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Cytogenetic analysis of esophageal squamous cell carcinoma cell lines by comparative genomic hybridization: relationship of cytogenetic aberrations to in vitro cell growth.

Cancer is characterized by autonomous growth of cells, and it is widely accepted that cell proliferation is primarily influenced by individual cell genetics. To elucidate the mechanisms of cancer cell proliferation, we studied differences in genetic aberrations for different type of tumors with different proliferation characteristics. We employed comparative genomic hybridization (CGH) to detect genetic aberrations in six cell lines of esophageal squamous cell carcinoma (ESCC). Three cell lines (YES-1, -2, and -3) grow in culture without fetal calf serum (group A), while others require serum to be maintained in vitro (group B). Both groups showed very similar cytogenetic aberrations: over-representations of 11q13 (6/6), 8q23-qter (5/6), Xq25-qter (5/6), 3q26-qter (4/6), 5p (4/6), 7p15-pter (4/6), 8q21.3-q22 (4/6), 17p (4/6), and 20q13 (4/6), and under-representations of 18q21-qter (6/6), 4q28-q33 (4/6), and 9p21 (4/6). Six amplification loci were mapped to chromosomal regions of 6q23 (1 case), 7p12 (2 cases), 9p21 (1 case), 11p11.2-12 (3 cases), 11q13 (2 cases), and 17p12 (2 cases). However, some differences were detected. DNA copy number increases at 7p12-p13, 11q14-q22, and 11q22-qter and under-representations of 4p, 8p, and 11p14-pter. In contrast, gains at 12p and 20p, and losses at 3p and 5q were detected only in group-B cell lines. These observations suggest that cytogenetic differences between the two groups may be linked to differences in cell growth characteristics in vitro, and that the genes in these chromosomal regions may play important roles in cell proliferation.

Carcinoma, Squamous Cell↗

Monitoring patients in complete cytogenetic remission after treatment of CML in chronic phase with imatinib: patterns of residual leukaemia and prognostic factors for cytogenetic relapse.

We monitored BCR-ABL transcript levels by quantitative real-time PCR in 103 patients treated with imatinib for chronic myeloid leukaemia in chronic phase for a median of 30.3 months (range 5.5-49.9) after they achieved complete cytogenetic remission (CCyR). The patients could be divided into three groups: (1) in 32 patients transcript levels continued to decline during the period of observation (nadir BCR-ABL/ABL ratio 0.015%); in five of these patients BCR-ABL transcripts became undetectable on repeated testing, (2) in 42 patients the transcript levels reached a plateau and (3) in 26 patients transcript numbers increased and the initial CCyR was lost. Three patients were not evaluable. Patients who remained in CCyR for at least 24 months appeared to have a low risk of subsequent cytogenetic relapse. We conclude that the pattern of 'residual' disease after achieving CCyR on imatinib is variable: some patients in CCyR show a progressive reduction in the level of residual disease, some reach a plateau where transcript numbers are relatively stable and others relapse with Ph-positive metaphases.

Adolescent↗

Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches.

We have characterized the cytogenetic alterations of the human embryonal cell line 293 by spectral karyotyping and G-banding analysis. To investigate its genomic stability, we compared the karyotypes of 293 and its daughter line EcR-293. Genotype profiling through short tandem repeats complemented the analysis. While displaying almost identical STR profiles and thus verifying their origin and their close relation, the two lines were remarkably different in their number of chromosomes and setup of aberrant chromosomes. However, the cell lines retained a stable karyotype in long term culture. The establishment of subclones from EcR-293, expressing inducible lacZ or MEN1 transgenes, only added minor changes to the karyotype. Our study shows that the cytogenetic constitution of a clonal cell line of the 293 origin appears to be sufficiently stable. However, care should be taken when comparing the properties of independent 293 lineages, since clonal variations might be substantial.

Cell Culture Techniques↗

Cytogenetic monitoring of human populations at risk in Egypt: role of cytogenetic data in cancer risk assessment.

