Investigation of new cytogenetic biomarkers specific to high-LET radiation using in vivo and in vitro exposed human lymphocytes.

PURPOSE: To find detectable cytogenetic biomarkers that can offer information about the radiation quality of in vivo exposure retrospectively. MATERIALS AND METHODS: Chromosome-type aberrations of peripheral lymphocytes of uterine cancer patients that received internal gamma- and external X-ray therapy or carbon beam therapy and of victims severely exposed to neutrons and gamma-rays in a criticality accident that occurred in Tokai-mura, Japan were analysed. Data obtained from in vitro irradiation experiments using 60Co gamma-rays and 10 MeV neutrons were compared with the in vivo exposure data. RESULTS: The ratio of acentric rings to dicentric chromosomes (termed RaD ratio) and that of excess fragments to dicentrics (termed EfD ratio) showed significant (p < 0.05) differences between the two groups of cancer patients, and these ratios for accidental victims were in between the values of the two groups of cancer patients. The in vitro studies using doses equivalent to 1 - 3 Gy of gamma-rays have confirmed that the EfD ratios were increased with the high LET (linear energy transfer) and RaD ratios decreased. CONCLUSION: The present data show that the RaD and EfD ratios can be used as cytogenetic biomarkers of exposure to high-LET radiation at least within a few years of exposure.

Refractory anemia with severe dysplasia: clinical significance of morphological features in refractory anemia.

Refractory anemia (RA) in myelodysplastic syndromes (MDS) are very heterogeneous diseases regarding their morphology, clinical features and survival. We proposed the new designations 'RA with severe dysplasia (RASD)' and 'RA with minimal dysplasia (RAminiD)'. In our criteria, RASD is considered present if a bone marrow (BM) examination shows Pseudo-Pelger-Huet anomalies of mature neutrophils > or =3% and/or micromegakaryocytes (mMgk) of megakaryocytes > or =10% in RA patients. RAminiD is defined as RA cases other than RASD. After the reclassification of 58 primary RA patients, the group was composed of 45 RAminiD and 13 RASD patients. The blast percentage in the BM and the frequency of cytogenetic abnormalities observed in the RASD patients were intermediate between those in the RAminiD and RAEB patients. The analysis of survival curves revealed differences among the three groups; the RASD patients had lower survival probabilities than those of the RAminiD group, and significantly higher probabilities than those of the RAEB group. (RAminiD vs RASD, P=0.06; RASD vs RAEB, P=0.004.) Our data indicate that in RA patients, RASD is a distinct subset of RA with an unfavorable clinical outcome.

New system for assessing the prognosis of refractory anemia patients.

Refractory anemia (RA) is a very heterogeneous disease regarding biological and clinical features. The International Prognostic Scoring System (IPSS) was useful for assessing the prognosis in the whole group of 219 myelodysplastic syndrome (MDS) patients. However, the IPSS was not sufficient in 132 RA patients. To predict survival and freedom from acute myeloid leukemia (AML) evolution, we investigated individual prognostic factors based on the clinical parameters (age, gender, morphologic features, cytopenias and cytogenetics) of 132 RA patients using univariate and multivariate analyses. Based on the results, we devised a new system for assessing the prognosis of RA patients. In our system, RA patients with pseudo-Pelger-Huët anomalies >/=3% were classified as high risk (12 patients); of patients without pseudo-Pelger-Huët anomalies >/=3%, those with intermediate/poor karyotype according to IPSS, Hb /=10% were classified as intermediate risk (57 patients); and those without high or intermediate risk were classified as low risk (67 patients). In our system, the analyses of both survival times and leukemia-free survival times revealed significant differences among the three groups (P < 0.0001).

Effect of high-level natural radiation on chromosomes of residents in southern China.

