Ring chromosomes in human neoplasias.

Though reported from a wide variety of human neoplasias, ring chromosomes, in general, are a rare finding in these diseases. The majority were detected by chance when tumors were screened for chromosomal aberrations. In most cases they are a part of highly complex karyotypic alterations and therefore part of unfavourable prognostic factors. However, in some tumor entities (e.g. tumors of mesenchymal origin) they are of such high prevalence (up to 70% of these tumors) and of such extraordinary specificity that they can even serve as cytogenetic hallmarks for differential diagnosis and for prognostic purposes. The well-known technical problems in malignant cells of achieving high banding quality to define all single chromosomal alterations have severely hampered clear identification of the chromosomes involved in rings until recently. Substantial progress of ring identification could only be achieved when molecular cytogenetic techniques became available. By these techniques it could not only be shown that certain breakpoint regions nonrandomly contribute to ring rearrangements which--at least in certain malignancies--are of basic importance, but also the molecular consequences of these changes could be defined in some cases. The present review summarizes a great number of reports on a total of 760 ring chromosomes in human neoplasias at different sites, but includes only cases with clearly identified rings. In addition, the molecular consequences of ring formation are addressed wherever pertinent information has recently been presented in the literature.

Continuous intraventricular pressure monitoring for diagnosis of normal-pressure hydrocephalus.

OBJECTIVES: Normal-pressure hydrocephalus (NPH) syndrome is treatable by implantation of a cerebrospinal fluid (CSF) shunt. However, diagnosis of NPH by clinical and radiological findings alone is unreliable, and co-existing structural dementia can contribute to low success rates after shunt implantation. The aim of our study was to investigate whether long-term results after shunt implantation in NPH improve when surgical candidates are selected by continuous intraventricular pressure monitoring (CIPM). PATIENTS AND METHODS: Ninety-two consecutive patients who were admitted with suspected NPH received CIPM for 48 h including an intraventricular steady-state infusion test to determine the resistance outflow. With positive CIPM, shunt implantation was performed and the patients were prospectively followed up for 1 to 10 years (median 6.5 years). RESULTS: CIPM was negative in 37 patients. Fifty-five patients had a positive CIPM and received CSF shunt. 96.1% of them improved from gait disturbance, 77.1% from cognitive impairment and 75.7% from urinary dysfunction. Clinical improvement remained during long-term follow-up in all but 3 patients who showed a decline at 4, 5 and 7 years, respectively. CIPM-related complications (ventriculitis) occurred in only one patient. CONCLUSION: CIPM is a safe and valuable tool to establish a reliable diagnosis of NPH and to identify promising surgical candidates.

The order of PNA-FISH-detected chromosomal telomere lengths in human T-cells is rather stable, even under the influence of strong mutagens.

The nucleo-protein structure of an intact telomere protects each chromosome from being recognized as a break and subsequently being degraded. The DNA component of this structure, the telomeric repeats, undergo attrition with every cell division, as well as in response to endogenous events, like oxidative stress. Exposure to exogenous damage promotes this process and leads to growth arrest, apoptosis and eventually to malignant transformation. It was thought that the shortest chromosome ends in humans are the most susceptible ones to become dysfunctional telomeres, and have therefore an important role in cell death and cancer. Here, we show that a stable hierarchy exists in the form of a telomere length profile of the whole human karyotype. This rank order is conserved between different human cell types and individuals, is maintained throughout a lifetime, and seems to be genetically determined. As a particular feature, this telomere length profile differs only marginally when normal human cell cultures and malignant transformed cells are compared. The profile is moreover stable when these different human cells are exposed to mutagens such as bleomycin or mitomycin C. From these findings, the question arises if also the stably long telomeres have a basic biological function.

FISH studies on the telomeric regions of the T-cell acute lymphoblastic leukemia cell line CCRF-CEM.

