynesian thyroid patients than among European thyroid cancer patients. After in vitro irradiation, the rise in frequency of dicentrics was similar in the Polynesian thyroid cancer group and the European thyroid patients and healthy donors. Four days after administration of 3.7 GBq 131I, the relative risk for a dicentric per cell was 1.3 (95% CI 1.0-1.5) times higher in Polynesian than in European patients. This can be explained by higher 131I retention in Polynesian compared with European patients. The results obtained revealed an increased frequency of cytogenetic abnormalities in Polynesian thyroid cancer patients compared with European control patients. CONCLUSION: These preliminary findings are compatible with possible previous environmental aggression and therefore imply a need for further investigations on larger series including, in particular, French Polynesian healthy donors. In addition to French Polynesians, Maori and Hawaiian control groups could be useful.

Baseline and treatment-induced chromosomal abnormalities in peripheral blood lymphocytes of Hodgkin's lymphoma patients.

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.

Multiple molecular mechanisms contribute to radiation sensitivity in mantle cell lymphoma.

Mantle cell lymphomas (MCL) are characterized by their aggressive behavior and poor response to chemotherapy regimens. We report here evidence of increased in vitro radiation sensitivity in two cell lines that we have generated from two MCL patients (UPN1 and UPN2). However, despite their increased radiation sensitivity, UPN2 cells were totally resistant to apoptotic cell death, whereas UPN1 cells underwent massive apoptosis 6 h after irradiation. The frequency of induced chromosomal abnormalities was higher in UPN1 as compared to UPN2. Distinct mechanisms have been found to contribute to this phenotype: a major telomere shortening (UPN1 and UPN2), deletion of one ATM allele and a point mutation in the remaining allele in UPN2, mutation of p53 gene (UPN1 and UPN2) with absence of functional p53 as revealed by functional yeast assays. After irradiation, Ku70 levels in UPN1 increased and decreased in UPN2, whereas in the same conditions, DNA-PKcs protein levels decreased in UPN1 and remained unchanged in UPN2. Thus, irradiation-induced apoptotic cell death can occur despite the nonfunctional status of p53 (UPN1), suggesting activation of a unique pathway in MCL cells for the induction of this event. Overall, our study demonstrates that MCL cells show increased radiation sensitivity, which can be the result of distinct molecular events. These findings could clinically be exploited to increase the dismal response rates of MCL patients to the current chemotherapy regimens.

Blastoid mantle cell lymphoma: evidence for nonrandom cytogenetic abnormalities additional to t(11;14) and generation of a mouse model.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32), which is associated with cyclin D1 hyperexpression and a poor prognosis. MCL cases have been shown to progress to a more aggressive disease but the molecular events responsible of this phenomenon have not been determined. We have established two cell lines from the pleural effusions of two patients with MCL that we have used for further cytogenetic characterization to better define the incidence and nature of secondary chromosome abnormalities using multicolor fluorescence in situ hybridization, whole chromosome paint, and specific probes. Both cell lines grew independently without growth factors. Using CCND1/IGH-specific probes, patient UPN1 was found to have a masked t(11;14). Numerous and complex chromosomal abnormalities were found in both cell lines affecting chromosomes 2, 8, 13, 18, 22, X, and Y. These abnormalities included 8p losses, suggesting the presence of an anti-oncogene in this region, rearrangements of 8q24, MYC gene, and translocations involving 8, X, and Y chromosomes, which might be significant in the pathogenesis of MCL progression. The use of the cell lines (UPN1) allowed us to generate a mouse model of human MCL, mimicking a disseminated lymphoma and leading to the death of the animals in 4 weeks. This blastoid MCL model could be of major interest to determine molecular events involved in MCL progression, allowing isolation of involved genes and their functional characterization, and to study the effects of new chemotherapy regimens in mouse models.

Premature chromosome condensation associated with fluorescence in situ hybridisation detects cytogenetic abnormalities after a CT scan: evaluaton of the low-dose effect.

The purpose of this study was to assess the cytogenetic effects of the X ray irradiation used during a CT scan in order to estimate the mean absorbed dose in circulating lymphocytes. Chromosomal aberrations were scored in blood lymphocytes of ten patients undergoing CT scans, by applying fluorescence in situ hybridisation (FISH) to metaphase cells and premature chromosome condensation (PCC) with chromosomes 1, 3 and 4 painting probes immediately after exposure. This generated a dosimetric index that reflects the dose to the circulating lymphocytes. By using PCC a significant increase in the frequency of chromosomal fragment was observed immediately after a CT scan. However, no significant increase in chromosomal aberration was detected in metaphase cells. The mean dosimetric index immediately after exposure was 0.057 Gy (95% CI: 0.052-0.082 Gy). This dosimetric index depends essentially on the size of the examined and exposed blood volumes. This dose is in close agreement with the dose length product (DLP) (Gy cm) (R = 0.80). It should be kept in mind when justifying requests for diagnostic CT scan especially in young patients. The presence of chromosomal fragments after a CT scan indicated the cytogenetic effect of a low dose. PCC associated with chromosome painting is a method for detecting the cytogenetic effect of a low dose immediately after exposure.