To study the effect of low-dose (rate) radiation on human health, we analyzed chromosomes of peripheral lymphocytes of residents in a high background radiation area (HBRA) and compared the results with those obtained from residents in a control area (CA) in Guangdong Province, China. Unstable types of chromosome aberrations (dicentrics and rings) were studied in 22 members of eight families in HBRA and 17 members of five families in CA. Each family consists of three generations. On average 2,600 cells per subject were analyzed. 27 adults and six children in HBRA and 25 adults and eight children in CA were studied with respect to translocations. On average 4,741 cells per subject were examined. We found an increase of the frequency of dicentrics and rings in HBRA, where the natural radiation level is three to five times higher than in the control area. But the increase of translocations in HBRA was within the range of individual variation in the controls.

A hematological remission by clonal hematopoiesis after treatment with recombinant human granulocyte-macrophage colony-stimulating factor and erythropoietin in a patient with therapy-related myelodysplastic syndrome.

We treated a patient with therapy-related myelodysplastic syndrome (MDS) with human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo). The patient achieved a hematological remission which continued for more than 16 months despite discontinuation of the treatment. Before the treatment with GM-CSF and Epo the patient had severe pancytopenia, required frequent red cell transfusions, and experienced episodes of severe infection, but after the therapy he no longer needed transfusion and no longer had the infection. While the patient remained in hematological remission, bone marrow examination revealed trilineage dysplasia, and cytogenetic analysis showed an abnormal karyotype [48, XY, +8, +der(1q5p)] in 100% of the metaphases examined. These findings suggest that the hematological remission of this patient may not result from the recovery of the non-clonal hematopoiesis of a normal clone, but result from the monoclonal hematopoiesis of a neoplastic clone.

Chromosome aberrations in bone marrow cells from Japanese patients with thorotrastosis.

Exposure of bone marrow cells to alpha-particle radiation causes various types of chromosome abnormalities and hematological malignancies. We performed chromosome analysis of hematopoietic stem cells from the bone marrow of 52 Japanese patients with thorotrastosis and 21 age-matched controls. The frequency of cells with stable chromosome abnormalities was significantly higher in the patients with thorotrastosis. Further studies found 14 clonal chromosome aberrations in cells from 11 patients (21.2%); clones observed in the cells from 2 of these patients had high frequencies of chromosome abnormalities. In one case, 68 to 100% of the cells analyzed had a large partial loss in the short arm of chromosome 1 and a translocation between the short arms of chromosomes 2 and 3 [46,XY,1p-,t(2p+;3p-)]. The cells from the other patient contained a clone with partial loss of both the short and long arms of chromosome 5 (46,XX,5p-,5q-). The frequency of this clone has been constant for the last 15 years (6-24%). We also analyzed bone marrow mononuclear cells from 17 of the patients for mutations of the TP53 tumor suppressor gene (formerly known as p53). However, no mutation was found in any of the cells, including those from the 2 patients with abnormal clones. Moreover, repeated medical examinations showed no evidence of leukemia or myelodysplasia in these patients. Our study suggests that exposure of bone marrow cells to alpha-particle radiation may induce clonal chromosomal aberrations at a high frequency.

Establishment of novel cell lines derived from two patients with chronic myelogenous leukemia in blast crisis; IMS-BC1 and IMS-BC2 which exhibit markedly different sensitivity to apoptosis.

We established two novel cell lines, designated as IMS-BC1 and IMS-BC2, from two patients with chronic myelogenous leukemia in blast crisis. The two cell lines were positive for CD13 and CD33 and negative for CD34 and HLA-DR by surface marker analysis. IMS-BC1 had four Philadelphia (Ph1) chromosomes and a breakpoint within the 3'-portion of M-bcr, and IMS-BC2 had five Ph1 chromosomes and two breakpoints within the 3'- and 5'-portions of M-bcr. Both cell lines' growth activities were moderately suppressed by IFN-alpha. The proliferation of IMS-BC2 was inhibited by IFN-gamma and apoptosis was induced within 72 h, while IMS-BC1 was resistant to IFN-gamma. Fibronectin inhibited the proliferation of the two cell lines at higher than 10 micrograms/ml, but only IMS-BC2 showed apoptosis. Transforming growth factor-beta inhibited the proliferation of IMS-BC2 resulting in apoptosis, while it inhibited that of IMS-BC1 moderately but failed to induce apoptosis. All-trans retinoic acid (ATRA) inhibited the proliferation of IMS-BC2 at very low concentration (10(-17) mol/l) and induced apoptosis at doses higher than 10(-9) mol/l within 72 h without terminal differentiation, while IMS-BC1 was completely resistant to ATRA. The two cell lines showed different responses to growth inhibitory cytokines and factors. These cell lines should prove useful in the analysis of mechanisms of apoptosis induced by growth inhibitory cytokines and factors.

