MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells.

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.

c-MYC deregulation is involved in melphalan resistance of multiple myeloma: role of PDGF-BB.

Oncogenes are important regulators of cancer growth and progression and their action may be modulated by proteins of the growth factor family, such as angiogenic cytokines, known to be strongly involved in neoplastic evolution. Reciprocal interactions between oncogenes and angiogenic modulators may represent, in haematological neoplasms, including multiple myeloma (MM), a possible mechanism of drug resistance. The aim of this work is to investigate in vitro and in vivo whether or not c-myc deregulation is involved in the melphalan resistance elicited by myeloma patients and consequently to clarify the role of the angiogenic factor PDGF-BB in modulating c-myc protein expression. Fifty-one MM patients on chemotherapy with melphalan were analyzed for structural alterations of the c-myc gene, c-Myc protein expression, as well as for serum PDGF-BB release. For the in vitro study, two M14-derived established cell clones, differing for the c-Myc protein expression (c-Myc low -expressing or constitutively expressing clones) were used. Our results show that PDGF-BB is able to up-regulate Myc expression and reduce melphalan sensitivity of tumor cell clones, constitutively expressing c-myc gene product. In addition, down-regulation of c-Myc protein induces the expression of PDGF-beta receptor molecules and reduces PDGF-BB release. In agreement with these results, in vivo data show that melphalan-resistant MM patients present overexpressed c-Myc protein and higher serum PDGF-BB receptor levels compared to minor responding patients.

Biological indicators of aggressiveness in T1 ductal invasive breast cancer.

BACKGROUND: Apoptosis plays an important role in the maintenance of tissue homeostasis. When defective, this process could contribute to the pathogenesis and the progression of tumors. On this basis, we investigated the combined effect of Bcl-2 and Bax expression, known regulators of apoptotic processes, in the activation of apoptosis in breast cancer. Their relationship with DNA content and proliferative activity was also studied in order to more accurately define breast cancer patients' prognosis and treatment. MATERIALS AND METHODS: In this study we investigated 76 T1 ductal invasive breast cancers and 76 normal epithelium samples for Bcl-2 and Bax expression by immunohistochemistry, for apoptosis by tunel assay and for DNA content and proliferative activity by flow cytometry. RESULTS: High levels of Bcl-2 were associated with prevention of apoptosis. Conversely high Bax expression was found to be related to apoptosis. DNA ploidy was strictly related to the proliferative activity. In addition most of the tumors showing high Bcl-2 expression were aneuploid. CONCLUSION: This report suggests that Bax over-expression could accelerate apoptotic cell death by counteracting the ability of Bcl-2 to inhibit apoptosis. These data also suggest that the ratio Bcl-2/Bax and their relationship with the activation of apoptosis could be used as predictive indicators of breast cancer patients' prognosis and response to conventional therapy.

p53 nuclear accumulation and multiploidy are adverse prognostic factors in surgically resected stage II colorectal cancers independent of fluorouracil-based adjuvant therapy.

To identify the prognostically highest risk patients, DNA content and p53 nuclear or cytoplasmic accumulation, evaluated by monoclonal antibody DO7 and polyclonal antibody CM1, were determined in 94 surgically resected stage II (Dukes B2) colorectal cancers, treated or not with adjuvant 5-fluorouracil-based chemotherapy. Sixty-one (65%) of the tumors were aneuploid, 16 (17%) of which had a multiploid DNA content; 50 (53%) displayed DO7 nuclear p53 accumulation, and 44 (47%) showed cytoplasmic CM1 positivity. In multivariate analysis, only multiploidy and p53 nuclear positivity emerged as independent prognostic indicators of a poorer outcome. Positivity for p53 was associated with shorter survival in 5-fluorouracil-treated and untreated patients. Therefore, in patients with Dukes B2 colorectal cancer, a biologic profile based on the combined evaluation of DNA multiploidy and p53 status can provide valuable prognostic information, identifying patients to be enrolled in alternative, more aggressive therapeutic trials.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27(kip1) levels.

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G(1) phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27(kip1) levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27(kip1) in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G(1) arrest and activates apoptosis by increasing p27(kip1) content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G(1) checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

c-Myb and Bcl-x overexpression predicts poor prognosis in colorectal cancer: clinical and experimental findings.