Somatic mutation plays a critical role in carcinogenesis. Numerous environmental agents can increase the probability that somatic mutation will occur. The use of genotoxicity testing is essential for assessing potential human toxicity so that hazards can be prevented. Cytogenetic monitoring of human populations exposed to chemicals has proved to be a useful tool for detecting the chemical mutagenic effects. Cytogenetic analyses of human chromosomes in peripheral lymphocytes allows direct detection of mutation in somatic cells. Different methods can be used for chromosomal analysis (conventional chromosomal analysis, sister chromatid exchange, micronucleus frequency detection). Micronucleus frequency can be detected either in peripheral blood lymphocytes or in exfoliated cells. Different examples of human population studies are presented. Several problems that are found in biomonitoring studies are discussed. These studies should help us learn about individual exposure assessment and biologically relevant doses, leading to quantitative assessment of human cancer risks.

Adult↗

Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): results of the European Working Party. Groupe Français de Cytogénétique Hématologique, Groupe de Français d'Hematologie Cellulaire, UK Cancer Cytogenetics Group and BIOMED 1 European Community-Concerted Action "Molecular Cytogenetic Diagnosis in Haematological Malignancies".

Acute promyelocytic leukemia (APL) is typified by the t(15;17), generating the PML-RAR alpha fusion and predicting a beneficial response to retinoids. However, a sizeable minority of APL cases lack the classic t(15;17), prompting the establishment of the European Working Party to further characterize this group. Such cases were referred to a workshop held in Monza, Italy and subjected to morphologic, cytogenetic, and molecular review, yielding 60 evaluable patients. In the majority (42 of 60), molecular analyses revealed underlying PML/RAR alpha rearrangements due to insertions (28 of 42) or more complex mechanisms, including 3-way and simple variant translocations (14 of 42). Metaphase fluorescence in situ hybridization (FISH) demonstrated that insertions most commonly led to formation of the PML-RAR alpha fusion gene on 15q. In 11 of 60 workshop patients, PLZF/RAR alpha rearrangements were identified, including 2 patients lacking the t(11;17)(q23;q21). In one case with a normal karyotype, FISH analysis revealed insertion of RAR alpha into 11q23, and PLZF-RAR alpha was the sole fusion gene formed. Two patients were found to have t(5;17), one with a diffuse nuclear NPM staining pattern and with NPM-RAR alpha and RAR alpha-NPM transcripts detected. In the other with an unbalanced der(5)t(5;17)(q13;q21) and a nucleolar NPM localization pattern, an NPM/RAR alpha rearrangement was excluded, and FISH revealed deletion of one RAR alpha allele. In the remaining 5 workshop patients, no evidence was found for a rearrangement of RAR alpha, indicating that in rare instances, alternative mechanisms could mediate the differentiation block that typifies this disease. This study highlights the importance of combining morphologic, cytogenetic, and molecular analyses for optimal management of APL patients and better understanding of the pathogenesis of the disease. (Blood. 2000;96:1297-1308)

Adult↗

[The role of DNA repair processes in the repair of cytogenetic disorders. The participation of slow to repair DNA damage in the repair of cytogenetic disorders].

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification.

Animals↗

Interphase cytogenetics as an adjunct in the cytodiagnosis of urinary bladder carcinoma. A comparative study of cytology, flow cytometry and interphase cytogenetics in bladder washes.

To determine the role of interphase cytogenetics as an adjunct in the cytodiagnosis of transitional cell carcinoma (TCC) in bladder washes, 40 separate samples were prospectively evaluated by conventional cytology, flow cytometry (FCM) and interphase cytogenetics using fluorescence in situ hybridization (FISH) to detect numerical chromosomal abnormalities involving chromosomes 8 and 12. The cases, all of which had subsequent histologic confirmation, were composed of 26 transitional cell carcinomas and 14 benign lesions. Cytology, FCM and FISH were concordant in 19 of 31 (61%) instances. The false-negative rates of the three parameters were as follows: cytology, 38.5%; FCM, 28.5%; FISH, 27%. The false-positive rates were 0%, 20% and 0%, respectively. The relatively lower sensitivity of cytology is attributed to sampling problems since 7 of the 10 false-negative cases were histologically high grade (grades 2 and 3) and should have been detected easily by conventional cytology if tumor cells were present. Thus, the sensitivity and specificity of FISH in detecting malignant cells of TCC in bladder washes is comparable to that of conventional cytology and FCM. When, as in the present study, there are insufficient numbers of cells for FCM analysis, the detection of numerical chromosomal abnormalities by FISH studies may serve as an adjunct in the diagnosis of TCC in bladder washes.

