Nucleotide-metabolism and chromosome alterations in human-malignant melanoma xenografts

The karyotypes of human melanomas exhibit multiple chromosome alterations. Recurrent deletions of 9p, 10q and 14q arms, which carry genes encoding for enzymes of purine metabolism, were also found in human gliomas, another neuroectodermal tumor previously studied for both cytogenetics and nucleotides metabolism. Postulating that this metabolism might also be modified in melanomas, the activities of eleven enzymes involved in catabolic and synthetic pathways of purine metabolism were measured, in addition to two enzymes of the pyrimidine synthesis. Assays were performed on six melanoma mestastases, five nodal and one cutaneous, after transplantation into nude mice. The purine metabolism was characterized by a more active catabolic than synthetic pathway, a possible imbalance between de novo and salvage pathways for adenylates synthesis, rather in favor of the de novo pathway, and a more active adenylate than guanylate synthesis. The skin metastasis exhibited quite different cytogenetic and metabolic patterns, when compared to the nodal metastases. Considering the relationships between cytogenetic and metabolic data, low activities of methylthioadenosine phosphorylase, adenosine kinase, adenosine monophosphate deaminase, nucleoside phosphorylase and 5'-nucleotidase were observed in melanomas, as well as frequent losses of 9p, 10q, Ip, 14q and 6q arms respectively carrying genes encoding for these enzymes, most of these rearrangements were confirmed by chromosome painting. The two enzymes exhibiting the highest activities were adenosine deaminase and adenylosuccinate lyase, encoded by genes mapped on chromosomes 20 and 22 respectively, frequently in excess in melanomas. Thus, for these tumors, the metabolic pattern roughly parallels the cytogenetic profile, even if the absence of case to case correlation suggests that gene dosage effect, if it occurs, is not the only parameter involved. The main enzymatic and cytogenetic difference between melanomas and gliomas, concerns both adenylosuccinate lyase activity and the balance of chromosome 22, high in melanomas and low in gliomas.     

Activity of thymidylate synthetase, thymidine kinase and galactokinase in primary and xenografted human colorectal cancers in relation to their chromosomal patterns

The relationship between chromosome anomalies and metabolic modifications in human colorectal cancers grafted into nude mice was studied. Two distinct chromosomal patterns have been demonstrated i.e., monosomic type (MT) characterized by multiple chromosome losses or deletions always involving chromosome 18, and trisomic type (TT) characterized by progressive gains of chromosomes. Grafted tumors conserve original karyotypes observed on corresponding primary tumors. Most changes involve the loss of chromosomes carrying genes encoding for enzymes of the de novo pathways and the gain of chromosomes carrying genes encoding for enzymes of the salvage pathways of nucleotide synthesis. In MT tumors the long arm (q) of chromosome 17 is frequently duplicated in association with a deletion of the short arm, forming an isochromosome 17q. The activities of 3 enzymes, thymidylate synthetase (TS) mapped on chromosome 18, thymidine kinase (TK) and galactokinase (GalK), both mapped on chromosome 17q, were studied. TS is a de novo enzyme and TK and GalK are salvage enzymes. A clear correlation could be demonstrated between tumor types and enzyme activities: MT tumors had lower TS and higher TK and GalK activities than TT tumors. These differences were too large to result from a gene dosage effect only. These data suggest that serial studies on grafted colorectal cancers give a better representation of metabolic disturbances than studies on fresh tumor samples, usually contaminated by non-cancerous cells.     

Cytogenetic abnormalities in human ependymomas

The karyotypes of 4 human ependymomas have been determined. In one ependymoma, translocations involving chromosomes 9, 17 and 22 were observed together with the loss of the normal chromosome 17. A second ependymoma had many chromosomal alterations that included a translocation between chromosomes 1 and 2 and re-arrangements involving chromosome 17. No major consistent alterations were detected in the remaining 2 cases. Our karyotypes do not resemble the few previously published karyotypes of ependymomas, but had features, such as the alterations involving chromosome 17, that were similar to those of other brain tumours in children.     