The aim of this study was twofold: to assess the relationship between c-Myb and Bcl-x expression and to evaluate the prognostic significance of their expression in colorectal carcinoma (CRC) patients. Analysis of tumors from 91 CRC patients for expression of c-Myb and Bcl-x revealed a significant relationship between these two proteins. Kaplan-Meier's analysis showed an increased risk of relapse and death in patients whose tumor specimens displayed high c-Myb levels and Bcl-x positivity. Similar results were also observed excluding Dukes' D patients. Molecular analysis using three c-Myb-overexpressing LoVo clones indicated that c-Myb overexpression was accompanied by up-regulation of Bcl-x(L) protein and mRNA. Tumors originating from these clones injected in nude mice were significantly larger than those formed in mice injected with parental or vector-transfected LoVo cells. Moreover, tumors derived from parental and control vector-transfected but not from c-Myb-overexpressing LoVo cells showed high frequency of apoptotic cells. These results provide direct evidence of an association between c-Myb and Bcl-x expression and suggest that expression of both molecules might be a useful prognostic marker in CRC.

Arrest of G(1)-S progression by the p53-inducible gene PC3 is Rb dependent and relies on the inhibition of cyclin D1 transcription.

The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G(1) arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G(1) arrest was Rb dependent. Furthermore, (i) the arrest of G(1)-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G(1)-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.

Evaluation of multiple bio-pathological factors in colorectal adenocarcinomas: independent prognostic role of p53 and bcl-2.

About 40% of patients with colorectal carcinoma will develop local or distant tumour recurrences. Integrated analyses of bio-pathological markers, predictive of tumour aggressiveness, may offer a more rational approach to planning adjuvant therapy. To this end, we analysed the correlation between p53 accumulation, Bcl-2 expression, DNA ploidy, cell proliferation and conventional clinico-pathological parameters by testing the prognostic significance of these variables in a series of 171 colorectal carcinoma patients with long-term follow-up. The relationships among the various bio-pathological parameters, analysed by multiple correspondence analysis, showed 2 different clinico-biological profiles. The first, characterised by p53 negativity, Bcl-2 positivity, diploidy, low percentage of cells in S-phase (%S-phase), a low Ki-67 score, is associated with Dukes' A-B stage, well differentiated tumours and lack of relapse. The second, defined by p53 positivity, Bcl-2 negativity, aneuploidy, high %S-phase and elevated Ki-67 score, correlates with Dukes' C-D stage, poorly differentiated tumours and presence of relapse. When these parameters were examined according to Kaplan-Meier's method, significantly shorter disease-free (DFS) and overall survival (OS) were also observed in patients bearing p53 positive and Bcl-2 negative tumours, in Dukes' B stage. In multivariate analysis, p53 accumulation and Bcl-2 expression emerged as independent predictors of a worse and better clinical outcome, respectively. Our results indicate that, in colorectal adenocarcinomas, a biological profile, based on the combined evaluation of p53 and Bcl-2, may be useful for identifying high risk patients to be enrolled in an adjuvant setting, mainly in an early stage of the disease. Int. J. Cancer (Pred. Oncol.) 84:545-552, 1999.

Effects of poly(ADP-ribose) polymerase inhibition on cell death and chromosome damage induced by VP16 and bleomycin.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein involved in cellular response to various genotoxic agents. To understand the role of PARP in the mechanisms which lead from specific DNA damage to cell death, we studied the effects of PARP inhibition in human lymphoblasts damaged with bleomycin (BLM) and VP16. These agents can induce DNA breakage but through different mechanisms, enabling the study of the different effects of PARP in inducing apoptosis in damaged cells. We demonstrate that in lymphoblasts VP16 treatment induces apoptosis to a greater extent than BLM treatment, and that PARP inhibition reduces VP16-induced apoptosis whereas it has no effect on BLM-induced apoptosis. After VP16 treatment with PARP inhibition, a reduction in the depletion of the proliferative compartment and a G2/M phase arrest are observed. Therefore, the increase in cell viability and the reduction in chromosome damage may both be the result of a prolonged DNA repair time. Hence, PARP appears to play a significant role in VP16-induced apoptosis and not in BLM-induced apoptosis. Since apoptosis is important in tumor treatment these findings might be useful when considering the combined employment of PARP inhibition with antineoplastic drugs.

