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.

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.

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.

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.

Chromosome translocation in residents of the high background radiation areas in southern China.

We performed a cytogenetical study using chromosome painting analysis on 9 residents of the naturally high background radiation areas (HBRA) and 8 residents of the control areas in southern China. The estimated dose (air kerma) of each resident measured by an electric pocket dosimeter showed 2.20-4.23 mGy/year in HBRA and 0.56-0.70 mGy/year in the control areas. A total of 14,096 cells (1,566 cells/case) in the former and 17,522 cells (2,190 cells/case) in the latter were analyzed. Children, both in HBRA and in the control areas, had translocations at low frequencies. The frequency of translocations among elder individuals varied widely and it was not possible to detect dose effect although it was detected in dicentrics. The effect of radiation on the induction of chromosome aberrations, which have a statistically potential risk of causing malignant or congenital diseases, seems to be less significant than those of metabolic factors and/or mutagenic agents (excluding radiation) even in HBRA in China.

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.

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).

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.

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.

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.

Induction of asymmetrical type of chromosomal aberrations in cultured human lymphocytes by ion beams of different energies at varying LET from HIMAC and RRC.

Frequencies of asymmetrical type of chromosome aberration were scored in cultured human blood lymphocytes irradiated with carbon and neon beams. Blood cells were irradiated with various doses to establish dose response curves for chromosome aberration frequency vs. dose, and chromosome preparation was made by conventional method. Dose response curves for the per cell frequencies of the dicentrics and centric rings as well as the excess amount of acentric fragments were described for 7 different qualities (LET = 22.4, 40.0, 41.5, 69.9, 70.0, 100.0 and 150 KeV/micrometer) of carbon and neon beams with three different energies, 135, 290 and 400 MeV/u. From the analysis of those dose response curves, the maximum effect was found in the region of LET value at near 70 KeV/micrometer together with linear expression in the response from all endpoints examined. The 135 MeV/u of carbons (69.9 KeV/micrometer) and neons(70.0 KeV/micrometer) showed linear response. The 290 MeV/u of carbons (100 KeV/m) and neons (150 KeV/micrometer) showed medium effects with different shape of response, linear with a plateau and upward concavity. The 2 carbon beams (41.5 and 40 KeV/micrometer) from 2 different accelerators showed much discrepancy in the response. RBE-LET relationship was also described by comparing the coefficient alpha of the 7 different dose responses. The peak (near 70 KeV/m) was localized close to that (80 KeV/m) for the survivals of dsb repair deficient cells (Eguchi-Kasai et al. 1998), but in different position from that previously reported in many other studies (100-200 KeV/mm). Identification of the RBEmax in the present study has yet to be definitive.

Bone marrow patterns in patients with aplastic anaemia and myelodysplastic syndrome: observations with magnetic resonance imaging.

Bone marrow magnetic resonance imaging (MRI) was obtained in 48 patients with myelodysplastic syndrome (MDS) (35 cases) or aplastic anaemia (AA) (13 cases). The lower thoracic and lumbar spine were evaluated on sagittal plane using a 1.5 Tesla superconducting MR unit with a surface coil. Pulse sequence of STIRs (TR 2000 msec, TI 160 msec, TE 20 msec) were applied. Four distinct patterns of signal intensity (SI) on the STIR images were classified as follows: pattern 1, homogeneously low SI; 2, marginally high SI; 3, heterogeneously high SI; 4, homogeneously high SI. In all 13 patients with AA, STIR images initially revealed pattern 1. In 25 of 35 cases with MDS patients, the STIR images were initially classified as pattern 3. The STIR images of 6 AA and 5 MDS patients with a clinical response to treatment showed pattern 2 similar to that of normal marrow distribution. The STIR images of MDS patients showed an abnormal distribution of SI. Significant signal changes in the STIR images can be observed in successive examinations of the patients, thus facilitating follow-up of the disease and treatment. MRI of the bone marrow provides a noninvasive means of grossly examining a large fraction and is a useful technique in patients with aplastic anaemia or myelodysplastic syndrome.

Sideroblastic anemia associated with multiple myeloma in Turner's syndrome.

A 67-year-old female was admitted to our hospital because of pancytopenia. Forty-six percent of erythroblasts in the bone marrow were ringed sideroblasts. Laboratory findings showed an IgG-kappa monoclonal gammopathy. She was diagnosed as having sideroblastic anemia associated with multiple myeloma in mosaic (45, X/46, XX/47, XXX) Turner's syndrome. There was no response to therapy. The chromosomal pattern of the patient was varied, and was accompanied by the development of refractory anemia with an excess of blasts from refractory anemia with ringed sideroblast 4 months after presentation. Cytogenetic studies suggested that the abnormal clone was restricted to the monosomic cell line.