Purine metabolism of human glioblastoma in vivo

The aim of this study was to identify targets for rational chemotherapy of glioblastoma. In order to elucidate differences in the biochemistry of tumor and normal human brain, in vivo pool sizes of purine nucleotides, nucleosides, and nucleobases and of purine metabolizing enzymes in biopsy material from 14 grade IV astrocytomas and 4 normal temporal lobe samples were analyzed. Specimens were collected during surgery using the freeze-clamp sampling technique and analyzed by high pressure liquid chromatography. Total purine nucleotides, adenylates, and guanylates in the tumors were 2186, 1865, and 310 nmol/g (wet weight), respectively, which corresponds to 61, 60, and 71% of normal brain tissue concentrations. Relative to normal brain the tumors had significantly lower ATP and GTP levels, essentially normal pool sizes of purine nucleosides and bases, unchanged activities of the salvage enzymes hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, and adenosine kinase (659, 456, and 98 nmol/h/mg protein, respectively) and 4-fold higher activities of IMP dehydrogenase (11.6 nmol/h/mg protein); the latter is the rate limiting enzyme for guanylate de novo synthesis. IMP pools in the tumors were 64% of values in normal brain. Modulation of the guanylate pathway in glioblastoma by inhibition of IMP dehydrogenase with tumor specific agents such as tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) appears to be a rational therapeutic approach. Preliminary in vitro experiments with normal and malignant tissue specimens from 2 additional patients revealed that significant amounts of the active metabolite thiazole-4-carboxamide adenine dinucleotide are formed from tiazofurin. At a concentration of 200 microM this drug was able to deplete guanylate pools in the tumors to a median of 54% of phosphate buffered saline treated controls. Flux studies with [14C]formate showed that tiazofurin strongly inhibited de novo synthesis of guanylates in glioblastoma to an average of 10% of controls. This effect was more pronounced in the tumors as compared to normal brain. No inhibition of salvage of [14C]guanine by tiazofurin could be observed in normal and malignant tissues. Supportive measures have to be considered to inhibit the highly active salvage enzyme hypoxanthine-guanine phosphoribosyltransferase that can partly antagonize a tiazofurin induced decrease in guanine nucleotides.     

Karyotypic abnormalities in tumours of the pancreas.

Short-term cultures from 20 pancreatic tumours, three endocrine and 17 exocrine, were cytogenetically analysed. All three endocrine tumours had a normal chromosome complement. Clonal chromosome aberrations were detected in 13 of the 17 exocrine tumours: simple karyotypic changes were found in five carcinomas and numerous numerical and/or structural changes in eight. When the present findings and those previously reported by our group were viewed in conjunction, the most common numerical imbalances among the 22 karyotypically abnormal pancreatic carcinomas thus available for evaluation turned out to be, in order of falling frequency, -18, -Y, +20, +7, +11 and -12. Imbalances brought about by structural changes most frequently affected chromosomes 1 (losses in 1p but especially gains of 1q), 8 (in particular 8q gains but also 8p losses), and 17 (mostly 17q gain but also loss of 17p). Chromosomal bands 1p32, 1q10, 6q21, 7p22, 8p21, 8q11, 14p11, 15q10-11, and 17q11 were the most common breakpoint sites affected by the structural rearrangements. Abnormal karyotypes were detected more frequently in poorly differentiated and anaplastic carcinomas than in moderately and well differentiated tumours.     

Cytogenetic and spectral karyotype analyses of benign and malignant cartilage tumours

To date, there have been few studies published on benign and malignant cartilage tumours using high resolution molecular cytogenetic techniques such as spectral karyotyping (SKY). In this study we have used a combination of chromosome banding, SKY and FISH to characterize the chromosomal pattern in 18 benign and malignant cartilage tumours and one small cell osteosarcoma with mesenchymal chondrosarcoma-like features. Clonal structural and/or numerical aberrations were detected in 14 of these tumours. All chondroblastomas and the chondromyxoid fibroma had diploid or near-diploid karyotypes with often relatively simple karyotypes. Although no consistent abnormalities were detected in the chondroblastomas, recurrent breakpoints were found at 2q35, 3q21-23, and 18q21. The chondromyxoid fibroma had an inv(6)(p25q13) as the sole anomaly, suggesting that this is a primary abnormality characteristic of this entity. The karyotypic findings in the chondrosarcomas were, as a rule, more complex than those in the benign tumours. A typical feature was the frequent occurrence of unbalanced rearrangements leading to genomic imbalances with losses and gains of certain chromosomes or chromosome regions. The following breakpoints were recurrent: Xq21, 6p10, 9p13, 20p11 and 22q11-12. Despite the use of high-resolution molecular cytogenetic techniques, we were not able to identify any consistent abnormalities in chondrosarcomas, suggesting that tumour-specific chromosome changes are not likely to be found in this group of tumours.     