The role of multiploidy as unfavorable prognostic variable in colorectal cancer.

OBJECTIVE: To analyze the prognostic value of DNA multiploidy in a prospective study on frozen surgical tissue samples from primary colorectal cancer. SUMMARY BACKGROUND DATA: Survival data from eleven prospective studies collectively comprising about thirteen hundred patients showed that aneuploidy correlated with a 5-year disease-free survival (DFS) significantly poorer than diploidy, and showed the limited prognostic value of results from retrospective studies employing paraffin-embedded material. METHODS: Multiple tumor samples of fresh/frozen surgical tissues from 120 colorectal cancer patients who had undergone radical surgery were taken for flow cytometric analysis of DNA content, and proliferative activity, shown as percentage of cells in S-phase (%S). The minimum follow-up of this series was 30 months. Univariate and multivariate analyses determined the independent significance of both clinical and biological variable on DFS. RESULTS: Values of %S equal to or higher than 17.3 correlated with a 5-year DFS poorer than values lower than 17.3 (44.5% vs 85.2% respectively; p = .03), even if only in patients younger than 64. The subgroup with multiploid tumors showed a significantly poorer 5-year DFS (44.5% vs. 62.6% in the non multiploid patients; p = .02). Subgrouping the Dukes'B stage alone by multiploidy, the difference in DFS was much more evident (31.2% vs. 68% respectively; p = .0004) and multivariate analysis showed multiploidy as the only significant variable. Above all, adjuvant therapy did not absolutely modify the unfavorable outcome of the multiploid Dukes'B patients. CONCLUSIONS: The prospective evaluation of ploidy allowed us to identify a very high-risk subgroup of patients with multiploid tumors. This biological characterization was easy to demonstrate and, above all in node-negative patients, reliable and very effective in terms of prognosis. The presence of multiploidy should result in a more aggressive therapeutic approach in the adjuvant setting.

c-myc antisense oligodeoxynucleotides enhance the efficacy of cisplatin in melanoma chemotherapy in vitro and in nude mice.

This study was designed to assess the efficacy of a new antimelanoma therapeutic strategy that relies on the use of a c-myc antisense 15-mer phosphorothioate oligodeoxynucleotide ([S]ODN), in combination with cisplatin (cis-diamminedichloroplatinum; DDP), which is currently used in the clinical management of melanoma patients. Proliferation and colony formation of melanoma cells were both inhibited by the DDP/c-myc antisense [S]ODN combination to a greater extent than that observed with either agent alone. Inhibition was most effective when DDP was followed by c-myc antisense [S]ODNs. Cell cycle flow cytometric analysis of cells exposed to the two agents either alone or in combination demonstrated that (a) c-myc antisense [S]ODNs induced an accumulation of cells in S phase and apoptosis in a fraction of the cells, detectable at day 5 after the beginning of treatment; (b) DDP induced a block in G2-M phase detectable at day 1, which was partially recovered, and apoptosis similar in extent to that induced by c-myc antisense [S]ODNs; and (c) DDP and c-myc antisense [S]ODNs together induced arrest in G2-M phase, which was maximum at day 3, i.e., delayed as compared to the block induced by DDP. The combination induced a higher percentage of apoptosis, evident at day 3 from the start of treatment, that correlated with a marked reduction in Bcl-2 expression. Mice bearing human melanoma xenografts and treated sequentially with DDP and c-myc antisense [S]ODNs showed a higher inhibition of tumor growth, reduction in the number of lung metastases, and increase in life span compared with those treated with either agent alone. Together, these data lend support to the development of anticancer therapies involving oncogene-targeted antisense ODNs and conventional antineoplastic drugs.

DNA ploidy, proliferative index and EGF-R status in 130 cases of resected gastric cancer--a multivariate analysis.

