A Case of Pleomorphic Adenoma of the Trachea

The extent of ultraviolet (UV) irradiation-induced DNA repairwas measured in bone marrow cells and peripheral lymphocytesof patients with refractory anemia with excess of blasts (RAEB).Bone marrow cells from RAEB, when exposed to a 2 J/m dose ofUV, exhibited 50% lower incorporation of tritiated thymidinethan those of control subjects. A similar finding was observedin the peripheral lymphocytes. These data suggest that bonemarrow cells and peripheral lymphocytes from RAEB are deficientin repair of UV-induced lesions by DNA. Furthermore, this impairedDNA repair efficiency in RAEB was not related to the presenceor absence of a karyotype abnormality.     

Chromosomal instability and radiosensitivity in myelodysplastic syndrome cells.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells. To investigate whether chromosomal instability and/or DNA repair defects are involved in the development of MDS, we measured the micronucleus (MN) frequency in peripheral blood lymphocytes exposed to various doses of X-rays, using a cytokinesis-block micronucleus assay. The spontaneous MN frequencies in RAEB and RAEB-T patients were significantly higher than those in normal individuals (P = 0.0224, P = 0.008, respectively). Also, the X-ray-induced MN frequencies in RA/RARS, RAEB, and RAEB-T patients were significantly higher than those in normal individuals (P = 0.007, P = 0.003, P = 0.003, respectively, at 2 Gy). In order to elucidate the cause of unusual radiosensitivity, we measured the expression levels of nucleotide excision repair (NER) genes in peripheral blood mononuclear cells using a RT-PCR method. Reduction of NER gene expression was found in only one of 10 patients with low risk MDS, but in four of 11 patients with high risk MDS. Our data suggest that chromosomal instability and DNA repair defects may be involved in the pathophysiology of disease progression of MDS.     

A remarkable effect of K-18 (IgG-melphalan complex) in a case of RAEB with hypoplastic marrow

A 63-year-old male with refractory anemia with excess of blasts (RAEB) and hypoplastic marrow was treated with K-18 (240 mg/day P.O.). On admission, peripheral blood revealed pancytopenia. Bone marrow specimen revealed severe hypocellularity with 18.9% of the blast cells. Ten months later, the blast cells in the bone marrow decreased to 3.8%, and complete remission (CR) was obtained. CR was eight weeks. Duration of response (CR + PR) continued for about eight months. K-18 is an antitumor agent with minimal side effects, and seems to be effective for RAEB with hypoplastic marrow.     

Deficiency of UV-induced excision repair in human thymocytes

The capacity of human thymocytes and of differentiated lymphocytes circulating in peripheral blood to perform unscheduled DNA synthesis (a measure of nucleotide excision repair) after UV irradiation was measured by radioautographic analysis. Only 4% of immature T lymphocytes, but 68% of circulating lymphocytes exhibited unscheduled DNA synthesis. When UV sensitivity of peripheral blood lymphocytes and thymocytes from the same donor were compared, the thymocytes, in each case, were significantly more UV sensitive than were the circulating lymphocytes. Peripheral blood lymphocytes from subjects undergoing halothane and morphine anesthesia during surgery showed 56% less excision repair capacity than those from unanesthetized donors. The difference occurred in the number of cells capable of repair rather than in the extent of repair synthesis per cell. Ultraviolet-induced unscheduled DNA synthesis occurred in only 3% of the thymocytes removed from rats killed by cervical dislocation. Therefore, the deficiency of excision repair was observed in rat thymocytes which had not been affected by anesthesia or surgical trauma. Since the thymus contains more than 90% immature T-cells, our results indicate that immature T-cells are deficient in nucleotide excision repair whereas the majority of mature peripheral blood lymphocytes exhibit such repair.     

DNA excision-repair deficiency of human peripheral blood lymphocytes treated with chemical carcinogens.

Human peripheral blood lymphocytes stimulated with concanavalin A for 72 hr have a 10-fold greater capacity to repair DNA damage induced by N-acetoxy-2-acetylaminofluorene than do unstimulated cells. The increased capacity of concanavalin A-activated cells to repair DNA is not observed after 24 hr in culture, a time at which stimulated cells have not begun to synthesize DNA. The maximum rate of repair synthesis obtained after treatment of stimulated cells with the "large patch"-inducing agent, N-acetoxy-2-acetylaminofluorene, is twice that obtained with methyl methanesulfonate, an agent inducing "small patch" repair. The difference between the maximum rates obtained with N-acetoxy-2-acetylaminofluorene and methyl methanesulfonate is 6-fold in a human lymphoblastoid line. Unstimulated lymphocytes show almost identical rates of repair after treatment with either N-acetoxy-2-acetylaminofluorene or methyl methanesulfonate. There is close correlation between the rate of N-acetoxy-2-acetylaminofluorene-induced repair synthesis and the loss of acetylaminofluorene adducts from DNA. Treatment of lymphocytes with methyl methanesulfonate leads to degradation of cellular DNA with the production of single-stranded regions. Such degradation is not observed with N-acetoxy-2-acetylaminofluroene. We conclude that the rate of excision repair is a function of the capacity of cells for DNA synthesis and that lymphocytes that do not synthesize DNA have a limited repair capacity and cannot be used to distinguish between large and small patch repair.     