Flow cytometric DNA analysis and chromosomal aberrations in malignant glioblastomas.

In this study we combined flow cytometry with fluorescence in situ hybridization to detect numerical aberrations in chromosomes. Fifty-nine human malignant gliomas were examined by flow cytometry for DNA-content and cell cycle analysis and for numerical aberrations of chromosome 1 by in situ hybridization using a chromosome specific centromere probe. Of the gliomas analysed, 42% were diploid and 58% showed aneuploid tumour cell populations. The DNA index was heterogeneous ranging from 1.0 to 2.3. The S-phase analysis showed proliferation activity from a very low range of 0.7% up to 17.0%. In general, diploid gliomas exhibited a lower S-phase activity than aneuploid gliomas. Of the aneuploid gliomas, 15% showed a peridiploid pattern with a DNA index mean of 1.1. In these peridiploid tumours a trisomy of chromosome 1 could be detected by fluorescence in situ hybridization (FISH). The frequency of trisomic chromosome 1 in malignant gliomas reflects a very slight increase in DNA index from diploid to peridiploid (DNA index 1.1). Comparison of chromosome numbers and DNA content gave good correlation. Also important, the results reflects the cell cycle, specifically the extent of S-phase activity. In general, cell proliferation of diploid and peridiploid gliomas is much less than in higher aneuploid gliomas. The analysis of DNA content may thus yield results with respect to the biological behaviour of tumours in general.     

Adenosine deaminase and adenosine kinase in rat hepatomas and kidney tumours.

Adenosine deaminase and adenosine kinase have been measured in rat liver, 12 transplantable hepatomas, regenerating, foetal and neonatal liver, adult and neonatal rat kidney and 2 transplantable kidney tumours. Adenosine, deaminase activity, relative to the normal liver value, was elevated 2-4 fold in hepatomas of rapid growth rate, was in the normal range in more slowly growing hepatomas and in regernerating liver, and was low in foetal and neonatal liver. Adenosine kinase activity was decreased, relative to rat liver values, in all the hepatomas; activity of this enzyme gave a negative correlation with tumour growth rate. Kinetic properties of the two enzymes were examined in partially purified preparations. Adenosine deaminases from both liver and rapidly growing hepatoma 3924A were subject to weak product inhibition by inosine. Adenosine kinase from liver and hepatoma 3924A was inhibited by the reaction products ADP and AMP, and the enzyme was also subject to excess substrate inhibition by concentrations of ATP in excess of 1 mM. In rat hepatoma cell lines growing in culture, the toxicity of adenosine correlated inversely with the ratio of adenosine deaminase activity to adenosine kinase activity. Chromatographic measurements showed that hepatoma cells incorporated less extracellular adenosine into their adenine nucleotide pools than did isolated liver cells. These results indicate that increased adenosine deaminase activity and decreased adenosine kinase activity may confer a selective advantage upon the cancer cell.     

Enzymological studies in chronic lymphocytic leukemia

Adenosine deaminase (ADA), 5'nucleotidase (5'NT), ecto-5'NT, purine nucleoside phosphorylase (PNP), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), adenosine kinase (AK), AMP-deaminase (AMPD) and adenylate kinase (AdKin) activities were assayed in peripheral blood lymphoid cells from 20 patients with B-cell type chronic lymphocytic leukemia (CLL). Significantly decreased mean activities of ADA, 5'NT, ecto-5'NT, PNP and AMPD were observed when comparing B-CLL lymphoid cells with control peripheral blood lymphocytes (PBL). AK and AdKin activities however, were found to be higher in B-CLL. Relatively wide ranges of ADA and 5'NT activity were observed. In patients with paraproteinaemia, 5'NT activity was found to be relatively high and in the range of the activities in normal PBL. ADA activity seemed to be slightly higher in patients without paraproteinaemia. No correlation could be found between the enzyme activities and the number of cells rosetting with sheep erythrocytes or bearing surface immunoglobulin (sIg). A relationship was suggested between 5'NT activity and Ig production.     