BACKGROUND/AIMS: The purpose of this study was to define the prognostic role of DNA ploidy, proliferative index and EGF-R status in resected gastric cancer. MATERIALS AND METHODS: Ten clinico-pathological parameters and three biological factors obtained from flow cytometry and immunohisto-chemistry were evaluated in a series of 130 gastric cancer patients who received surgical treatment, including 28 stage IV cases (21.6%), using paraffin-embedded and fresh specimens in 77.7% and 22.3% of the cases, respectively. These variables were first analyzed and tested for correlation within the whole series and then weighted against survival in 117 applicable cases through univariate and multivariate analyses. RESULTS: Aneuploidy was significantly related to higher proliferative activity, EGF-R expression and deeper stomach wall infiltration. Higher proliferative activity was significantly related to deeper stomach wall infiltration and larger tumor diameter. The latter showed a significant relationship to EGF-R expression. Univariate analysis showed the significant variables for survival to be DNA ploidy, pT, pN, M, stage, histological type according to Lauren and tumor diameter. Multivariate analysis calculated on these significant variables using the Cox multiple stepwise regression model detected three factors which independently influence survival: pathological stage (p < 0.00001), histological type according to Lauren (p < 0.002) and DNA ploidy (p < 0.03). CONCLUSIONS: DNA ploidy was shown to be a significant prognostic parameter in resected gastric cancer after pathological stage and histological type according to Lauren. The prognostic roles of proliferative activity and EGF-R status require further investigation.

EGF-R expression in ductal breast cancer: proliferation and prognostic implications.

We studied epidermal growth factor receptor (EGF-R) expression in relation to steroid receptor status, flow cytometric DNA content and S-phase fraction (%S) in a selected case series of 129 ductal primary operable breast cancer to determine the possible role of EGF-R in prognosis assessment. EGF-R expression was positively related with proliferation activity, suggesting that EGF-R could be involved in the regulation of breast cancer cell growth. We found about 80% of highly proliferating DNA aneuploid tumors in the EGF-R positive category, while the EGF-R negative tumors showed a lower frequency of highly proliferating DNA aneuploid tumors (57%), confirming the important role of EGF-R in breast cancer aggressiveness and progression. No relationship between EGF-R expression and steroid receptor status was observed. To better understand how EGF-R and estrogen receptor (ER) operate together to stimulate breast cancer cell growth, we analyzed the %S in the two groups of ER negative (ER-) and ER positive (ER+) tumors, stratifying the patients on the basis of EGF-R tumor positivity. Here breast tumor proliferation activity seems mainly to be induced by the stimulus of EGF-R, the %S values of the EGF-R negative tumors in the ER- and ER+ groups being 6.1 and 6.9%, respectively. Instead, the median %S of EGF-R positive tumors was 10% in the ER- class and 14% in the ER+ group. The analysis of the percentages of 5-year patient disease free survival were 84% for patients with EGF-R negative tumors and 61% for patients with EGF-R positive lesions, respectively. The data reported here further show the crucial role of EGF-R in breast cancer cell growth and that the EGF-R overexpression is indicative of a poor prognosis.

nm23 influences proliferation and differentiation of PC12 cells in response to nerve growth factor.

The nm23 genes codify nucleoside diphosphate kinases, which have been shown to be involved in the regulation of microtubule dynamics. We have demonstrated previously that the association between the Nm23-M1 protein and cytoskeletal beta-tubulin correlates with cell differentiation. It is known that microtubules and microtubule-associated proteins are fundamental elements regulating neuronal differentiation. In the present study, we have investigated the ability of nm23 to influence nerve growth factor-induced PC12 cell differentiation. To this end, we have altered PC12 intracellular levels of nm23-M1 by means of sense and antisense transfections. In the presence of nerve growth factor, overexpression of nm23 delays cell cycle transition, rapidly induces neurite outgrowth, and increases the expression of neurofilament and microtubule proteins. On the contrary, down-regulation of nm23 enhances cell proliferation and inhibits neuronal differentiation. These findings indicate that neuronal cell proliferation and differentiation can be modulated by nm23 expression levels.

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.

Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice.

BACKGROUND: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. PURPOSE: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice, METHODS: The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths and the median number of long metastases in treated and control mice bearing NG xenografts. c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. RESULTS: Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced celluLar levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of long metastases, and an increase in life span. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. CONCLUSIONS: treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-Myc inhibits their growth and is associated with the induction of apoptosis.

Adriamycin resistance modulation induced by lonidamine in human breast cancer cells.