Carcinoma, Transitional Cell↗

Cytogenetic and interphase cytogenetic characterization of atypical chronic lymphocytic leukemia carrying BCL1 translocation.

Conventional chromosome analysis (CCA) and fluorescent in situ hybridization (FISH) studies, using a 390-kb yeast artificial chromosome probe spanning the area of multiple breakpoints of the BCL1 locus at 11q13, were performed on 57 patients fulfilling the French-American-British criteria for the diagnosis of atypical B-cell chronic lymphocytic leukemia (CLL). To better define the incidence of 13q deletions and trisomy 12, FISH analysis was also performed using a cosmid probe that recognized a DNA sequence between the Rb gene and the D13S25 locus at band 13q14 and a chromosome 12-specific pericentromeric probe. All patients were characterized by cytoimmunological and hematological studies. Fourteen cases displayed three fluorescent signals in 41-98% interphase cells when hybridized to the BCL1 yeast artificial chromosome probe, documenting the presence of BCL1 translocation (BCL1-positive cases). The presence of t(11;14)(q13;q32) was ascertained in 12 cases using CCA and by dual color interphase FISH using the BCLI probe and a 14q telomere probe in 2 karyotypically normal cases. The remaining 43 cases had two signals in more than 95% interphase cells (BCL1-negative) and did not have the t(11;14) at CCA. Although 13q14 deletions were seen by means of CCA in only 5 of 14 BCL1-positive cases, hemizygous or homozygous deletions at band 13q14 were detected by FISH in 11 of 14 BCL1-positive cases, as compared with 17 of 43 BCL1-negative cases (P = 0.01). A subclone with trisomy 12 in addition to BCL1 translocation and del(13q14) was present in four BCL1-positive cases. We arrived at the following conclusions: (a) FISH with this BCL1 YAC probe is an efficient method for the detection of the t(11;14) and of the corresponding involvement of the BCL1 locus in this lymphoproliferative disorder; (b) the majority of BCL1-positive atypical CLLs by French-American-British criteria may carry 13q14 deletions; (c) the recognition of this cytogenetic subset of atypical CLL, sharing some immunological and cytogenetic features with mantle cell lymphoma, may be important, because these patients usually present isolated peripheral blood and marrow lymphocytosis, with or without mild to moderate spleen involvement, and may require early cytotoxic treatment.

Chromosomes, Artificial, Yeast↗

[Biomarkers of cytogenetic damage in humans and risk of cancer. The European Study Group on Cytogenetic Biomarkers and Health (ESCH)].

Cytogenetic assays in peripheral blood lymphocytes (PBL) have been used extensively in the last decades to survey human exposure to genotoxic agents. The conceptual basis for this approach has been the hypothesis that the extent of genetic damage in PBL reflects critical events for carcinogenic processes in target tissues. The predictive value of these tests for subsequent cancer risk has been recently evaluated by two cohort studies of cancer mortality and incidence carried out in Italy and in five North European countries. In this paper we report the update of both cohorts. In the new follow-up, a total of 64 cancer deaths out of 2019 subjects in the Italian cohort and 127 new cases of cancer out of 3182 subjects in the Nordic cohort were observed. The cytogenetic endpoints studied were CA (chromosomal aberrations), SCE (sister chromatid exchanges) and MN (micronuclei). In order to take into account the interlaboratory variation of absolute values, the results were trichotomized within each laboratory into three strata: low (1-33 percentile), medium (34-66 percentile), or high (67-100 percentile). The association between chromosomal damage and cancer risk was evaluated through SMR (standardized mortality ratio) for Italy and SIR (standardized incidence ratio) for the Nordic countries. National mortality/incidence cancer rates (age, sex and calendar-year specific) were used as reference. A linear trend of SMRs and SIRs according to CA level was found in both cohorts for the "All Cancers" cause (p < 0.01). In the Italian cohort it was also possible to analyze some specific cancer sites: a significant increase of SMR among subjects with a high level of CA with respect to the general population was found for lung cancers and lymphatic and hematopoietic tissue cancers. Contrariwise, no association between cancer mortality/incidence and SCE or MN frequency was observed. Findings from this study support the existence of an association between CA frequency and cancer risk.

Adult↗

Clinical cytogenetics and molecular cytogenetics.

The short report will be focused on helping our students to understand commonly used conventional and cutting edge cytogenetic techniques and their clinical applications, the advances and drawbacks of each technique, and how to pick the right test(s) for a specific patient in order to achieve a proper diagnosis efficiently and economically.