Induction of centrosome and chromosome aberrations by imatinib in vitro.

Imatinib (STI571, Gleevec/Glivec) is a potent selective tyrosine kinase inhibitor and is used successfully in the treatment of chronic myeloid leukemia (CML). While karyotype alterations, in addition to the Philadelphia chromosome, are a common phenomenon of progressing CML, the observation of BCR-ABL-negative leukemic clones with distinct aberrant karyotypes under an imatinib regimen is not yet understood. Here we test the hypothesis that such tumor clones may be induced de novo from normal cells by imatinib. In vitro experiments with varying drug concentrations (5-20 microM) were performed on normal human dermal fibroblasts (NHDF), Chinese hamster embryonal and Indian muntjak fibroblasts. After 3 weeks of treatment, analysis of cell cultures by centrosome immunostaining and conventional cytogenetics revealed that imatinib induced centrosome and chromosome aberrations in all cultures in a significant dose-dependent and species-independent manner. Moreover, the results of NHDF long-term culture experiments demonstrated that aberrant phenotypes, emerging under imatinib treatment for 12 weeks, were not reversible after prolonged propagation omitting the drug. These observations suggest a causative role of imatinib in the origin of centrosome and karyotype aberrations (genetic instability) and thus may explain the emergence of clonal chromosomal abnormalities in BCR-ABL-negative progenitor cells under imatinib therapy.     

Intratumoral genetic heterogeneity in pilocytic astrocytomas revealed by CGH-analysis of microdissected tumor cells and FISH on tumor tissue sections

Pilocytic astrocytomas are the most frequent gliomas of childhood. The majority of cases show cytogenetic normal karyotypes. Although in diffuse gliomas TP53 gene mutations or deletions occur with significant frequency, the role in pilocytic astrocytomas remains unclear. Histomorphologically different areas of 14 pilocytic astrocytomas were microdissected and analyzed for genetic aberrations and heterogeneity. CGH analysis revealed gains of chromosome arm 4q and 6q mainly in areas of classic biphasic pattern, whereas pleomorphic areas presented gains of chromosome 6 and 8q. Using two-color fluorescence in situ hybridization (FISH) the spatial distribution of aneuploidies for chromosomes 7, 8, 17 and the TP53 gene were assessed on parallel sections. FISH analysis revealed a significant percentage of cells with interspersed heterozygous deletions of TP53 in all tumors (14/14), ten cases showed also monosomy 17. Besides gains of chromosomes 7 and 8, losses of these chromosomes were detected in the majority of tumors. In conclusion, pilocytic astrocytomas show a genetic heterogeneity associated with variations of histologic structure as well as an intratumoral heterogeneity observed on single cell level by FISH.     

Pyrimidine nucleotide synthesis is more extensive in poorly differentiated than in well-differentiated human gastric carcinoma

In tissues obtained from patients undergoing gastrectomy, the activities of 12 enzymes involved in pyrimidine nucleotide synthesis: cytidine triphosphate (CTP) synthetase, deoxycytidine monophosphate (dCMP) deaminase, thymidine monophosphate (dTMP) kinase, uridine (Urd), deoxycytidine (dCyd) and thymidine (dThd) kinases, Urd, deoxyuridine (dUrd) and dThd phosphorylases, cytidine (Cyd) and dCyd deaminases, and DNA polymerase were examined in the eight-well-differentiated and 12 poorly differentiated gastric cancer tissues and the ten normal tissues. These cases were clinically advanced and serosal invasions were evident. Activities of these enzymes were higher in the poorly differentiated tissues than the well differentiated type and in the normal tissues. Significant differences were noted between the poorly differentiated and well-differentiated types, in dTMP kinase (P less than 0.02), dThd kinase (P less than 0.05), dThd phosphorylase (P less than 0.01), and DNA polymerase (P less than 0.05). The authors' findings show that the level of pyrimidine nucleotide synthesis, in both de novo and salvage pathways, is higher in the poorly differentiated gastric cancer tissues than in the well-differentiated type and suggest that antitumor drugs have an increased susceptibility in cases of poorly differentiated gastric carcinoma.     

