Diagnostic application of a capture based NGS test for the concurrent detection of variants in sequence and copy number as well as LOH.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

Loss of heterozygosity for chromosome 11 in adenocarcinoma of the stomach.

Loss of heterozygosity (LOH) at several chromosomal loci is a common feature of the malignant progression of human tumors. In the case of chromosome 11, LOH has been well documented in several types of solid neoplasms, including gastric carcinoma, suggesting the presence of suppressor gene(s) at 11p15 and 11q22-23. Little is currently known about the molecular events occurring during the development of gastric cancer. To define the regions of chromosome 11 involved in gastric cancer progression, we used high-density polymorphic markers to screen for LOH in matched normal and tumor tissue DNA from 60 primary gastric carcinomas. We found that 21% of the tumors showed LOH simultaneously at 11p15 and 11q22-23, 41% had LOH at 11p15, and 30% had LOH at 11q22-23. We confirm that the minimal critical area of LOH for 11p15.5 is the approximately 2-Mb region between loci D11S1318 and D11S988. However, when we analyzed the pattern of LOH according to the country of origin of the patient, LOH for 11q22-23 alone was found only in cases from Italy. The minimal critical region of LOH at 11q22-23 is identical to that identified for other solid tumors, suggesting that the same putative tumor suppressor gene(s) contained within this region is involved in the pathogenesis of several common human tumors.

Heterogeneous protooncogene amplification correlates with tumor progression and presence of metastases in gastric cancer patients.

In order to evaluate the relevance of protooncogene alterations in gastric cancer and to specifically relate these alterations to types and stages of the neoplasia, we studied oncogenes of possible interest in gastric tumors with different clinical parameters. Fifty DNAs from primary gastric adenocarcinoma were analyzed, by the Southern blotting technique, for the presence of amplification or rearrangements of seven different protooncogenes: c-myc, c-erbB2, c-Ki-ras, c-Ha-ras, c-N-ras, hst, and c-mos. All the tumors analyzed were histologically classified and staged. Amplification of the following genes was found: c-myc (2 of 50), hst (3 of 50), c-erbB2 (3 of 50), and c-Ki-ras (5 of 50). The simultaneous amplification of hst (3 cases), c-myc (1 of 3), or c-Ki-ras (2 of 3) was observed. Analysis of DNAs from atrophic and metaplastic gastric mucosa (which can be regarded as preneoplastic lesions) of the 10 patients showing gene amplification demonstrated that this was limited to neoplastic cells. Considering protooncogene amplification in general (i.e., involving different genes and occurring to different degrees) and clinical parameters of tumors, we found a statistically significant association between amplification and both tumor progression and presence of metastases. Therefore, at least for the genes analyzed, amplification is a relatively infrequent phenomenon and represents a late event in the temporal development of gastric cancer.

K-ras and p53 gene mutations in pancreatic cancer: ductal and nonductal tumors progress through different genetic lesions.

We studied K-ras and p53 gene mutations in a panel of 57 primary pancreatic cancers including ductal and nonductal tumors. DNAs were obtained from formalin-fixed, paraffin-embedded material. Target sequences were amplified by polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis and sequencing. Both K-ras and p53 genes were frequently mutated in ductal cancers (25 of 35, 71.4%; 18 of 35, 51.4%, respectively). K-ras mutations were confined to the second position of codon 12 where base transitions and transversions were equally observed. p53 changes were mainly missense mutations. Transitions and transversions were found equally with a prevalence of G:C-->A:T changes among transitions. No gene alterations were present in the 6 exocrine nonductal tumors and (with one exception) in the 12 endocrine tumors analyzed. Our results indicate that mutated K-ras and p53 genes can cooperate in the establishment of ductal pancreatic cancers, whereas other genetic events have to be present in nonductal tumors. Moreover, K-ras alterations may represent an early event in ductal tumorigenesis, as suggested both by the high gene mutation frequency and by the presence of mutations in low-grade tumors. On the contrary, p53 gene changes seem to represent an event required for the malignancy progression of ductal tumors from lower to higher grades.