[Validation of biological dosimetry in patients conditioned with total body irradiation: conventional cytogenetics and in situ hybridization(FISH)]. / Validation de la dosimétrie biologique chez des patients conditionnés par irradiation corporelle totale: étude cytogénétique conventionnelle et hybridation in situ (FISH).

PURPOSE: Validation of biological dosimetry versus physical dosimetry in malignant haemopathy patients conditioned by total body irradiation (TBI) before bone marrow transplantation (BMT). PATIENTS AND METHODS: The scoring of chromosomal aberrations in peripheral lymphocytes irradiated in vivo was used to perform the biological dosimetry. The data were compared to those obtained with healthy volunteers' total blood exposed to in vitro irradiation with linear accelerator doses (0.2, 0.5, 0.75, 1, 2, 3, 4 and 5 Gy) for dose-response curves. In experimental animal models, can in vivo and in vitro responses be considered as being the same? All the published human data are based on retrospective dose evaluation with very large uncertainties on the dose precisely delivered to the subject. TBI before BMT was taken as a model where the dose calculation results from the physical method, with homogeneous beam and dose delivered precisely along the entire organism. In vivo response allows us to validate biological dosimetry in 15 adult patients (female + male), before (D = 0 Gy) and after the first fraction of 1.8 Gy, delivered by a linear accelerator (18 MV, dose-rate of 15.8 cGy/min-1). Two methods, conventional cytogenetics (CCG) and fluorescent in situ hybridization (FISH painting) of chromosome 4 were respectively used to analyze the unstable chromosome aberrations and stable chromosome aberrations. RESULTS: Healthy volunteer lymphocytes, before irradiation, yielded 0.1% dicentrics and 0.3% translocations of chromosome 4, with 2.5% for the whole genome. Patients before irradiation had 2% dicentrics and 11.48% chromosome 4 translocations for the whole genome. In the 15 patients, for a physical dose of 1.8 Gy, the evaluated biological dose was 1.93 Gy (95% CI: 1.85-2.05 Gy) with conventional cytogenetics and 2.06 Gy (95% CI: 1.75-2.15 Gy) with FISH. CONCLUSION: These results, in which the biologically estimated dose is in complete agreement with the dose calculated by physical dosimetry in the homogeneous irradiation model, suggest the validation of biological dosimetry in TBI conditioning.

Biologic dosimetry in thyroid cancer patients after repeated treatments with iodine-131.

UNLABELLED: To estimate a cumulative dosimetric index that reflects the dose to the circulating lymphocytes after repeated treatments with 131I, biologic dosimetry was applied to 18 patients with differentiated thyroid carcinoma and neck relapse or lung metastases. METHODS: Chromosomal aberrations were scored in peripheral blood samples that were obtained before and 4 days after each administration of 3.7 GBq 131I according to two methods, conventional cytogenetics and chromosome 4 painting. RESULTS: The mean dosimetric index was equal to 0.5 Gy by both methods after the administration of 3.7 GBq 131I. Repeated administrations of 131I delivered the same dose each time, resulting in a cumulative dose from 1-3.5 Gy in the patients who had two to seven treatments. However, the estimated dose, based on the number of chromosomal aberrations on Day 4 and, above all, from the third treatment on, was considerably lower than the real dose absorbed by the lymphocytes. This may be linked to the phenomenon of apoptosis, which results in a loss of information during the course of repeated irradiation. CONCLUSION: Both chromosomal painting and conventional cytogenetics underestimate the cumulative dose after repeated 131I treatments. A complementary test measuring apoptosis may improve the dose estimates.

A new immunoradiometric assay (IRMA) system for thyroglobulin measurement in the follow-up of thyroid cancer patients.

A new commercially available kit for thyroglobulin (Tg) measurement [immunoradiometric assay (IRMA) system based on monoclonal antibodies] was used in 479 patients with thyroid carcinoma. The effective working range was 1 ng/ml, and results were strongly correlated with our homemade radioimmunoassay (RIA). This IRMA method is less susceptible to interferences of auto-antibodies than our RIA. During thyroxine (T4) treatment, the Tg level was undetectable in 98% of patients after total thyroid ablation, in 91% after total thyroidectomy and in 42% after lobectomy only. In this situation, Tg was found in all patients with large metastases and in 88% of those with small metastases. Following T4 withdrawal, Tg was detectable in all patients with neoplastic disease and in 13% of those in complete remission after total thyroid ablation. In conclusion, Tg measured with this IRMA method appears to be a reliable marker of differentiated thyroid carcinoma.