Chromosomes↗

Morphological and cytogenetic changes in therapy-related leukemia developed in a t(8;21)-acute myeloid leukemia (M2) patient: sequential cytogenetic and molecular analyses.

A patient with acute myeloid leukemia (AML)-M2 with t(8;21)(q22;q22) achieved complete remission with remission-induction chemotherapy followed by consolidation and intensification chemotherapies. T(8;21)(q22;q22) disappeared, but chimeric AML1/MTG8 was continuously detected in bone marrow cells. Following the development of therapy-related leukemia after 1 year, evolution of therapy-related AML-M4 with t(11;17)(q23;q25) and the rearrangement of the MLL gene were observed, while AML/MTG8 disappeared. After reinduction and following intermittent chemotherapies, a subsequent alternative transformation to AML-M2 occurred after detection of t(3;21)(q21;q22), with a break in the AML1 gene shown by interphase fluorescence in situ hybridization analysis. This leukemia transformed to AML-M4 after t(9;22)(q34;q11), with a minor BCR/ABL rearrangement, and then finally to AML-M2. This therapy-related leukemia was resistant to chemotherapy. These findings indicate that alterations in cytogenetic and molecular events caused by chemotherapeutic agents contribute to the sequential evolution of new leukemic clones with different morphology.

Adult↗

Karyotypes and cytogenetic diversity of the genus Cobitis (Pisces, Cobitidae) in Poland: a review. Cytogenetic evidence for a hybrid origin of some Cobitis triploids.

This work summarized the results of comparative chromosomal study of Cobitis fish from 13 locations in Poland. At least two distinct species C. taenia and C. elongatoides were cytogenetically recognized. The populations of C. taenia were detected in only three lakes located in northern Poland. One hybrid diploid, four triploid and four tetraploid forms were recognized among the samples. These hybrid forms or biotypes of Cobitis could not be distinguished by external characteristics. Based on the species-specific chromosome markers such as NORs, CMA3-positive and C-bands, it is hypothesized that C. taenia, C. elongatoides, and one or two other species have taken part in the origin of polyploids. Two different hybrid diploid-polyploid Cobitis complexes were distinguished. One was composed of C. taenia, two triploid hybrid forms and two tetraploids. The other complex included C. taenia and C. elongatoides, which have taken part in the origin of coexisting hybrid forms: diploid hybrid C. taenia-elongatoides, four different triploid and two tetraploid biotypes. The results obtained indicate the hypothetical mode of origin of some polyploid Cobitis forms.

Animals↗

Mosaic tetrasomy 9p in a girl with multiple congenital anomalies: cytogenetic and molecular-cytogenetic studies.

We report on a 16-year-old girl with tetrasomy 9p mosaicism. Clinical investigations disclosed a malformation syndrome with craniofacial abnormalities, dysplasia of the right clavicle, short neck with cervical ribs, patella dislocation, Dandy-Walker malformation, mental retardation and blindness. Karyotype analysis of blood lymphocytes indicated an additional marker in the size of a C-group chromosome with a large heterochromatic block in 88% of the investigated metaphases. The origin and structure of this additional marker could not be determined by chromosome banding. Application of fluorescence in situ hybridisation and comparative genomic hybridisation identified the origin of the marker chromosome, demonstrating the effectiveness of molecular-cytogenetic investigations in the diagnosis of structural and numerical chromosome abnormalities.

Abnormalities, Multiple↗

Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban.

The cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10(-7)-10(-3) M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10(-3) M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 micrograms/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 micrograms/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 +/- 0.14/cell and 13.40 +/- 0.20/cell after treatment with Gardona (2 micrograms/ml) and Dursban (4 micrograms/ml), respectively, compared with 8.2 +/- 0.19/cell and 7.6 +/- 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.

Animals↗

Biologic and clinical significance of cytogenetic and molecular cytogenetic abnormalities in benign and malignant cartilaginous lesions.

