Occurrence of acute myeloid leukemia in hydroxyurea-treated sickle cell disease patient.

Hydroxyurea (HU) has been widely used in sickle cell disease. Its potential long-term risk for carcinogenesis or leukemogenic risk remains undefined. Here, we report a 26 y old African-American female with Sickle Cell Disease (SCD) who developed refractory/relapsed acute myeloid leukemia (AML) 6 months after 26 months of HU use. That patient's cytogenetics and molecular genetics analyses demonstrated a complex mutation profile with 5q deletion, trisomy 8, and P53 deletion (deletion of 17p13.1). P53 gene sequence studies revealed a multitude of somatic mutations that most suggest a treatment-related etiology. The above-mentioned data indicates that the patient may have developed acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) as a direct result of HU exposure.

Outcomes of first-line treatment for chronic lymphocytic leukemia with 17p deletion.

Although uncommon in treatment-naive patients with chronic lymphocytic leukemia, deletion 17p is a high-risk disease characteristic. We analyzed and reported outcomes for 63 patients with deletion 17p chronic lymphocytic leukemia who received first-line therapy at our institution; at time of first treatment, 81% had unmutated immunoglobulin heavy chain variable gene and 58% had complex karyotype. Forty-nine patients (76%) received first-line fludarabine, cyclophosphamide, rituximab-based therapy, 6 (11%) received rituximab-based and 8 (13%) received lenalidomide-based treatment. Overall, the complete plus nodular partial remission rate was 33%; on multivariable model, higher complete plus nodular partial remission rate was observed in patients with less than 50% cells positive for deletion 17p, and a higher probability of achieving at least a partial remission was observed with fludarabine, cyclophosphamide, rituximab-based treatment. After a median follow up of 33 months (range 1-89 months), the estimated median progression-free survival was 14 months (95% confidence interval 10-18) and estimated median overall survival was 63 months (95% confidence interval 43-83). In multivariable analysis, factors independently associated with longer progression-free survival were response to treatment and absence of complex karyotype. Achievement of complete plus nodular partial remission rate and mutated immunoglobulin heavy chain variable gene were independently associated with longer overall survival in multivariable model. Complex karyotype was associated with increased risk for Richter's transformation. New first-line strategies and agents must aim at both improving response and maintaining remission in patients with deletion 17p, particularly in the presence of complex karyotype.

Gamma-irradiation induces HER-2/neu overexpression in breast cancer cell lines and sensitivity to treatment with trastuzumab.

PURPOSE: Overexpression of human epidermal growth factor receptor-2 (HER-2/neu) in breast cancer patients is a prerequisite for treatment with trastuzumab. In the present study, we demonstrate by fluorescence in situ hybridization (FISH) analysis that HER-2/neu gene amplification and chromosome 17 (CEP17) polysomy can be induced by irradiation in human breast cancer cell lines with low basal level of HER-2/neu. MATERIALS AND METHODS: The irradiation-induced HER-2/neu gene amplification and CEP17 polysomy enhanced HER-2/neu at the protein level in both human MDA-MB-231 and MDA-MB-435 breast cancer cell lines which was determined by immunohistochemistry and fluorescence analysis and was correlated with mRNA levels. RESULTS: Irradiation affected to a high degree the responsiveness of both cell lines to in vitro treatment with trastuzumab. The direct antiproliferative effect of trastuzumab, as well as its capacity to induce natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), was considerably higher in the irradiated tumor cells compared to their non-irradiated counterparts. CONCLUSION: Our data demonstrate that irradiation induces HER-2/neu gene amplification and CEP17 polysomy thereby enhancing expression of this protein in breast cancer cell lines rendering them susceptible to treatment with trastuzumab. They also suggest that patients with HER-2/neu negative inoperable tumors undergoing local radiation therapy may benefit from treatment with trastuzumab.

Chronic alcohol exposure induces genome damage measured using the cytokinesis-block micronucleus cytome assay and aneuploidy in human B lymphoblastoid cell lines.