Increased fragile sites and sister chromatid exchanges in bone marrow and peripheral blood of young cigarette smokers

Cigarette smoking is considered to be the single most important acquired cause of cancer mortality. Studies of chromosome aberrations, sister chromatid exchanges, and fragile sites in peripheral blood or bone marrow are useful methods to detect the effects of the environmental mutagens or carcinogens found in cigarette smoke. The effects of smoking on the immature cells in the bone marrow have not been studied. Here, we examine the peripheral blood and bone marrow in 18 smokers (15 females and 3 males) with a median age of 25 years (range, 21-40) and an average cigarette use corresponding to 6 pack years. In both bone marrow cells and peripheral blood lymphocytes, we were able to show a significantly increased frequency of sister chromatid exchanges in smokers with a 5 or more cigarette pack year history, but not in those who smoked less than 5 pack years. We also found a higher frequency of sister chromatid exchanges in peripheral blood lymphocytes than in bone marrow cells. In addition, the peripheral lymphocytes of smokers demonstrated (a) a significantly higher frequency of fragile sites, (b) an increased number of metaphases with extensive breakage; and (c) elevated expression of fragile sites at the cancer breakpoints 3p14.2, 11q13.3, 22q12.2, and 11p13-p14.2 and at the oncogene sites bcl 1, erb B, erb A, and sis. Our results suggest that chromosomal DNA of peripheral blood lymphocytes is sensitive to cigarette smoking. Studies of the chromosomal changes in these cells provide an index of the mutagenic damage caused by these exogenous agents in individual patients and the ability of individuals to repair that damage, and might predict susceptibility to malignant events.     

Prognostic Features in the Myelodysplastic Syndromes: Importance of Morphological Atypia in the Marrow Cell Lineages

In order to clarify the relationship between myelodysplastic morphologic features of marrow cells and prognoses and 1.0 define other prognostic factors, 124 patients with the FAB criteria of myelodysplastic syndrome (MIX) were analysed. These included 57 patients with refractory anemia (RA), 5 RA with ring sideroblasts (RARS), 25 RA with excess of blasts (RAEB), 14 chronic myelomonocytic: leukemia (CMML) and 23 with RAEB in transformation (RAEB in T). Univariate analysis of all MDS patients or those of RA demonstrated that the following factors, which were not reported or fully investigated previously, were significantly associated with prognosis. These included neutrophil alkaline phosphatase (NAP) score (significant only for all MDS), the percentage of marrow erythroblasts and lymphocytes present, the percentage of cells with morphological abnormalities in individual cell lineages and the number of cell lineages showing atypia (significant for all MDS and RA).

Multiple regression analysis showed that (%) of marrow erythroblasts, NAP score, hemoglobin levels and number of marrow granulocytes with atypia were significant for predicting the prognosis of all MDS patients while the number of marrow megakaryocytes and granulocytes with atypia were significant for prognosis in the subgroup with RA.     

Development and field-test validation of an assay for DNA repair in circulating human lymphocytes

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.     

Bestatin treatment of myelodysplastic syndromes (MDS) and the effects of bestatin on hematopoiesis in MDS

A high remission rate (75%) was achieved in a preliminary study using bestatin in patients with refractory anemia with excess blasts (RAEB) and RAEB in transformation (RAEB-t). One of 2 patients with RAEB-t and 3 of 6 patients with RAEB obtained complete response. Two patients with RAEB achieved good response, but one with refractory anemia failed. Clonogenic marrow cell culture studies in patients with myelodysplastic syndromes have demonstrated intrinsic hematopoietic stem cell abnormalities, in particular defective erythroid colony formation. After bestatin treatment, these abnormalities as well as hematologic findings were markedly improved. The results suggest that bestatin has an enhancing effect on burst promoting activity production of helper (CD4 positive) T lymphocytes and the effect on hematopoiesis of bestatin may be mediated by T lymphocytes.     