So far, the problem of an influence of translocations on the telomeres of the involved chromosomes has not been addressed yet in human cells. Therefore, the telomeres of a karyotypically rather well characterized T-cell acute lymphoblastic leukemia (T-ALL) cell line (CCRF-CEM) with several marker chromosomes were examined using peptide nucleic acid (PNA) telomere FISH probes to compare the telomere length of these markers with that of the chromosome arms of their origin. In addition, chromosome libraries, centromeric probes, and subtelomeric DNA probes were used to further define the marker chromosomes. Two markers could be newly defined and a concise karyotype of the cell line could be obtained by these detailed examinations: 42-47,X,-X,del(5) (q35?),t(5;15)(q14;q13.2),t(8;9)(p11;p24),del(9)(:p13-->qter)/inv(9)(pter-->p12::q21-->p12::q21-->qter),+13,+20,+der(22)(p+ [HSR?])[cp]. The relative telomere length of all chromosomes showed considerable interchromosomal, intercellular, and inter-passage variation. However, it could be shown, that in four different passages of the examined cell line the observed differences between relative telomere lengths of the markers and the chromosomes of their origin, with two exceptions (short arms of del/inv9 and der22), were not significant. On the other hand, because of its mentioned variability, telomere length alone is not sufficient to reliably define the derivation of markers.

Studies on the action of mitomycin C and bleomycin on telomere lengths of human lymphocyte chromosomes.

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.

Are telomeres a specific target for mutagenic attack by cytostatics in neoplastic cells?

Damage to telomeres induced by cytostatic therapy theoretically could generate telomere shortening and, subsequently, induce an additional genomic instability in neoplastic cells. Model experiments were carried out to examine this hypothesis. Cells of the T-ALL derived cell line CCRF-CEM were exposed to various different concentrations of Bleomycin (BLM) or Mitomycin C (MMC) for various times. Telomere lengths of metaphase chromosomes of the exposed cells were compared with those without this exposure (controls). In addition, telomerase activity was determined with a TRAP assay under the given conditions using the BLM experiments as a model. Although slight changes of total telomere length could be found in single experiments, the differences between exposed and non-exposed cells were not significant. Also, a considerable telomerase activity was shown which, however, did not substantially differ between exposed and non-exposed cells. From these data it may be concluded that, at least in the examined cell line, telomeres are not a preferential target for this kind of mutagenic attack.

Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells.

The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future.

Comparative analysis of imbalances in genomic DNA and mRNA expression levels in chondrosarcoma-derived cell line FSCP-1.

Malignant cell transformation results from multiple biological alterations including chromosomal abnormalities, oncogene activation, loss of suppressor gene function and a imbalance in cell regulating processes. The aim of our study was to combine gene expression and genomic analysis to evaluate the cellular phenotype of a chondrosarcoma cell line, which is potentially a useful in vitro model system for physiological and/or neoplastic chondrocytes. cDNA-array, quantitative PCR and comparative genomic hybridization (CGH) technologies were used to analyze gene expression profiles of chondrosarcoma cell line FSCP-1 in correlation to changes of DNA copy number on corresponding chromosomal sections. Gene expression analysis revealed similarities, but also great differences in between the chondrosarcoma cell line and physiological chondrocytes. In particular the proliferative activity was up-regulated and molecules involved in matrix synthesis and turnover down-regulated. CGH analysis revealed a heterogeneous pattern of DNA gains or losses. The c-myc oncogene, located on 8q24.12-q24.13, was the only gene with a marked up-regulation located on a chromosome section with a gain of DNA copy number. The inability of the chondrosarcoma cell line FSCP-1 to maintain an adequate matrix turnover as well as a notable proliferative activity is similar to neoplastic chondrosarcoma in vivo. The limited correlation between the CGH analysis and the gene expression pattern supports the notion that also in neoplastic cells most genes are not primarily regulated by the gene dosage, but by cellular regulation pathways. However, genes such as c-myc might represent significant exceptions potentially relevant for the clinico-biological behavior of the neoplasms.

Comparative genomic hybridization (CGH): ten years of substantial progress in human solid tumor molecular cytogenetics.