Excessive alcohol consumption is associated with an increased risk of a variety of cancers. The specific association between alcohol consumption and increased risk of breast cancer has been a consistent finding in numerous studies to date; however, the biological mechanism remains unknown. One possibility is that alcohol induces chromosome instability and aneuploidy events commonly seen in cancer. The cytokinesis-block micronucleus cytome assay was used to assess the ability of alcohol to induce DNA damage and aneuploidy in two human B lymphoblastoid cell lines--WIL2-NS and GM13705. The cells were treated chronically with physiologically relevant levels of alcohol (0.36 and 1.35% v/v) for a period of 6 weeks. Results demonstrate that in these cell lines, chronic treatment with alcohol induces micronuclei, nucleoplasmic bridges and nuclear buds, indicative of the various genome damaging events of chromosome loss and breakage, asymmetric chromosome rearrangement and gene amplification, respectively. Using chromogenic in situ hybridisation, we measured chromosome 17 aneuploidy in these cell lines. Results from this assay indicate that chronic treatment of alcohol induces aneuploidy (measured as chromosome 17 aneuploidy) in both cell lines. The results from this study support the hypothesis that alcohol is a probable cause of cancer initiation by inducing chromosomal instability and aneuploidy, which may be a result of multiple indirect mechanisms.

Comparative genomic hybridization study of arsenic-exposed and non-arsenic-exposed urinary transitional cell carcinoma.

To compare the differences in DNA aberrations between arsenic-exposed and non-arsenic-exposed transitional cell carcinoma (TCC), we analyzed 19 arsenic-exposed and 29 non-arsenic-exposed urinary TCCs from Chi-Mei Hospital using comparative genomic hybridization. DNA aberrations were detected in 42 TCCs including 19 arsenic-exposed and 23 non-arsenic-exposed TCCs. Arsenic-exposed TCCs had more changes than unexposed TCCs (mean+/-SD, 6.6+/-2.9 vs. 2.9+/-2.2). Arsenic exposure was significantly associated with the number of DNA aberrations after adjustment for tumor stage, tumor grade and cigarette smoking in multiple regression analysis. The most frequent DNA gains, which were strikingly different between arsenic-exposed and non-arsenic-exposed TCCs, included those at 1p, 4p, 4q and 8q. A much higher frequency of DNA losses in arsenic-exposed TCCs compared with non-arsenic-exposed TCCs was observed in 10q, 11p and 17p. Chromosomal loss in 17p13 was associated not only with arsenic exposure, but also with tumor stage and grade. The p53 immunohistochemistry staining showed that chromosome 17p13 loss was associated with either p53 no expression (25%) or p53 overexpression (75%). The findings suggest that long-term arsenic exposure may increase the chromosome abnormality in TCC, and 17p loss plays an important role in arsenic-induced urinary carcinogenesis.

Mutagen sensitivity and neoplastic progression in patients with Barrett's esophagus: a prospective analysis.

BACKGROUND: Defects in DNA damage recognition and repair have been associated with a wide variety of cancers. We conducted a prospective study to determine whether mutagen sensitivity, as determined by an in vitro assay, was associated with the future development of cancer in patients with Barrett's esophagus, which is associated with increased risk of progression to esophageal adenocarcinoma. METHODS: We measured sensitivity to bleomycin in peripheral blood lymphocytes in a cohort of 220 patients with Barrett's esophagus. We followed these patients for 1,230 person-years (range, 3 months to 10.1 years; median, 6.4 years), using development of cancer and aneuploidy as end points. A subset of these patients was evaluated for inactivation of tumor-suppressor genes CDKN2A/p16 and TP53 [by mutation and loss of heterozygosity (LOH)] in their Barrett's segments at the time of, or before, the bleomycin test, and the patients were stratified by CDKN2A/p16 and TP53 status in an analysis of mutagen sensitivity and progression. RESULTS: Bleomycin-sensitive patients were found to be at significantly greater risk of developing aneuploidy (adjusted hazard ratio, 3.71; 95% confidence interval, 1.44-9.53) and nonsignificantly greater risk of cancer (adjusted hazard ratio, 1.63; 95% confidence interval, 0.71-3.75). Among patients with detectable LOH at the TP53 locus (on chromosome 17p), increasing bleomycin sensitivity was associated with increased risk of developing cancer (P(trend) < 0.001) and aneuploidy (P(trend) = 0.005). CONCLUSIONS: This study supports the hypothesis that sensitivity to mutagens increases the risk of neoplastic progression in persons with Barrett's esophagus, particularly those with 17p LOH including TP53.

Okadaic acid: chromosomal non-disjunction analysis in human lymphocytes and study of aneugenic pathway in CHO-K1 cells.