低剂量甲氨蝶呤对DNA的损伤及甲酰四氢叶酸对其保护作用

背景与目的：研究多次低剂量应用甲氨蝶呤所致BALB/c小鼠淋巴细胞及骨髓细胞DNA的损伤,并探讨甲酰四氢叶酸对其损伤的保护作用。材料与方法：应用甲氨蝶呤0.5 mg/kg腹腔注射,每周1次,共8周;用单细胞凝胶电泳方法检测其外周血淋巴细胞及骨髓细胞DNA的损伤情况。结果：单用甲氨蝶呤组细胞尾长及拖尾频率与对照组相比均有显著差异,而同时应用等量的甲酰四氢叶酸,其细胞尾长及拖尾频率与对照组相比无显著差异。结论：SCGE技术可敏感地检测多次应用低剂量甲氨蝶呤造成的DNA损伤,并能反映甲酰四氢叶酸对甲氨蝶呤所致的DNA损伤的保护作用。     

Mutagen-induced disturbances in the DNA of human lymphocytes detected by antinucleoside antibodies.

The alkylating mutagens N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, and N-nitroso-methylurea induced immunoreactivity to antinucleoside antibodies in human peripheral blood lymphocytes in vitro. This could also be detected in lymphocytes taken from a patient soon after i.v. administration of cyclophosphamide. The immunoreactivity response, which indicates denatured DNA or DNA single-strand breaks, was scored by immunofluorescent or immunoperoxidase techniques. Examination of blood from 10 normal subjects showed that 32 +/- 4% (S.E.) of resting peripheral blood lymphocytes were immunoreactive to antinucleoside antibodies. We have shown that these naturally occurring immunoreactive lymphocytes are largely accounted for by a subpopulation of thymus-derived lymphocytes bearing the Fc receptor for immunoglobulin M. The presence of these cells did not interfere with the use of peripheral blood lymphocytes for in vitro measurement of additional immunoreactivity caused by alkylating mutagens. The response proved to be dose dependent; up to 90% of lymphocytes could be rendered immunoreactive. Parallel studies with HeLa cells showed a similar dose-response relationship between mutagen action and immunoreactivity. With some agents, the immunoreactivity technique detected effects at lower concentrations than could be detected by HeLa cell survival studies. With N-nitrosomethylurea, measurement of DNA repair synthesis by [3H]thymidine autoradiography showed that in HeLa cells these two parameters of response to DNA damage increased in parallel. Our results provide a new basis for detecting the action of alkylating mutagens on human lymphocytes in vitro or in vivo.     

Characterization of clonogenic cells in refractory anemia with excess of blasts (RAEB-CFU): response to recombinant hematopoietic growth factors and maturation phenotypes

The proliferative and maturation abilities of bone marrow progenitors in patients with refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) have previously been investigated in vitro using impure sources of colony stimulating activity. Here we report studies that were concerned with defining growth factor responses of RAEB progenitors (RAEB-CFU) in colony culture using pure hematopoietic growth factors. Marrow cells of 10 RAEB patients were cultured with recombinant IL3, GM-CSF, G-CSF, M-CSF and EPO. Factor dependent colony growth of four patients was examined in detail cytologically. The analysis revealed notable deficiencies in the colony forming spectrum as compared with normal marrow: although granulocytic colonies were formed in all of these four RAEB cases, macrophage colonies could not be induced in 1/4 cases and eosinophilic and erythroid colony formation could not be propagated in 2/4 cases with the proper stimuli. These findings are indicative of the intrinsic incapabilities of RAEB-CFU to mature along certain differentiation pathways in response to the growth factors. We then determined the surface phenotypes of RAEB-CFU using MoAbs Vim-2 (myelomonocytic) and B13C5 (CD34) following dual labeling and fluorescence activated cell sorting and subsequent culture of the separately sorted BI3C5+/Vim-2+, BIC5+/Vim-2-, BI3C5-/Vim-2+ and BIC5-/Vim-2- cells. In normal marrow most clonogenic cells were recovered from the BI3C5+/Vim-2- fraction. In contrast, in RAEB marrow increased proportions of the colony forming cells were BI3C5+/Vim-2+, BI3C5-/Vim-2+, or BI3C5-. The altered distribution of surface immunophenotypes of RAEB-CFU provides further evidence for the imbalance of maturation in the progenitor cell compartment. The results are discussed in view of the concept that the inabilities of the RAEB hematopoietic precursors to mature in response to the hematopoietic growth factors are partial and variable, but may culminate in a progressive loss of the differentiation competence of the progenitors when leukemia evolves.     