Data from ten years of research using comparative genomic hybridization (CGH) for the detection of chromosomal alterations in human solid tumors are concisely reviewed. By use of a basic methodology with some variations more or less specific patterns of genomic imbalances were found in a large number of tumors of various entities. Specific gains and losses of genomic material have not only opened the way to the detection of a series of cancer-related genes but also to clinical implications. Not only several areas of basic oncogenetic research, but also differential diagnosis, prognosis of disease progression, and therapeutic decisions have profited by CGH.

Loss of 9p21 is embedded in a complex but consistent pattern of genomic imbalances in oral squamous cell carcinomas.

35 oral squamous cell carcinomas examined previously by comparative genomic hybridization (CGH) exhibited 5 up to 47 copy number alterations (CNAs). 13 of those cases showed a loss of parts of the short arm of chromosome 9, band p21 being affected in all of these cases. A highly complex but strikingly consistent pattern of genomic imbalances with an average 31.5 CNAs per tumor was associated with this deletion, and gains clearly dominated over losses of genomic material. Comparable patterns, however, could also be found in tumors with a high number of CNAs (24 CNAs) but without the deletion. Low numbers of imbalances were accompanied by low consistency of the CNA patterns. None of these latter cases showed the deletion 9p21. 66.7% of the dim(9p21)-positive tumors were of class pT4 (vs. 22% in dim(9p21)-negative cases), 77% of stage III or IV (vs. 47% in the group without the deletion), but only 8% of the dim(9p21)-positive tumors were classified as grade 3 (vs. 41% in the negative group). Other clinicopathologic features like prevalence of relapse, or survival time could not be as clearly associated with the deletion. For instance, short relapse-free survival was clearly associated with a high number of CNAs, rather independent of presence or absence of dim(9p21) in the affected tumor. From these findings it is concluded that previously found associations of 9p21 deletion with clinical parameters can reasonably be estimated only in the context of the pattern and complexity of the genomic imbalances accompanying this chromosomal loss in the examined tumors.

Thin section arrays for I-FISH analysis of chromosome-specific imbalances in squamous cell carcinomas of the head and neck.

Thin section arrays of 20 head and neck squamous cell carcinomas were studied by I-FISH for gains (including amplification) and losses of specific genomic segments. These arrays allow the examination not only of a number of tumor sections but also of the surrounding margins and of inconspicuous control tissue in one experiment. All tumor sections examined significantly differed from the inconspicuous control tissues by containing more or less extensive cell populations with aberrant signal constitutions. In no case, however, did the aberrant population constitute the whole area of the section. Gains of signals were strikingly more frequent than were losses. All tumors showed significant gains of the segments examined, the highest differences between tumor and control sections were found for the segments 9q34 and 8q24, followed by 5p15.3 and 11q13. Amplifications were most frequently found of 11q13: 8 of the 20 tumors showed amplifications in more than 20% of the nuclei, while no nucleus with more than four signals was found in any of the control tissues (control: 0%). Amplifications of the target sequences on chromosomes 8 (14 tumors) and 9 (8 tumors) were observed in low but significant percentages of nuclei, no significant cell population was detected with an amplification of 5p15.3. Fourteen tumors exhibited a significant loss of 13q14, and only 8 tumors a significant loss at any other site. In the tumor margin sections, in most cases, the margins apparently were also affected by the one or the other of the genomic changes of the pertinent primary tumor. Nevertheless, there were, in some cases, also large differences depending on the way of analysis, but also on the specific signal constitution considered. Tumor stages T3 and T4 tended to have higher frequency of nuclei with gains of 5p15.3, 8q24, and 11q13 as compared to T2 tumors and less gains of 9q34 and loss of 13q14. With the exception of 8q24 and 13q14 alterations there was also a trend to higher percentages of aberrant nuclei in the margin of T3-4 tumors vs. T2 tumors.

Comparative M-FISH and CGH analyses in sensitive and drug-resistant human T-cell acute leukemia cell lines.