Evidence of chromosomal instability in prostate cancer determined by spectral karyotyping (SKY) and interphase fish analysis

The way in which cytogenetic aberrations develop in prostate cancer (CaP) is poorly understood. Spectral karyotype (SKY) analysis of CaP cell lines has shown that they have unstable karyotypes and also have features associated with chromosomal instability (CIN). To accurately determine the incidence of de novo structural and numerical aberrations in vitro in CaP, we performed SKY analysis of three independent clones derived from one representative cell line, DU145. The frequent generation of new chromosomal rearrangements and a wide variation in the number of structural aberrations within two to five passages suggested that this cell line exhibited some of the features associated with a CIN phenotype. To study numerical cell-to-cell variation, chromosome 8 aneusomy was assessed in the LNCaP, DU145, and PC-3 cell lines and a patient cohort of 15 CaP primary tumors by interphase fluorescence in situ hybridization (FISH). This analysis showed that a high frequency of numerical alteration affecting chromosome 8 was present in both in vitro and in CaP tissues. In comparison to normal controls, the patient cohort had a statistically significant (P<.05), greater frequency of cells with one and three centromere 8 copies. These data suggest that a CIN-like process may be contributing towards the generation of de novo numerical and structural chromosome abnormalities in CaP.     

Low cytidine deaminase levels in human hematopoietic cell lines

Purine and pyrimidine enzyme profiles of human cell lines have been investigated. A novel observation was the finding that most of the cell lines showed very low or undetectable levels of cytidine (deoxycytidine) deaminase, while they possessed pyrimidine 5'-nucleotidase, cytidine and deoxycytidine kinase activities. Most cell lines showed high levels of adenosine deaminase and purine nucleoside phosphorylase activities and low levels of purine 5'-nucleotidase. We propose that high adenosine deaminase and purine nucleoside phosphorylase activities and low cytidine deaminase activity may be of importance for immature hematopoietic cells in order to ensure a balanced synthesis of the DNA precursors.     

Activity of the enzymes of DNA metabolism in the blood of patients with breast cancer

Age-related changes in the activity of thymidine- and adenosine-metabolizing enzymes were studied in healthy females and those with breast cancer aged 46-70 years. A significant increase in activity of thymidine kinase, adenosine deaminase and 5'-nucleotidase and a decrease in that of thymidine phosphorylase were registered in blood serum of breast cancer patients of all age brackets. Adenosine deaminase activity in blood serum and lymphocytes of breast cancer patients was found to significantly change after surgery. A direct correlation was established between pretreatment thymidine phosphorylase activity and histological type of tumor, on the one hand and results of chemotherapy, on the other. The applicability of enzyme level assay for evaluating response to pre- and postoperative medication was studied.     

Mitotic instability associated with late genomic changes in bone and soft tissue tumours

One source of genomic instability in tumours is abnormal mitotic segregation of chromosomes. Evaluation of chromosome segregation and cytogenetic aberrations in 28 bone and soft tissue neoplasms revealed few mitotic disturbances in benign lesions, whereas most of the malignant tumours, except for chondrosarcomas, showed anaphase bridges and/or multipolar mitoses. Only cases with chromosomal imbalances exhibited these defects and they were not present in any of the cases with sole primary changes, indicating that mitotic instability is established relatively late in mesenchymal tumour development. Most cases with multipolar mitoses exhibited abnormal centrosome configurations. However, induction of supernumerary centrioles in vitro failed to produce mitotic abnormalities in normal cells, indicating that additional disturbances of the cell division machinery are required for the generation of mitotic multipolarity.     