Base transitions are the most frequent genetic changes at P53 in gastric cancer.

We searched for P53 mutations in gastric carcinoma by analyzing tumor DNAs from 29 patients. We detected 13 different somatic mutations in 15 patients (52%) and a biallelic polymorphism in exon 6 (5 heterozygous subjects). The somatic mutations were mainly localized in the sequences corresponding to the highly conserved domains of the protein. Twelve samples showed a single base change: 11 missense and 1 nonsense mutations. Three samples showed deletions leading to a frame shift, to the in-frame loss of 2 amino acids, and to the deletion of a splicing site. All point mutations, except one, were transitions, and 91% of them were G:C-->A:T changes. We previously analyzed this panel of tumors for allelic loss at the 17p13 chromosomal region, where the P53 gene had previously been located: the results showed an increasing incidence of allelic loss in late-stage tumors. On the contrary, in the present study no trend between P53 mutations and tumor stages was found. This observation indicates that mutation events precede allelic loss in gastric cancer. Half (54%) of the mutations occurred in samples without allelic loss, suggesting that specific mutated alleles, acting in a dominant negative fashion, can alter in vivo the P53 protein function.

A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.

In December 1997, the National Cancer Institute sponsored "The International Workshop on Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition," to review and unify the field. The following recommendations were endorsed at the workshop. (a) The form of genomic instability associated with defective DNA mismatch repair in tumors is to be called microsatellite instability (MSI). (b) A panel of five microsatellites has been validated and is recommended as a reference panel for future research in the field. Tumors may be characterized on the basis of: high-frequency MSI (MSI-H), if two or more of the five markers show instability (i.e., have insertion/deletion mutations), and low-frequency MSI (MSI-L), if only one of the five markers shows instability. The distinction between microsatellite stable (MSS) and low frequency MSI (MSI-L) can only be accomplished if a greater number of markers is utilized. (c) A unique clinical and pathological phenotype is identified for the MSI-H tumors, which comprise approximately 15% of colorectal cancers, whereas MSI-L and MSS tumors appear to be phenotypically similar. MSI-H colorectal tumors are found predominantly in the proximal colon, have unique histopathological features, and are associated with a less aggressive clinical course than are stage-matched MSI-L or MSS tumors. Preclinical models suggest the possibility that these tumors may be resistant to the cytotoxicity induced by certain chemotherapeutic agents. The implications for MSI-L are not yet clear. (d) MSI can be measured in fresh or fixed tumor specimens equally well; microdissection of pathological specimens is recommended to enrich for neoplastic tissue; and normal tissue is required to document the presence of MSI. (e) The "Bethesda guidelines," which were developed in 1996 to assist in the selection of tumors for microsatellite analysis, are endorsed. (f) The spectrum of microsatellite alterations in noncolonic tumors was reviewed, and it was concluded that the above recommendations apply only to colorectal neoplasms. (g) A research agenda was recommended.

Variable mutation frequencies in coding repeats of TCF-4 and other target genes in colon, gastric and endometrial carcinoma showing microsatellite instability.

Frameshift mutations in genes containing mononucleotide repeats are often observed in cancers exhibiting a high frequency of microsatellite instability (MSI-H). Several tumor types, including colorectal, gastric, and endometrial carcinomas, display this phenotype in a significant proportion of cases. We recently showed in a large series of MSI-H colorectal tumors that approximately 40% of them exhibited frameshift mutations in an (A)9 tract within the coding region of the TCF-4 gene, a crucial member of the APC/beta-catenin/TCF pathway. In the present study, we have examined MSI-H cancers from other primary tumor sites for mutations in this new target gene. Two of 22 (9%) MSI-H primary gastric cancers and none of 23 MSI-H endometrial primary tumors and cell lines were found to have a 1 bp deletion in the TCF-4 repeat. In the same series of tumors we also looked for frameshift mutations in other coding repeats localized within the TGF beta-RII, BAX, IGFIIR, hMSH3 and hMSH6 genes. Our results suggest that the TCF-4 gene, in a similar manner to some of these latter genes, is differentially altered in MSI-H tumors from different primary sites.

p53 codon 72 polymorphism does not affect the risk of cervical cancer in patients from northern Italy.