Okadaic acid (OA) is the main marine toxin implicated in the diarrhetic shellfish poisoning (DSP) in humans after consumption of contaminated bivalve molluscs. We have previously shown that OA was an in vitro aneugenic compound that induced chromosome loss via micronuclei formation in CHO-K1 cells. The aims of this study were to investigate the chromosomal non-disjunction (ND) potential of OA in human lymphocytes and the pathways involved for aneuploidy in CHO-K1 cells. Firstly, we analysed the formation of micronuclei and the non-disjunction for chromosomes 1 and 17 in binucleated human lymphocytes cells with the cytokinesis-blocked micronucleus (CBMN) assay coupled to a fluorescent in situ hybridization (FISH) technique with centromere-specific DNA probes. We showed that OA statistically increased the frequency of micronucleated lymphocytes in the dose range from 20 to 35 nM. However, FISH analysis did not reveal any increase in the non-disjunction for both chromosomes whatever the concentration between 2.5 and 35 nM. However, a significant increase in ND for the chromosome 17 was found at 1 nM. Secondly, in CHO-K1 cells, we investigated the dose and time dependent effects of OA: (i) on cell cycle progression, (ii) on mitotic-phase arrest and (ii) on mitotic spindle and centrosome abnormalities. The results showed that OA induced a progressive accumulation of mitotic CHO-K1 cells in prometaphase, an induction of multipolar mitotic spindle with centrosome amplification and the formation of multinucleated cells. We concluded that OA did not induce chromosome non-disjunction but should more likely induced chromosome loss in human lymphocytes. Moreover, our results obtained in CHO-K1 suggest that OA induced aneuploidy by preventing the chromosome attachment to the mitotic spindle and by amplifying the centrosome. The mode of action of the toxin in relation to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A) and the mitosis process is discussed.

Therapy-related erythroleukemia caused by the administration of UFT and mitomycin C in a patient with colon cancer.

A 54-year-old man with colon cancer underwent hemicolectomy. He received postoperative adjuvant chemotherapy with UFT (tegafur/uracil at a 1 : 4 molar ratio) and mitomycin C (MMC) for 3 years. Three years and 4 months after the start of chemotherapy, pancytopenia was noted. Bone marrow aspiration smear demonstrated an increased number of immature erythroblasts, including megaloblasts and myeloblasts. Chromosomal analysis demonstrated structural and numerical abnormalities of 5, 7, 15, and 17. Therapy-related erythroleukemia, acute myeloid leukemia (AML), M6, was diagnosed. The disease progressed after 5 months, and the patient was received chemotherapy with cytosine arabinoside, aclacinomycin, and granulocyte colony-stimulating factor (CAG), and showed a partial hematological response. Careful monitoring for the generation of therapy-related leukemia is needed when UFT and MMC are used for postoperative adjuvant chemotherapy for colorectal cancer.

Balanced chromosome abnormalities inv(16) and t(15;17) in therapy-related myelodysplastic syndromes and acute leukemia: report from an international workshop.

The Workshop identified 48 unselected patients with therapy-related myelodysplastic syndrome or acute myeloid leukemia (t-MDS/t-AML) and inv(16), and 41 patients with t(15;17) after chemotherapy (CT) and/or radiotherapy (RT) for a malignant or nonmalignant disease. The primary diseases were: breast cancer, 33 patients; lymphomas, 24 patients; various other solid tumors, 30 patients; and nonmalignant diseases, 2 patients. The general type of previous therapy was RT only in 10 patients with an inv(16) and in 12 patients with a t(15;17), alkylating agents plus topoisomerase II inhibitors in 24 patients with an inv(16) and in 18 patients with a t(15;17), topoisomerase II inhibitors only in 5 patients with an inv(16) and in 2 patients with a t(15;17), alkylating agents only in 6 patients in each subgroup, and other types of chemotherapy in 3 patients in each subgroup. Most CT-treated patients (69%) also received RT. The latency period to development of t-MDS/t-AML was short: a median of 22 months in patients with inv(16) and 29 months in patients with t(15;17). Twenty-six patients (54%) with an inv(16) and 17 patients (41%) with a t(15;17) had additional cytogenetic abnormalities, which were unrelated to age and survival in both subgroups. Trisomy of chromosomes 8, 21, and 22 and del(7q) were the most frequent additional abnormalities in the inv(16) subgroup, whereas +8, -5, and del(16q) were most frequent in the t(15;17) subgroup. The disease was overt t-AML in 38/48 patients (79%) with an inv(16) and in 38/41 patients (93%) with a t(15;17). Thirty-three of 39 intensively treated patients (85%) with an inv(16) obtained a complete remission, whereas 24 of 35 intensively treated patients (69%) with a t(15;17) obtained a complete remission. The median overall survival of intensively treated patients was 29 months in both cytogenetic subgroups. In the inv(16) subgroup, patients younger than 55 years of age had a longer survival when compared with older patients (P = 0.006). The study supports the observation that t-MDS/t-AML with inv(16) and t(15;17) is often associated with prior therapy with topoisomerase II inhibitors; however, a notable finding was the high frequency of treatment with only radiotherapy, 29% of t(15;17) and 21% of inv(16). Response rates to intensive chemotherapy in this study were comparable to those of de novo disease.