The effect of Lonidamine (LND), an energolytic chemosensitizing agent, on the MDR (multidrug resistant) phenotype of a human breast cancer cell line (MCF-7) has been studied. The intracellular adriamycin (ADR) accumulation and distribution, the plasma membrane potential and the P170 glycoprotein phosphorylation, have been analysed after LND treatment. The analysis of the subcellular localisation of ADR in both wild type and resistant MCF-7 cells treated with ADR or ADR + LND revealed that LND induced an ADR intracellular redistribution in both cell lines. MCF-7 ADR resistant cells exposed to LND (50 micrograms/ml) showed a change in the electrical charges distribution across the plasma membrane and a time-dependent reduction of P170 phosphorylation (70% at 24 hr). These effects were associated with a marked increase in intracellular ADR accumulation in resistant cells.

DNA ploidy, proliferative index, and epidermal growth factor receptor: expression and prognosis in patients with gastric cancers.

BACKGROUND: The 5-year survival rate of patients with stomach cancer is usually around 20%. The clinico-pathological features that are presently used to assess patient prognosis are not sufficient to define gastric tumor behavior. Therefore, an accurate analysis of different biological characteristics of gastric cancer cells could allow the course of disease to be predicted and may help to improve treatment strategies. EXPERIMENTAL DESIGN: The prognostic values of DNA ploidy, proliferative activity and epidermal growth factor receptor (EGF-R) expression were studied in gastric tumors from a series of 63 patients. DNA ploidy and proliferative activity, evaluated in terms of DNA index (DI) and proliferative index (PI), respectively, were determined by flow cytometry on paraffin-embedded tumor tissues. EGF-R expression was detected by immunohistochemistry on paraffin-embedded tumor sections of the same specimens. The clinico-pathological and the biological parameters were then correlated, and the patients overall survival was calculated using a chi-square test and the Kaplan-Meier method. RESULTS: DNA ploidy abnormal cell clones were found in 44% of cases (median DI = 1.4, range 1.04-2.5). Aneuploid tumors showed high PI more frequently than diploids (71% versus 36%, p = 0.01). The analysis of the expression of EGF-R revealed that 88% of aneuploid tumors were positive for receptor expression. On the contrary, diploid tumors showed the presence of EGF-R only in 56% of cases (p = 0.01). DI, PI, and EGF-R expression were not related to histological grade. Conversely, the three biological parameters were significantly correlated to clinical stage and tumor invasion. The Kaplan-Meier survival curves showed a 73% 5-year survival rate in patients with diploid tumors whereas only 33% of patients with aneuploid lesions had a good prognosis (p = 0.001). CONCLUSIONS: We demonstrate that DNA ploidy, PI, and EGF-R expression are closely related to some pathological and clinical characteristics in gastric cancer. The close relationship between aneuploidy, EGF-R positive expression, node involvement, and tumor invasion suggests that these parameters may be indicators of high malignancy. Finally, the results also show that aneuploidy and EGF-R-positive expression are indicative of a worse prognosis in gastric cancer patients. The study of these parameters might allow a more accurate stratification of patients, so that a targeted therapeutic protocol may be defined.

Gastric cancer cell DNA content correlates with early and late results after gastrectomy.

The DNA content of 59 adenocarcinomas of the stomach in patients who had undergone subtotal or total gastrectomy more than 5 years before was measured. The DNA measurements were done by flow cytometry performed on Propidium Iodide--stained cells disaggregated from paraffin-embedded tissues. Fifty-nine evaluable good quality histograms of DNA ploidy patterns were obtained. The Proliferative Index (PI) was determined in 35 cases. The remaining 24 cases didn't show a reliable reading histograms. Of the 59 tumors, 33 (56%) were diploid and 26 (44%) were aneuploid; 19 showed a high PI (> or = 3.8%) and 16 a lower one. A statistically significant difference was found between the two groups (diploid/aneuploid and low/high PI) compared to the prognostic values known as T, N and Stage. 65% of the T3-T4 cancers, 54% of the N1-N2 lesions and 58% of the stage III and IV were found to be aneuploid. 73.7% of the 19 tumors presenting high PI, showed an aneuploid pattern. A high PI was found in 71.4% of the T3-T4 tumors. 77.4% of patients of the diploid group (any stage) survived at 5 years against 36% of those presenting aneuploid patterns. Patients with PI > or = 3.8% showed a 42.1% 5-year survival rate. A 94.4% 5-year survival rate of diploid and early stage cancers was documented against a 33.5% of aneuploid and advanced stage cancers.