Translocation (8;21) and its variants in acute nonlymphocytic leukemia. The relative importance of chromosomes 8 and 21 to the genesis of the disease.

Chromosome analysis was performed in 25 patients with acute nonlymphocytic leukemia (ANLL), mostly of the M2 type. Eighteen had the standard translocation, t(8;21)(q22;q22), four had complex translocations involving 1p36, 11p13, 17p11, and 17p23, respectively, with chromosomes 8 and 21, and the remaining three patients had simple translocations, one with t(3;21)(p14;q22) and two with t(16;21)(p11;q22), without involving chromosome 8. Chromosome abnormalities additional to t(8;21) and its variants that were most frequently observed were -X, -Y, and del(9). Complex translocations are thought to be derived from the standard translocation and to be essentially similar in nature. The finding that chromosome 21 was involved in all of the standard, simple, and complex translocations, and that chromosome 8 was not involved in simple variants suggest a greater weight of chromosome 21 in the relative importance of the two chromosomes to the genesis of ANLL.

The Philadelphia chromosome. Considerations based on studies of variant Ph translocations.

The nature of the Philadelphia (Ph) translocation and the process of its formation were studied by attempting various chromosome banding analyses of variant Ph translocations among 210 patients with Ph-positive chronic myelocytic leukemia examined at the National Institute of Radiological Sciences, Chiba. The following assumptions could be drawn from the results of the analyses: 1) The involvement of specific regions of chromosomes #9 and #22, q34 and q11, respectively, is an indispensable condition of the Ph translocation. 2) The so-called variant Ph translocations are all complex and are derived from a standard Ph translocation. 3) The Ph translocations, both standard and complex ones, are not always stable. The complex translocations are subject to further chromosome evolution, as is the conversion of the standard translocation to complex translocations. There seems to be no fundamental difference between the standard and complex Ph translocations, with the latter being merely a more progressed form of the former. Analyses at the molecular level of the same cases employed in this study are yielding results that support the above assumptions.

Radiation exposure and chromosome abnormalities. Human cytogenetic studies at the National Institute of Radiological Sciences, Japan, 1963-1988.

The results of human cytogenetic studies performed at the National Institute of Radiological Sciences (NIRS), Chiba, Japan for about 25 years are described. The studies were pursued primarily under two major projects: one involving people exposed to radiation under various conditions and the other involving patients with malignant diseases, especially leukemias. Whereas chromosome abnormalities in radiation-exposed people are excellent indicators of radiation exposure, their behavior in bone marrow provide useful information for a better understanding of chromosome abnormalities in leukemias and related disorders. The role of chromosome abnormalities in the genesis and development of leukemia and related disorders is considered, suggesting a view for future studies in this field.

Chromosomal in situ hybridization and Southern blot analyses using c-abl, c-sis, or bcr probe in chronic myelogenous leukemia cells with variant Philadelphia translocations.

The Philadelphia (Ph) chromosome is a cytogenetic hallmark of chronic myelogenous leukemia (CML). Whereas the majority of Ph-positive CML patients show the standard Ph translocation involving chromosomes 9 and 22, t(9;22)(q34;q11), the minority of cases exhibit a variant type of Ph translocation involving these two and other chromosomes (complex type) or those involving #22 and chromosomes other than #9 (simple type). To get an insight into the nature of variant Ph translocations and the process of their formation, we examined the localization of the c-abl and c-sis oncogenes and the breakpoint cluster region (bcr) gene by chromosomal in situ hybridization in ten variant Ph translocations of CML including five simple and five complex ones as initially interpreted. In situ hybridization showed that c-abl localized to band 9q34 and c-sis localized to band 22q12-q13 were translocated on the Ph and on one of the rearranged chromosomes other than #9, respectively, in all the variant translocations examined. On the other hand, bcr localized to band 22q11 was translocated on various chromosomes but mostly on chromosome 9. Parallel Southern blot analyses on DNA from leukemic cells of five patients including two with simple translocations and three with complex ones revealed rearrangements of bcr with breakpoints occurring mostly in a 5' portion of 5.8-kb BamHI/BglII sequences, which are quite similar to those detected so far in CML cases with the standard Ph translocation. The present findings strongly suggest that variant Ph translocations of CML are all complex, and some of them are formed stepwisely from the standard translocation.