Cell lines of human T-cell acute lymphoblastic leukemias (T-ALL) have gained high interest for study of mechanisms of cytostatic drug resistance. However, they should also be suited to examine the validity and reliability of molecular cytogenetic techniques in detecting genomic alterations in neoplastic cells. Therefore, comparative genomic hybridization (CGH) and 24-color-fluorescence-in-situ-hybridization (M-FISH) were applied to eight sublines of CCRF-CEM leukemia cells selected in vitro for drug resistance and to their drug-sensitive parental counterparts. All cell lines were characterized by altered chromosome numbers and by a variety of chromosomal structural aberrations as shown by M-FISH. The great majority of anomalies detected by this technique were confirmed by CGH. Interestingly, a considerable number of the rearrangements found were imbalanced. Amplifications of 5q13 in the six methotrexate-resistant cell lines, a del(9)(p21pter) in all lines examined, and a gain of chromosome 20 in 9 of the 10 lines examined were readily detected by both techniques. The same held true for losses of chromosomes 17 and 18 in the near tetraploid cell lines which could also be confirmed by CGH. Some imbalances of genomic material detected by CGH were, however, not observed by means of M-FISH, possibly due to the limited extension of the corresponding chromosomal segment involved or the small subpopulation of cells affected. On the other hand, reciprocal translocations, balanced isochromosomes, and small deletions remained mainly undetected by CGH. A comparison of chromosomal alterations in drug-resistant and parental cell lines showed not only amplifications of chromosomal segments harboring well-known drug resistance genes, e.g., the dihydrofolate reductase gene, but also chromosomal changes which may involve novel genes associated with drug resistance. Thus, the present study has clearly unveiled the strengths and weaknesses of both techniques which can excellently complement each other. Their combination allowed a distinct improvement of the definition of the complex karyotypes of drug-resistant cell lines.

Increased translocation frequency of chromosomes 7, 11 and 14 in lymphocytes from patients with neurocysticercosis.

Neurocysticercosis (NCC) has been associated with a high frequency of DNA damage in human circulating lymphocytes and more recently with the development of hematological malignancies. Chronic inflammation, a common feature of helminthic infections, has been proposed to play a key role in carcinogenesis induced by parasites. However, this mechanism is more likely to occur during local tumorigenesis rather than in systemic neoplasia such as that reported for patients with NCC. As an alternative, constant antigen stimulation, which is a feature of chronic NCC, may increase the frequency of aberrations in chromosomes that harbor regions constantly rearranged during T and B lymphocyte maturation, e.g. chromosomes 7 and 14. Therefore, in this study we determined the frequencies of aberrations in chromosomes 7, 11 and 14 in lymphocytes from 10 NCC patients and 10 controls and compared them with the frequency observed in chromosomes 1, 2 and 4 in the same cell samples. Chromosome aberrations were analyzed using a chromosome painting technique. Although the genome painted by probes for chromosomes 1, 2 and 4 was almost twice as large as that painted by probes for chromosome 7, 11 and 14, translocations involving the later (median 7.6 per 1000 metaphases) were more frequent than those occurring in chromosomes 1, 2 and 4 (median 2.5 per 1000 metaphases, P = 0.002). These results suggest that persistent antigen stimulation can cause chromosome instability in lymphocytes from patients with NCC and should be considered as an additional mechanism whereby parasites may induce cancer.

DNA amplification on chromosome 7q in squamous cell carcinoma of the tongue.

Squamous cell carcinoma of the head and neck exhibit a highly variable picture of chromosomal aberrations. In the present study the clearly defined anatomical region of the tongue was analyzed for potentially specific patterns of chromosomal alterations. Fresh tumor samples from 18 patients afflicted by squamous cell carcinoma of the tongue constituted the clinical basis of the present investigation. The tumor samples were analyzed on the basis of comparative genomic hybridization (CGH), a molecular cytogenetic FISH-approach. Gains in DNA copy numbers were detected as the predominant imbalance on chromosomes 7q (9/18), 3q (48/18), 16p (7/18) and 20q (7/18). The regions of minimal overlap on these chromosomes were mapped to 7q11.2q11.3 and 3q26. A conspicuous finding was the frequent detection of amplifications in the 7q11 region. Gains in the 7q region have been rarely reported in CGH studies of tumors derived from different regions of the head and neck. Amplifications on 7q could thus be specifically linked with the tongue region and could correlate with specific clinical factors of this tumor entity.