A case-control study was performed to investigate the risk of cervical cancer associated with p53 polymorphism at codon 72, encoding either arginine or proline. It has been recently suggested that the arginine isoform increases the susceptibility to invasive cervical cancer; however, data remain controversial. The polymorphism was examined by both allele-specific PCR and RFLP analysis in 101 patients with primary cervical cancer and in 140 healthy women of the same age and from the same geographical area. The distribution of p53 genotypes in cervical cancer patients and in controls was not significantly different (P = 0.445), and homozygosity for arginine at residue 72 was not associated with an increased risk for cervical cancer (odds ratio, 0.81; 95% confidence interval, 0.47-1.42; P = 0.52). Similarly, different genotype distribution and increased risk were not observed when patients versus controls were analyzed according to human papillomavirus status and cancer histotype. Therefore, no evidence of association between homozygosity for p53 arginine and cervical cancer was found in our population sample.

p53 gene mutations and protein nuclear accumulation are early events in intestinal type gastric cancer but late events in diffuse type.

We screened for p53 alterations in 71 early gastric cancers of differing histological types and growth patterns, 18 advanced cancers of diffuse type, 19 dysplastic lesions, and 12 extensive intestinal metaplasia cases. Tumors were investigated for gene mutations (exons 5-8) with PCR-based denaturing gradient gel electrophoresis and sequencing techniques, and for protein accumulation with immunohistochemical methods. Nontumor samples were studied with immunohistochemistry alone. Of the early cancers, intestinal tumors showed a much higher p53 mutation frequency (41%) than did diffuse cancers (4%). When comparing early and advanced tumors of the same type, we observed a similarity in mutation frequency (41 versus about 50%) for intestinal tumors, and a significant increase for diffuse tumors (from 4 to 33%). Immunopositive case distribution between tumor types and stages paralleled that of mutated cases. Immunohistochemical and genetic analysis gave concordant results for all samples with gene mutations. Eighteen of the 65 (28%) nonmutated tumors displayed significant immunoreactivity. Early tumors that massively penetrated the submucosa, i.e., the early tumors for which prognosis is worst, showed the highest frequency both of p53 gene mutation and of nonmutated protein accumulation. Twelve of 19 dysplastic lesions showed significant immunoreactivity, whereas intestinal metaplasias proved unreactive in all but a few cells. Our results yield two implications: that p53 alterations have a crucial and early role in gastric carcinogenesis of intestinal type, likely acting at the transition step between metaplasia and dysplasia; and that the alterations are mainly associated with tumor progression in cancer of diffuse type.

Intestinal and diffuse gastric cancers arise in a different background of Helicobacter pylori gastritis through different gene involvement.

Investigation of extensively sampled nontumor gastric mucosa from 205 early gastric cancers showed Helicobacter pylori colonization in 85% of cases, including 100% of diffuse and 78% (83% in 97 cases with Swiss rolls) of glandular or mixed cancers. Intestinal metaplasia, including its type III variant, was prominent in the mucosa associated with glandular and mixed (but not diffuse) early cancers. Both glandular (usually called "intestinal") and diffuse-type cancers showed admixtures of intestinal and gastric tumor cell phenotypes. Both p53 gene mutations and p53 protein immunostaining were essentially restricted to glandular or mixed cancers and associated dysplastic lesions. Their appearance in the advanced stage of diffuse cancer was partly due to a change of the histologic pattern from glandular to diffuse during progression of some tumors. Loss of laminin, beta I integrin, or zonula adherens junctions was a common finding in both early and advanced diffuse cancer. It is concluded that two main pathways operate in gastric carcinogenesis, both starting from H. pylori gastritis and both leading to phenotypically variable, often mixed gastric/intestinal tumor growth. However, only one of the two pathways involves intestinal metaplasia, its type III variant, p53 gene alteration, and dysplasia to end in glandular cancer. In the other pathway, diffuse cancer apparently arises directly from hyperplastic, sometimes atypical necks of mostly nonmetaplastic gastric glands, through primary involvement of genes affecting cell-cell and cell-matrix junctional proteins.