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.

Chromosomal insertion and amplification of human papillomavirus 16 DNA sequences in a cell line of argyrophil small cell carcinoma of the uterine cervix.

The chromosomal location of human papillomavirus (HPV) 16 DNA sequences integrated in a cell line derived from argyrophil small cell carcinoma of the uterine cervix was determined by means of fluorescence in situ hybridization (FISH). The HPV 16 DNA sequences were integrated near a fragile site and the location of the c-myc oncogene at 8q24.1. Amplification of the integrated viral sequences resulted in an abnormally banded region. The amplified HPV 16 DNA sequences were also detected in every interphase nucleus by FISH.

Transformation of bilineal hybrid acute leukemia to acute lymphoid leukemia: a case report with serial analyses of cytogenetics and gene rearrangement.

A 17-year-old male with bilineal hybrid acute leukemia is described. Two-color flow cytometric analysis of blast surface phenotype revealed that there were two groups of blasts which showed either CD 10+ CD 19+ CD 13- CD 33- or CD 10- CD 19- CD 13+ CD 33+, but not both. He developed a complete remission by treatment with vincristine, prednisolone, adriamycin, and L-asparaginase. After 8 months, however, leukemia relapsed and lymphoid blasts were dominant. Cytogenetic analysis at presentation showed 46,XY,t(3;9)(p21;p22), and at relapse it showed 46,XY,t(1;3;9)(1pter----1q32::3p25----3pter;3 qter----3p21::9p22----9pter; 9qter----9p22::3p21----3p25::1q32----1q ter),t(2;19)(p21;q13). Analysis of the heavy chain joining region at diagnosis showed three hybridizing bands, all rearranged, but at relapse only one rearranged band. Analysis of the constant region for the beta T-cell receptor gene (TCR beta) both at diagnosis and at relapse showed one rearranged and one germline band, suggesting that rearrangement of one allele of TCR beta of not only lymphoid but also myeloid blasts occurred. It is considered that the target cell of lymphoid leukemia cells and that of myeloid leukemia cells at diagnosis were the same, which differentiated to two lineages, and the clone which evolved from lymphoid lineage proliferated at relapse.

[Prominent lymphadenopathy and double Ph1 chromosomes as initial and recurrent manifestations of chronic myelogenous leukemia in blast crisis: report of a case and review of the literature].

A 60-year-old woman was admitted because of fatigue. Physical examination revealed prominent peripheral lymphadenopathy, marked tonsillar swelling and hepatosplenomegaly. The leukocyte count was 68,900/microliters with 75% lymphoid blasts and 5% basophils. The karyotype of the blood cells was 46, XX, Ph1/47, XX, Ph1, +Ph1. The diagnosis of CML in blast crisis was made. After chemotherapy using adriamycin, cyclophosphamide, vincristine, and prednisolone (CHOP), lymphadenopathy and splenomegaly reduced and lymphoid blasts disappeared from the blood and bone marrow. At that time only single Ph1 (46, XX, Ph1) clone was detected in her bone marrow. Four months later, hematological relapse accompanied by lymphadenopathy occurred and DNA analysis of the blasts showed the rearrangement of bcr gene. The simultaneous chromosomal analyses of the blood, bone marrow and lymph node revealed that almost all cells examined had the karyotype "47, XX, Ph1, + Ph1". In spite of repeated chemotherapy the patient did not improve and died. This case suggests a relationship between lymphadenopathy and double Ph1 chromosomes in CML.

Granulocyte colony-stimulating factor-dependent growth of an acute myeloblastic leukemia cell line.

We report herein the establishment and characterization of a granulocyte colony-stimulating factor (G-CSF)-dependent acute myeloblastic leukemia (AML) cell line. The cell line, designated as OCI/AML 1a, has been cultured in the presence of G-CSF and has shown exponential growth for over two years. The cells growing in suspension culture resembled myeloblasts on the basis of morphologic, cytochemical and surface phenotypic analyses. Other CSFs, interleukin-3 and granulocyte-macrophage colony-stimulating factor did not support the growth of OCI/AML 1a cells so well as G-CSF. The effect on the growth of OCI/AML 1a cells of G-CSF was almost completely abolished by neutralizing monoclonal anti-G-CSF antibody. These findings showed that OCI/AML 1a cells required G-CSF for growth. OCI/AML 1a cell line will be valuable for studies of the biological nature, proliferation and differentiation of leukemic cells. Furthermore, OCI/AML 1a cells should be useful for determining the mechanism by which G-CSF induces the growth of hemopoietic cells.