FISH monitoring of 100 courses of human leukemias: the cytogenetic viewpoint.

Interphase fluorescence in situ hybridization (I-FISH) analyses were performed from 2 up to 13 times along the course of 100 human leukemias (36 chronic myeloid leukemias, 38 acute myeloblastic leukemias, 17 acute lymphoblastic leukemias, and 9 additional hematopoietic neoplasias) in order to control clonality, evolution, and disappearance of the basic cytogenetic changes. The relevance of these data to confirm clinical remission or to detect minimal residual disease and/or relapse was evaluated. Fifty-four patients were monitored following hematopoietic stem cell transplantation, and 46 cases after chemotherapy. Various chromosome or aberration specific DNA probes were applied for follow-up in the time frame of 1 month up to 13 years. From the cytogenetic point of view, the aim was to determine the power of resolution of the I-FISH technique in the detection of clinically significant changes in the course of disease and its usefulness in daily routine cyto-genetics as compared with classical cytogenetics. In addition, reliability standards of the various DNA probes should be established. In 75 patients with remissions during the entire period of I-FISH monitoring no conspicuous signal constitution was detected. Of 25 relapses or progresses of disease, which were clinically confirmed, 22 were reliably detected by I-FISH, in only 1 case I-FISH monitoring failed to detect the aberrant clone. In 2 patients conventional karyotype analysis confirmed the relapse by detecting complex chromosomal aberrations, but specific probes for I-FISH confirmation were not available. These data suggest that I-FISH analyses in the follow-up of leukemias is a simple and in most cases sufficiently sensitive and highly reliable way of monitoring the outcome of therapy. It may well serve to close the gap between conventional karyotyping and the highly sensitive molecular techniques.

Chromosome painting for cytogenetic monitoring of occupationally exposed and non-exposed groups of human individuals.

The suitability of a three-color fluorescence in situ suppression hybridization technique was examined for monitoring five different groups of individuals: 30 occupied in radiology, 26 occupied in nuclear medicine or radiation physics, 32 patients with breast cancer, 26 occupied with military waste disposal, all presumably exposed to low doses of radiation or chemical mutagens and a non-exposed control group (N=29). The average frequency of breaks constituting the various aberrations did not significantly differ between the groups of medical radiation appliers and the control group. However, breast tumor patients and military waste disposers, as groups, showed a higher aberration rate than did healthy controls. Stable rearrangements mainly characterized the groups of controls, tumor patients, and radiation appliers, while a higher proportion of unstable aberrations was found in the chemically exposed individuals. Individuals with an increased frequency of aberrations could be detected within each examined group, which clearly determined the average values of the whole group. With respect to interchromosomal distribution of the breakpoints constituting the found aberrations and the involvement of the labeled chromosomes in rearrangements, the observed values were very close to the expected ones in the controls. A rather similar trend of deviations from expectation was observed in all other groups. Chromosome 4 was slightly over-affected, while chromosome 2 was slightly underrepresented in all analyzed groups (except tumor patients). Rearrangements of the labeled chromosomes with the unlabeled ones exceeded expectation. In conclusion, chromosome painting if included in further attempts of human population monitoring will broaden the basis of argumentation with respect to health risks introduced by mutagen exposure.

The cytogenetic view of standard comparative genomic hybridization (CGH): deletions of 20q in human leukemia as a measure of the sensitivity of the technique.

BACKGROUND: The limits of the resolving power of comparative genomic hybridization (CGH) have been given as 10-20 Mbp if at least 50% of the studied neoplastic cell population carried the corresponding aberration. MATERIAL AND METHODS: Genomic DNA of five cases of hematologic neoplasias, in all of which--among other anomalies--deletions of different size of chromosome 20q were found by GTG banding and confirmed by FISH analyses, was subjected to CGH. RESULTS: CGH revealed four types of del(20q), and, in addition, detected a tiny terminal del(3p) in one of the cases. The size of the smallest deleted segment, clearly visible by eye on the CGH metaphase image, was estimated to range between 5 and 7 Mbp. CONCLUSION: Visual determination was shown to have a stronger resolving power in CGH than software used for the analysis in one case, while in another one, the results obtained from the ratio profiles would have been considered insignificant without the knowledge of the hybridization pattern on the corresponding CGH metaphase images. The potential of the standard CGH technique not only to detect, but visualize small segmental aneusomies as well, suggests that its resolution actually mirrors the resolution of banding techniques.