Genetic pattern, histological structure, and cellular phenotype in early and advanced gastric cancers: evidence for structure-related genetic subsets and for loss of glandular structure during progression of some tumors.

Gastric cancer shows remarkable heterogeneity in histological pattern, cellular phenotype, and genotype. Tumor subsets identified by varying procedures have shown limited reciprocal correlation and have failed to provide a sound rationale for the characterization and classification of all tumors. Based on a case series of 130 gastric cancers that covered both early (70 cases) and advanced (60 cases) stages and that represented most histological types and structural patterns, this study investigated (1) microsatellite instability and p53 gene mutation by means of PCR-based molecular techniques and (2) p53 protein accumulation or tumor cell immunophenotype by means of immunoperoxidase procedures. It was found that microsatellite instability and p53 gene mutation involve two distinct subsets of both early and advanced-stage glandular (intestinal) cancer, and that, contrastingly, they leave purely diffuse cancers unaffected. Mixed cancers, namely, those in which glandular admixed with diffuse growths, showed scarce microsatellite instability at all stages, whereas prominent p53 gene mutation and p53 protein accumulation was limited to the advanced stage alone. No significant correlation was found between tumor cell immunophenotype and either genotype or histotype, although some correlation with particular structural patterns was detected. Comparison of intramucosal with invasive growths within any given tumor suggested that invasive cancers with diffuse-type growth arise in part from mucosal cancers of glandular or mixed structure through progressive loss of intercellular junctional systems. It is concluded that at least two genetically distinct subsets of glandular cancer, one with microsatellite instability and the other with p53 lesions, should be separated both from purely diffuse cancer and, at least in the advanced stage, from mixed cancer. Available evidence suggests distinct clinicopathologic profiles for such tumor entities.

Primary retroperitoneal mucinous cystoadenocarcinomas: an immunohistochemical and molecular study.

Special immunohistochemical stains for the identification of gastroenteropancreatic antigens in two cases of primary retroperitoneal mucinous cystoadenocarcinomas (PRMC) show that these tumours have patterns similar to ovarian mucinous tumours. Markers of pyloric type gastric mucosa differentiation (M1, cathepsin E, concavavalin A, pepsinogen II) are mostly positive in benign and borderline areas with endocervical type differentiation, while immunoreactivity for intestinal cell markers (M3SI and CAR-5) and for DU-PAN-2 is present mainly in frankly malignant areas, regardless of differentiation type. DNA analysis shows a point mutation of K-ras oncogene at codon 12 (GGT to CGT) in one case. The immunohistochemical and genotypic similarity of PRMC and ovarian mucinous tumours may indicate similar mechanisms in their histogenesis.

Intraductal papillary-mucinous tumours represent a distinct group of pancreatic neoplasms: an investigation of tumour cell differentiation and K-ras, p53 and c-erbB-2 abnormalities in 26 patients.