Enzymic capacities of purine de Novo and salvage pathways for nucleotide synthesis in normal and neoplastic tissues

The enzymic capacities of the de novo and the salvage pathways for purine nucleotide synthesis were compared in rat in normal, differentiating, and regenerating liver, and in three hepatomas of widely different growth rates. The activities of the key de novo and salvage enzymes were also determined in mouse lung and Lewis lung carcinoma, in human kidney and liver, and in renal cell carcinoma and hepatocellular carcinomas. A precise and reproducible assay was worked out for measuring the activities of adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) in crude liver and hepatoma systems. Kinetic studies on the salvage enzymes were carried out in the crude 100,000 X g supernatant fluid from normal liver and rapidly growing hepatoma 3924A. In both tissue extracts, Michaelis-Menten kinetics was observed for adenine phosphoribosyltransferase and HGPRT. The reciprocal plots for 5-phosphoribosyl-1-pyrophosphate (PRPP) of liver and hepatoma enzymes gave apparent KmS of 2 microM for adenine phosphoribosyltransferase and 4 microM for HGPRT, showing two orders of magnitude higher affinities for PRPP than that of the rate-limiting enzyme of de novo purine synthesis, amidophosphoribosyltransferase (EC 2.4.2.14) (Km = 400 to 900 microM). The apparent Km values for adenine of liver and hepatoma adenine phosphoribosyltransferase were 0.6 to 0.9 microM, respectively. For both liver and hepatoma HGPRT, the reciprocal plots for hypoxanthine and guanine yielded the same Km of 3 microM. The specific activities of purine phosphoribosyltransferases were markedly higher than that of amidophosphoribosyltransferase in rat thymus, spleen, testis, bone marrow, colon, liver, kidney cortex, lung, heart, brain, and skeletal muscle, but were lower in the small intestine. In hepatomas and regenerating and differentiating liver, the activities of the salvage enzymes were 2.1- to 32-fold higher than that of amidophosphoribosyltransferase. The purine phosphoribosyltransferase activities were also higher than that of amidophosphoribosyltransferase in Lewis lung carcinoma (8.2- to 32-fold), human renal cell carcinoma (3.5- to 22-fold), and hepatocellular carcinoma (3.4- to 30-fold). The high activities and the high affinity to PRPP of the purine phosphoribosyltransferases might explain the lack of linkage of the behavior of these enzymic activities with proliferation in normal, regenerating, differentiating, or neoplastic tissues. In contrast, the specific activity of the amidophosphoribosyltransferase, which is lower than that of the salvage enzymes, is linked with transformation as it is increased in all examined tumors.4     

Enzyme activities controlling adenosine levels in normal and neoplastic tissues

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5′-nucleotidase (5′-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5′-NT, and PNP in tumor tissue than in neighboring mucosa (p>0.01 for ADA, AK, and PNP; p>0.05 for 5′-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5′-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.     

Chromosomal changes during development and progression of prostate adenocarcinomas

Chromosomal copy number changes were investigated in 16 prostate carcinomas, 12 prostatic intraepithelial neoplasias (PIN; 4 low-grade and 8 high-grade) adjacent to the invasive tumour areas, and 5 regional lymph node metastases. For this purpose, comparative genomic hybridization (CGH) was performed and a copy number karyotype for each histomorphological entity was created. CGH on microdissected cells from non-neoplastic glands was carried out on 3 different cases to demonstrate the reliability of the overall procedure. None of the non-neoplastic tissue samples revealed chromosome copy number changes. In PIN areas, chromosomal imbalances were detected on chromosomes 7, 8q, Xq (gains), and on 4q, 5q, 8p, 13q and 18q (losses). In the primary tumours, recurrent (at least 25% of cases) gains on chromosomes 12q and 15q, and losses on 2q, 4q, 5q, Xq, 13q and 18q became apparent. Losses on 8p and 6q as well as gains on 8q and of chromosome 7 were also detected at lower frequencies than previously reported. The pooled CGH data from the primary carcinomas revealed a novel region of chromosomal loss on 4q which is also frequently affected in other tumour entities like oesophageal adenocarcinomas and is supposed to harbour a new tumour suppressor gene. Gains on chromosome 9q and of chromosome 16 and loss on chromosome 13q were observed as common aberrations in metastases and primary tumours. These CGH results indicate an accumulation of chromosomal imbalances during the PIN-carcinoma-metastasis sequence and an early origin of tumour-specific aberrations in PIN areas.