A culture technique for chromosome analysis in human myeloid leukemias.

A simple culture technique for the study of cells from myeloid leukemias and other blood disorders of myeloid series is described. The procedure is the same with that of the ordinary cultures, except for the addition of colony stimulating factor. A large number of mitotic cells can be obtained, and they are quite suitable for banding treatments. Clear banding patterns are consistently obtained.

Acute nonlymphocytic leukemia following lung cancer in a patient with a constitutional supernumerary chromosome.

A patient with a constitutional bisatellited supernumerary marker chromosome developed a large cell lung carcinoma and subsequent acute nonlymphocytic leukemia (ANLL) of the M2 type showing an (8;21) translocation and del(9). The ANLL-M2 appeared to be independent of the lung carcinoma. The presence of the supernumerary chromosome might have been associated with the development of the two diseases.

Heritable fragile sites and cancer: fra(16)(q22) in lymphocytes of an acute nonlymphocytic leukemia patient with inv(16)(p13q22).

Fragile site testing was performed on normal peripheral blood lymphocytes from three acute nonlymphocytic leukemia patients who carried inv(16)(p13q22) in malignant cells. Cultures were treated with BrdU, distamycin A, Hoechst 33258, or folic acid deprivation to induce fragile site expression. One patient was found to be a carrier of fra(16)(q22), but the expression was observed only by Hoechst 33258 treatment.

Heritable rare fragile sites in patients with leukemia and other hematologic disorders.

Fragile site studies were performed on a total of 126 patients with leukemia and other hematologic disorders including myelodysplastic syndrome (MDS) and polycythemia vera (PV). Compared with an incidence (6.0%) of heritable rare fragile sites in the healthy population, the frequency was not higher in the patient group (3.2%), as a whole. However, two cases of fra(17)(p12) in MDS appeared fourfold larger than expected for this group of patients. In one case, a homozygous carrier of fra(17)(p12) in PV was also very rarely expected from its population incidence. These findings suggested a possible role of rare fragile sites, at least in the etiology of these preleukemic or myeloproliferative disorders.

Distamycin A-inducible fragile sites and cancer proneness.

To determine the baseline frequency of autosomal rare fragile sites in cancer patients, we conducted a population cytogenetic study of 370 patients with leukemias, solid tumors, and other neoplastic disorders. Twenty carriers of rare fragile sites were detected in this patient group. The rare autosomal fragile sites were at fra(8)(q24), fra(11)(p15), fra(16)(p12.1), fra(16)(q22), and fra(17)(p12). All of these fragile sites were found to be distamycin A inducible. Compared with a population incidence in healthy subjects (44 of 845, 5.21%), the overall incidence of distamycin A-inducible fragile sites was not higher in the patient group (20 of 370, 5.41%). Analysis of these individual fragile sites and particular diseases, however, suggests that the distamycin A-inducible fragile sites may play a role in the etiology of leukemia, myeloproliferative disorders, and benign tumors.

Dynamic analysis of chromosome aberrations in three victims of the Tokai-mura criticality accident.