Deficient nucleotide excision repair activity in protein extracts from normal human lymphocytes

DNA repair activity in human peripheral blood lymphocytes (PBL)has been investigated by various techniques. Here, we reportthe use of an in vitro assay in order to assess nucleotide excisionrepair activity (NER). The mechanism of this major repair processrelies on two broad steps: first, recognition, incision andexcision of the damaged DNA; second, repair synthesis on thegapped DNA. Briefly, damaged plasmids were incubated with wholecell extracts which allows one to quantify DNA repair synthesis.When NER was determined on plasmid DNA damaged with UV-lightor cisplatin, PBL extracts showed no repair synthesis for unstimulatedlymphocytes. Using a new in vitro assay measuring only the damage-specificDNA incision activity in cell extracts, we found that the incisionstep in the repair reaction was blocked in unstimulated PBL.By mixing PBL with XP (group A, B, C, D) extracts, no restorationof NER activity was observed. In addition, these lymphocytesalso lacked DNA replication activity as determined with pre-incisedplasmid substrate. However, a phytohemagglutinin treatment ofPBL led to an extent of repair synthesis similar to that observedwith extracts from lymphoblastoid cells. When lymphocytes wereincubated in 20% serum medium with and without phytohemagglutinin,the repair activity increased dramatically after 24 h. Duringthe activation of lymphocytes, the extent of repair synthesiswas proportional to the percentage of cells in S phase of thecell cycle. Our results suggest that the blockage of the cellcycle in G0/G1 in PBL may be responsible for their lack of NERactivity.     

The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes.

The Wilms' tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.     

Analysis of prognostic factors in patients with refractory anemia with excess of blasts (RAEB) reclassified according to WHO proposal

The WHO classification subdivides the FAB RAEB category into RAEB-1 (bone marrow (BM) blasts <10%, peripheral blasts <5%) and RAEB-2 (bone marrow blasts >10% and peripheral blasts >5%). We reclassified according to WHO criteria 228 RAEB patients and analysed them in terms of haematological, karyotypic and prognostic features. We used the database of 680 MDS patients referred to our Institution from 1990 to 2000. Clinical features at presentation, such as sex, age, leukocyte count, polymorphonuclear cell count (PMN), platelet count, haemoglobin level, presence of one or more lineage dysplasia were tested in univariate and multivariate analysis in the two groups of RAEB-1 and RAEB-2 reclassified patients. In multivariate analysis we identified prognostic significant factors in the two patient groups, which consisted of age >70 years and platelet count <100 x 10(9)l(-1) for RAEB-1 category, while for RAEB-2 group parameters negatively influencing survival and risk of progression were haemoglobin <10g/dl, platelet count <100 x 10(9)l(-1), bone marrow blastosis >15% and complex karyotype. We also found differences in cytogenetic data (more balanced translocations and complex karyotypes in RAEB-2 group, p=0.02), and in survival (23.3 months in RAEB-1 vs. 16.1 months in RAEB-2 group, p=0.001). WHO classification provides valuable prognostic information for RAEB patient population, and can identify those subjects with more unfavourable prognosis who should be offered alternative therapeutic strategies.     

Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level.

Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.     

Clonal Analysis of Agnogenic Myeloid Metaplasia

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder that leads to a sustained proliferation of megakaryocytes and an increase of reticulin fibers within the bone marrow. Blood and bone marrow samples from patients with advanced AMM with fully developed myelofibrosis as well as cases in the cellular phase of the disease were investigated for clonality. Clonality was studied by X-linked restriction length polymorphism in conjunction with DNA methylation patterns. Granulocytes and total bone marrow cells proved to be monoclonal in origin whereas at least a minor portion of the peripheral lymphocytes were not clonally derived. Our findings indicate that the cellular phase of AMM as well as the fully developed disease progressed to myelofibrosis represent a monoclonal proliferation of pluripotent hematopoietic stem cells.     

Lymphoproliferative diseases of fowl: chromosome breaks caused in lymphocytes by JM-V herpesvirus.

Chromosome preparations were made of bone marrow cells and peripheral lymphocytes isolated from chicks that developed leukemia following infection with JM-V herpes-virus. Karyotypic analysis revealed a high frequency of chromosome breaks and aneuploidy, as well as some pulverization of chromosomes. The number of chromosome breaks began to increase at 2-3 days post infection, and by 5 days post infection it reached 12.7% of bone marrow cells and 17.2% of peripheral lymphocytes. Similarly, the number of aneuploid metaphase figures increased rapidly and reached 12% of bone marrow cells and 19% of peripheral lymphocytes at 5 days post infection. Some specificity was observed in the chromosomes that were affected.