Intraductal papillary growth of mucin producing hypersecreting, columnar cells characterizes a group of rare pancreatic exocrine neoplasms which we propose to call intraductal papillary-mucinous tumors (IPMT). We analysed the histopathology of 26 IPMT in relation to gastro-enteropancreatic marker expression, genetic changes and biology. Four IPMT showing only mild dysplasia were considered to be adenomas. Nine tumours displayed moderate dysplasia and were regarded as borderline. Severe dysplasia-carcinoma in situ changes were found in 13 IPMT which were therefore classified as intraductal carcinomas. Six of these carcinomas were frankly invasive and two of these had lymph node metastases. The invasive component resembled mucinous non-cystic carcinoma in all but one tumour which showed a ductal invasion pattern. Immunohistochemically, an intestinal marker type was found in most carcinomas, while gastric type differentiation prevailed among adenomas or borderline tumours. K-ras mutations (seven at codon 12 and one at codon 13) were found in 31% of IPMT (2 adenomas, 1 borderline, 5 carcinomas). Nuclear p53 overexpression was detected in 31% of IPMT (6 carcinomas and 2 borderline IPMT) and correlated with p53 mutations (one at exon 8 and the other at exon 5) in two carcinomas. p53 abnormalities were unrelated to K-ras mutation. c-erbB-2 overexpression was observed in 65% of IPMT, with various grades of dysplasia. Twenty-two of 24 patients are alive and well after a mean post-operative follow-up of 41 months. Only two patients, both with invasive cancer at the time of surgery, died of tumour disease. It is concluded that pancreatic IPMT encompass neoplasms which, in general, have a favorable prognosis, but are heterogeneous in regard to grade of dysplasia and marker expression. Adenoma, borderline tumour, intraductal carcinoma and invasive carcinoma can be differentiated. p53 changes but not K-ras mutation or c-erbB-2 overexpression are related to the grade of malignancy. Most IPMT differ in histological structure, marker expression and behaviour from ductal adenocarcinoma.

K-ras gene mutations: an unfavorable prognostic marker in stage I lung adenocarcinoma.

Activation of K-ras gene by point mutations, a common finding in lung adenocarcinomas, has been suggested to decrease patient survival. We investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (81/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel electrophoresis analysis of specific sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33, 84.8%) or 13 (5/33, 15.2%) of the gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiolo-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours (p = 0.016). Multivariate analysis showed that age of patient (p = 0.001) and K-ras mutation status (p = 0.04) were the only independent factors influencing survival significantly. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.

Somatic frameshift mutations in the Bloom syndrome BLM gene are frequent in sporadic gastric carcinomas with microsatellite mutator phenotype.

BACKGROUND: Genomic instability has been reported at microsatellite tracts in few coding sequences. We have shown that the Bloom syndrome BLM gene may be a target of microsatelliteinstability (MSI) in a short poly-adenine repeat located in its coding region. To further characterize the involvement of BLM in tumorigenesis, we have investigated mutations in nine genes containing coding microsatellites in microsatellite mutator phenotype (MMP) positive and negative gastric carcinomas (GCs). METHODS: We analyzed 50 gastric carcinomas (GCs) for mutations in the BLM poly(A) tract aswell as in the coding microsatellites of the TGFbeta1-RII, IGFIIR, hMSH3, hMSH6, BAX, WRN, RECQL and CBL genes. RESULTS: BLM mutations were found in 27% of MMP+ GCs (4/15 cases) but not in any of the MMP negative GCs (0/35 cases). The frequency of mutations in the other eight coding regions microsatellite was the following: TGFbeta1-RII (60 %), BAX (27%), hMSH6 (20%),hMSH3 (13%), CBL (13%), IGFIIR (7%), RECQL (0%) and WRN (0%). Mutations in BLM appear to be more frequently associated with frameshifts in BAX and in hMSH6and/or hMSH3. Tumors with BLM alterations present a higher frequency of unstable mono- and trinucleotide repeats located in coding regions as compared with mutator phenotype tumors without BLM frameshifts. CONCLUSIONS: BLM frameshifts are frequent alterations in GCs specifically associated with MMP+tumors. We suggest that BLM loss of function by MSI may increase the genetic instability of a pre-existent unstable genotype in gastric tumors.