Emergence of unusual cytogenetic abnormalities under interferon-alpha therapy in patients with chronic myelogenous leukemia.

New treatments that may change the course of a disease or have potential carcinogenicity can result in the emergence of new cytogenetic or clinical disorders. We report here the cytogenetic evolution of 52 cases of Philadelphia (Ph)-positive myelogenous leukemia (CML) receiving interferon-alpha (IFN-alpha) therapy compared with that of 59 Ph-positive CML cases treated with busulfan (BU) or hydroxyurea (HY). Twenty-one percent of the CML patients receiving IFN-alpha displayed unusual secondary abnormalities, among which alteration of the long arm of chromosome 3, del(7), and del(9) were recurrent. The frequency of these unusual secondary changes was significantly higher than in CML cases after Bu or Hy treatment (P = 0.02). Three of the 11 IFN-alpha-treated CML patients displayed cytogenetic evolution in the chronic phase and, in two cases, the cytogenetic findings were transient, inasmuch as they disappeared upon withdrawal of IFN-alpha. In addition, a majority of cytogenetic abnormalities involved chromosome 3 at bands 3q21 and 3q26, which corresponds to the locus of EVI1, a gene implicated in the development or progression of human myeloid leukemias. Possible explanations include: toxicity of IFN-alpha by effect on bone marrow stroma, immune-modulating effects of IFN-alpha, and mutagenic effects of IFN-alpha. The mechanisms underlying these cytogenetic changes remain to be elucidated. However, our data suggest that IFN-alpha induces additional cytogenetic abnormalities even in the chronic phase through its immune-modulating effects and that these unusual cytogenetic abnormalities do not alter the history of CML.

[Chromosome damage in the course of laryngeal squamous cell carcinoma]. / Uszkodzenia chromosomów w przebiegu plaskonablonkowego raka krtani.

The aim of the article is a review of own cytogenic studies on laryngeal cancer confronted with the literature data. Spontaneous and bleomycin-induced chromosome instability was analysed in peripheral blood lymphocytes in relation to genetic risk of cancer incidence and progression. Comparative genome hybridization (CGH) was applied to demonstrate gains and losses of DNA copy number in tumour and non-tumour laryngeal mucosa. The profiles of imbalances of DNA copy number were shown to differ between metastazing and non-metastazing tumours. Preliminary data indicate a frequent loss of Y chromosome in tumour cells. The loss of heterozygosity at chromosome p53 locus (17p) has been shown to be more frequent than at chromosome locus coding 16 gene (9p). Altogether, the experiments have proven that a dynamics of chromosome aberrations is highest at the stage of metastasis.

Targeted breakage of paracentromeric heterochromatin induces chromosomal instability.

Current models suggest that genomic instability is crucial in the accumulation of the multiple alterations required for tumorigenesis. However, the nature of the initial damage responsible for the origin of genomic instability remains poorly understood. In this investigation we demonstrate that the nucleotide analog 2,6-diaminopurine (DAP) can be used to induce highly focused damage to the large blocks of paracentromeric heterochromatin on chromosomes 1, 9 and 16. A large fraction of cells exposed to DAP exhibit undercondensation of alpha and classical heterochromatin which persists into metaphase. Subsequent chromosome breakage was observed for one of the target chromosomes by preferential exclusion of chromosome 16 fragments into micronuclei (P < 0.0001). The specificity of DAP-induced chromosomal breakage enabled us to utilize it as a reagent to demonstrate that paracentromeric heterochromatin is a sensitive target for the induction of persistent genomic instability. We observed a 100-fold increase in mutagenesis affecting a chromosome 16 marker (APRT) compared with marker loci on chromosomes 17 (TK) or X (HPRT). We previously reported that APRT- mutants were recovered at a high rate upon selection in DAP in a process involving recombinationally mediated loss of heterozygosity that extends from the telomere to the boundary region of the paracentromeric heterochromatin. Karyotypic analysis of DAP-resistant APRT- mutant clones demonstrated extensive genomic instability, particularly evidence of multiple and sequential events affecting chromosome 16. These data suggest that the heterochromatic breakage observed cytogenetically immediately following DAP exposure is also responsible for the initiation of persistent genomic instability.

Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes.

Risk assessment from exposure to spindle inhibitors should take into account the possibility of threshold concentration-response curves for aneuploidy induction. We analysed concentration-dependent induction of chromosome non-disjunction by well known spindle poisons (colchicine, carbendazim, mebendazole and nocodazole) and a reference clastogen, methyl methanesulphonate (MMS) in vitro in human lymphocytes; and integrated these findings with earlier results of chromosome loss in micronuclei. Chromosome non-disjunction was estimated on cytokinesis-blocked lymphocytes after simultaneous fluorescent in situ hybridization labelling with two chromosome-specific centromeric probes (chromosomes 1 and 17). The frequencies of spontaneous non-disjunction showed important inter-individual variations and were surprisingly high (7.04-15.39%). Lower concentrations of aneugens did not induce a statistically significant increase of non-disjunction frequencies over the respective control levels, whereas higher concentrations clearly induced a concentration-dependent increase in the non-disjunction frequencies with the four aneugens tested. On the contrary, even at high concentrations, MMS induced a slight increase in the frequency of non-disjunction but without being statistically significant when compared with the control frequencies. We estimated the inflection points, the first statistically significant concentrations, the last non-statistically significant concentrations and the number of events from concentration-response curves of chromosome non-disjunction and chromosome loss. A threshold-type of concentration-response for non-disjunction is highly probable for colchicine and nocodazole. For carbendazim and mebendazole the inflection point fell above the first statistically significant concentrations. But since it is obvious from dose-response curves where the inflection point/threshold lies, it appears that the model might be picking up some irregularities (possibly due to experimental variability in the dose-response curve at concentrations greater than the threshold). For accurate estimation of the threshold, analysis of more concentrations or more cells might be needed. Our data strongly indicate that in cultured human lymphocytes chromosome non-disjunction is a major mechanism of aneuploidy induction by spindle inhibitors and since non-disjunction occurs at lower concentration than chromosome loss, the aneuploidy threshold should be estimated on the basis of non-disjunction rather than on micronuclei frequencies (chromosome loss).

Molecular mechanisms of spontaneous and induced loss of heterozygosity in human cells in vitro.

The human TK6 lymphoblast cell line is heteroallelic at the thymidine kinase (TK) locus, with one functional and one nonfunctional allele. Cells that have undergone loss of heterozygosity (LOH) at TK can be selected and cloned in an in vitro assay. In order to study the extent of LOH, we have analyzed a total of 166 thymidine kinase-deficient mutants that arose either spontaneously or following induction by X-ray or ethyl methane sulfonate (EMS) using DNA probes in and around the TK gene on chromosome 17. Two distinct groups of mutants with different doubling times were identified. Among slow-growth mutants, the predominant change for both spontaneous and induced mutants was LOH that generally extended through the entire TK gene to both proximal and distal markers on 17q. While the majority of both spontaneous and X-ray-induced normal-growth mutants showed LOH, this was considerably more localized in scale for X-ray-induced mutants, which rarely involved the distal marker. LOH was rare among EMS-induced normal-growth mutants. LOH was never observed with a 17p marker, indicating that nondisjunctional events were not involved in any of the mutant clones examined. Densitometric analysis of the LOH mutants indicated mitotic recombination was a likely mechanism in more than half the spontaneous LOH mutants in both groups, whereas most induced mutants appeared to arise from simple deletions.

Chromosome aberrations induced by etoposide (VP-16) are not random.

The clastogenic effect of etoposide, an anti-cancer chemotherapeutic drug, was investigated in vitro on lymphocytes of 5 healthy donors. The analysis of the first division metaphases arising after mutagenesis in G1 phase shows that chromosome-type aberrations are much more frequent than chromatid-type lesions. The distribution in relation to chromosome lengths of the 439 breakpoints that were accurately identified is not random: chromosomes 1, 11 and 17 are most frequently involved, while chromosomes 4, 5 and X are seldom affected. This non-random distribution may be related to chromosome structure, since R-band-rich chromosomes are significantly more affected than G-band-rich chromosomes.