Cartilaginous neoplasms are often histologically and therapeutically challenging. Predicting biologic behavior can be difficult. In this study, 120 nonneoplastic, benign, and malignant cartilaginous lesions from 103 patients were cytogenetically analyzed in a 6-year period after short-term culture. For selected cases, fluorescent in situ hybridization (FISH) techniques using chromosome-specific probes were performed on metaphase/interphase preparations and on paraffin-embedded tissue sections. Clonal abnormalities of chromosomes 2, 3, 5, 7, 8, and 12 were most frequently observed. Involvement of chromosomes 5, 8, and 12 may be etiologically significant because of the gene localizations for the human cartilage link protein, Langer-Giedion syndrome (a rare syndrome characterized by multiple exostoses), and type II collagen (a major component of normal cartilage) respectively, to these three chromosomes. That chromosome 7 abnormalities were observed only in malignant tumors is of diagnostic value. The identity of three marker chromosomes and the significance of trisomy 7 (a finding of controversial meaning), were determined with FISH. That the presence of chromosome aberrations and increasing histologic grade strongly correlated (p = 0.001) is of prognostic importance. Moreover, complex aberrations were observed nearly exclusively in high-grade tumors (p = 0.001). The data show that nonrandom chromosome loci are aberrantly affected in cartilaginous lesions and that these abnormalities may be of significant histopathogenetic consequence. In addition, these chromosome abnormalities appear to be diagnostically and prognostically valuable in classifying and grading chondromatous neoplasms.

Adult↗

Cytogenetic and molecular cytogenetic analysis of sacral chordoma.

A specific or characteristic anomaly has not yet been identified in chordoma. Cytogenetic analysis of a sacral chordoma from a 70-year-old male revealed the following chromosomal complement: 42,XY,add(1)(p11), -3,der(4)t(4;?;?18;?)(q12;?;?;), -6, -9, -14,der(16) t(4;?;16)(q12;?;q11), der(17)add(17)(p12) t(17;18)(q11;p11),der(18)del(18)(p11)add(18)(q?)[6]/42, idem,der(9)t(6;9)(q11;p11)[4]/46,XY[10]. In situ hybridization with chromosome-specific paint probes assisted in identifying several of the rearrangements formerly designated as marker chromosomes. Although some of the anomalies observed appear unique to this case, others are similar to those previously reported in chordoma.

Aged↗

Cytogenetic hazards from agricultural chemicals. 4. Sequential screening in the barley progeny test for cytogenetic activity of some systemic fungicides and a metabolite.

Four systemic fungicides and a metabolite: Benomyl, Carbendazim, Thiophanate-methyl, Dexon and dimethyl phenylenediamine were screened in the barley progeny test for cytogenetic activity. All affected germination, seedling growth, mitotic and meiotic activity, pollen fertility and seedset in the M1 generation to different degrees; however, no chlorophyll mutations were induced and the M1 effect were much reduced in the M2 progeny. Though the results compare well with those from studies on mammalian cells, the use of these fungicides may not affect the plant breeder and the user of grain.

Chromosome Aberrations↗

Isolation of immunoresistant human glioma cell clones after selection with alloreactive cytotoxic T lymphocytes: cytogenetic and molecular cytogenetic characterization.

Intratumoral heterogeneity and genetic instability within gliomas may allow intrinsically immunoresistant (IR) cells to escape alloreactive cytotoxic T lymphocyte (aCTL) cellular immunotherapy. The potential existence of aCTL-resistant variants prompted us to investigate whether cellular immunotherapy resistant glioma models could be isolated. To generate the models, repeated intermittent or continuous selective pressure (ISP or CSP) with multiple aCTL populations was applied to a low-passage glioblastoma cell explant, 13-06-MG, obtained from a patient at initial diagnosis. IL-6 and IL-8 secretion was greater in coincubates of aCTL cells with 13-06-ISP and 13-06-CSP immunoselected cells than those with 13-06-MG parental cells. Initially, the immunoselected cells were less sensitive to aCTL lysis; however, the reduced aCTL-sensitivity was not maintained upon further selection. We therefore isolated IR clones from continuously immunoselected cells (13-06-CSP). The frequency of IR clones was 1-6 cells per 10,000 immunoselected cells. Two clones selected for further study, 13-06-IR29 and 13-06-IR30, resisted aCTL lysis in the absence of immunoselective pressure. Cytogenetic analyses revealed structural anomalies and genomic imbalances unique to the IR clones. Based on these findings, a hypothetical model is proposed that traces the origin of the IR clones to a clonal variant within the 13-06-CSP and 13-06-MG populations.

Brain Neoplasms↗