Rapid assessment of replication error phenotype in gastric cancer.

Forty gastric tumors were investigated for microsatellite instability at the D2S119 and L-myc loci. These tumors and 143 other gastrointestinal cancers were previously analyzed for instability at several different microsatellites. By evaluating previous and present results, repeated sequences were selected that frequently underwent replication errors (RERs). To coamplify these sequences, the following multiplex polymerase chain reactions (PCRs) were performed: 1) D2S119/L-myc/D18S59; 2) D2S119/L-myc/D3S1076; and 3) D2S177/L-myc/BAT-RII. Therefore, the 40 gastric tumors in the present survey were rescreened using multiplex PCRs. Each multiplex allowed detection of nearly all RER+ tumors (80% for multiplex 3 and 87% for multiplexes 1 and 2) that had been previously identified by amplifying 9 different loci with independent reactions. Moreover, for multiplexes 1 and 2, the size differences between normal and RER alleles were sufficient to be detected by electrophoresis on conventional polyacrylamide gels after DNA staining with ethidium bromide. This approach allows a rapid and easy assessment of RER phenotype in gastric tumors.

DNA amplification in human gastric carcinomas.

We recently identified a genomic domain at chromosome 10q26 that is highly amplified in the gastric carcinoma cell lines KATO III and SNU-16 and contains the BEK/K-sam gene, which encodes several growth factor receptors. A contiguous segment of 200 kb spanning this gene was amplified in five of 139 (3.6%) primary gastric carcinomas, all of them classified as poorly differentiated tumors. There was no amplification of this genomic region in a variety of other solid tumors. The overall frequency of gene amplification among the gastric carcinomas rose to 19.4% when MYC, ERBB2, and INT2 were included in the analysis, with significant association with advanced tumor stage. Amplification of various genomic regions in solid tumors may be more frequent than previously estimated.

Study on aneuploidy and p53 mutations in astrocytomas.

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Detection of K-ras mutations by denaturing gradient gel electrophoresis (DGGE): a study on pancreatic cancer.

In pancreatic neoplasias mutations in the first exon (codon 12) of K-ras gene occur at high frequency and seem to have a diagnostic significance. We set up the DGGE conditions to search for these mutations in pancreatic tumor sample DNAs. All samples were directly classified by simply comparing their DGGE patterns with those of control cell lines carrying known K-ras base substitutions. We found a mutation frequency of 73% in pancreatic adenocarcinoma, whereas no mutations were observed in benign lesions. The non-isotopic method we used turned out to be rapid and sensitive. DGGE could therefore be utilized for the detection of K-ras mutations in pancreatic lesions, to evaluate their actual or potential malignancy. In general, DGGE could be useful for K-ras gene screening on pathological tissue samples.

Effects of topoisomerase II inhibitors on gastric cancer cells characterized by different genetic lesions.

Gastric cancer is poorly-responsive to widely used antitumour drugs, the efficacy of which is thought to be related to the capacity of triggering apoptosis. This process requires a series of gene products including a functional p53 protein. We tested the effects of two DNA topoisomerase II poisons, etoposide and doxorubicin, on gastric cancer cell lines with different genetic lesions. We characterised MKN74 and MKN28 cells for p53 gene status and for the expression of p53 and p21 proteins, as well as of topoisomerase II alpha and beta isoforms. After drug treatments, the cells were analysed for drug cytotoxicity, colony forming ability, cell cycle distribution and presence of apoptotic features. Our findings demonstrated that both etoposide and doxorubicin have a potent anti-proliferative effect on gastric cancer cells. Cell death kinetics was different in the two cell lines, MKN74 cells being more sensitive than MKN28 to the drugs. MKN74 cells, although harboring a wt p53 gene, were unable to undergo a massive apoptosis following etoposide treatment. The response of this cell line might be related to the topoisomerase II beta isozyme, the expression of which proved to be undetectable.