Comparative genomic hybridization-aided unraveling of complex karyotypes in human hematopoietic neoplasias.

The information obtained by conventional cytogenetics (CC) in human leukemias is sometimes limited, in particular by complex karyotypes with many marker chromosomes. While CC is restricted to metaphases with a good quality, interphase fluorescence in situ hybridization (I-FISH) is also capable of analyzing specific anomalies in the interphase nuclei. Comparative genomic hybridization (CGH) gives additional information about the imbalanced karyotype changes in the whole genome. The aim of this study was to assess the contribution of CGH to the unraveling of complex GTG karyotypes, which are difficult to evaluate by banding analysis, and to compare these results with those by CC and FISH. Thirteen bone marrow samples and one sample obtained from peripheral blood of 13 leukemia patients were examined by CC, FISH and CGH. The GTG banding analysis showed complex karyotypes with many marker chromosomes. The most frequent abnormalities were numerical and structural aberrations on chromosomes 5 and 7. In 12 of the 14 samples, the CGH analysis was able to detect chromosomal imbalances with losses of material on chromosome 5 and 7 as the most frequent aberrations. In all 14 samples, additional FISH analyses were performed. For most of the studied neoplasias, a close correlation between CC, FISH and CGH data was observed. CGH was considerably helpful in adding additional information to classical karyotyping, if the low quality or number of metaphases was insufficient for a reliable CC analysis. Even in cases where whole chromosome painting could be applied, it added information on the breakpoints of the observed rearrangements. In only 2 of the studied 14 samples, neither CGH nor I-FISH could improve the result of karyotyping. CGH, nevertheless, can be regarded as a powerful additional technique in leukemias with unsuccessful CC, incomplete, or complex karyotypes with many marker chromosomes. A systematic analysis by three techniques such as CC, FISH and CGH guarantees an optimal genetic characterization of the neoplasias.

Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma.

INTRODUCTION: Cytokines of the gp130-family, particularly interleukin(IL)-6, play a crucial role in the propagation of malignant plasma cells. MATERIALS AND METHODS: The role of IL-6 and other gp130-cytokines was studied in the human plasma cell line INA-6 in vitro and in INA-6 xenografts. The proliferative response to gp130-cytokines was evaluated and activated components of gp130-signaling pathways were identified by Western blotting and DNA binding studies. Specifically, expression of IL-6 and receptors for IL-6 and leukemia inhibitory factor were analysed by RT-PCR and ELISA. RESULTS: The plasma cell line INA-6 was cultured for several years remaining strictly dependent on exogenous IL-6. Other gp130-cytokines had no significant effect on INA-6 cell proliferation in vitro. Due to an activating mutation in the N-ras gene, mitogen-activated protein kinases (MAPK) were constitutively phosphorylated. In contrast, signal transducer and activator of transcription(STAT)-3 activation was dependent on stimulation with IL-6. Blocking of either one of these pathways resulted in a significant decrease of INA-6 cell proliferation. Remarkably, INA-6 xenografts did not require exogeneous IL-6 for proliferation in vivo. Instead, an autocrine IL-6 loop and, in certain tumor sublines, responsiveness to additional gp130-cytokines was induced during in vivo growth. CONCLUSION: Activation of the gp130 signal transducer is mandatory for INA-6 cell growth in vitro and in vivo. Both the MAPK and the Jak/STAT pathway are operative in malignant plasma cells and either one is essential for plasma cell growth. The INA-6 cell line provides a preclinical model to study growth regulation of human plasmacytoma cells and to evaluate novel therapeutic strategies.