PURPOSE: To investigate the dynamics of chromosome aberrations in the blood cells of three workers severely exposed to neutrons and gamma-rays in a criticality accident that occurred in Tokai-mura, Japan, in 1999. MATERIALS AND METHODS: The change with time of the frequency of' chromosome aberrations in the three patients was examined using a new analysis to score drug-induced prematurely condensed ring chromosomes (PCC-R) and a conventional meta-phase analysis. RESULTS: The frequencies and cellular distributions of PCC-R, dicentrics and rings did not change significantly among the samples obtained at 9-48h after the accident while the first depletion of lymphocytes occurred. The distributions of these aberrations in the cells of two patients showed a slight overdispersion compared with a Poisson distribution reflecting neutron and non-uniform exposures. The dose response curve of rings paralleled that of dicentrics, but not PCC-R. The half-lives of PCC-R (8.5 months) and of rings (8.7 months) were shorter than that of dicentrics (13.5 months). CONCLUSIONS: In the three patients of the Tokai-mura accident, lymphocytes in the circulating and extravascular pools had reached equilibrium at 9h, and highly damaged lymphocytes did not selectively move away from the circulatory system during the first rapid depletion of lymphocytes after exposure. Data on the in vivo half-life of PCC-R as well as dicentrics and rings obtained in the present study may be useful for retrospective dosimetry.

Non-fluorescent chromosome painting using the peroxidase/diaminobenzidine (DAB) reaction.

PURPOSE: To develop and validate non-fluorescent chromosome painting for bright-field microscopy using the peroxidase/diaminobenzidine (DAB) reaction. MATERIALS AND METHODS: Peripheral blood lymphocytes were taken from patients with uterine cancer who had received heavy-ion radiation therapy. Chromosome slides were treated with RNase and pepsin, denatured mildly, hybridized with a biotinylated DNA probe specific for whole-chromosome 4 and stained using the peroxidase/DAB reaction with an avidin-biotin amplification. The slides were analysed under a bright-field microscope and an atomic force microscope. The detection rate of chromosome aberrations by DAB painting was compared with that obtained by dual analysis of Giemsa staining and FISH painting. RESULTS: When chromosomes 4 were painted, 11.5% of unstable aberrations were detected by DAB painting, while 10.8% of them were found by dual analysis of Giemsa staining and FISH painting. CONCLUSION: A DAB painting method that can effectively detect rearranged aberrations was established. It has advantages over FISH painting: the preparations can be analysed by bright-field microscope, can be preserved permanently and are suitable for analysis by an automated system.

Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy.

Heavy ion radiation (high linear energy transfer, LET, radiation) induces various types of chromosome aberration. In this report, we describe a new method employing an atomic force microscope (AFM) for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. Metaphase mouse chromosomes with chromatid gap or chromatid breaks induced by heavy ion irradiation were marked under a light microscope. Then the detailed structure of chromosomes of Giemsa-stained or unstained samples was visualized by the AFM. The height data of chromosomes obtained by AFM provided useful information to distinguish chromatid gaps and breaks. A fibrous structure was observed on the unstained chromosome, the average width of which was about 45.8 nm in the image of AFM. These structures were considered to be 30-nm fibers on the chromosome. The structure of the break point regions induced by neon- or carbon-ion irradiation was imaged by AFM. In some cases, the fibrous structure of break points was detected by AFM imaging after carbon ion irradiation. These observations indicated that AFM is a useful tool for analysis of chromosome aberrations induced by heavy ion radiation.

Cytogenetical dose estimation for 3 severely exposed patients in the JCO criticality accident in Tokai-mura.

A dose estimation by chromosome analysis was performed on the 3 severely exposed patients in the Tokai-mura criticality accident. Drastically reduced lymphocyte counts suggested that the whole-body dose of radiation which they had been exposed to was unprecedentedly high. Because the number of lymphocytes in the white blood cells in two patients was very low, we could not culture and harvest cells by the conventional method. To collect the number of lymphocytes necessary for chromosome preparation, we processed blood samples by a modified method, called the high-yield chromosome preparation method. With this technique, we could culture and harvest cells, and then make air-dried chromosome slides. We applied a new dose-estimation method involving an artificially induced prematurely condensed ring chromosome, the PCC-ring method, to estimate an unusually high dose with a short time. The estimated doses by the PCC-ring method were in fairly good accordance with those by the conventional dicentric and ring chromosome (Dic+R) method. The biologically estimated dose was comparable with that estimated by a physical method. As far as we know, the estimated dose of the most severely exposed patient in the present study is the highest recorded among that chromosome analyses have been able to estimate in humans.