Intracellular movement of green fluorescent protein-tagged phosphatidylinositol 3-kinase in response to growth factor receptor signaling.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein-tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein-tagged fragments of p85 we show that binding to the receptor requires the NH(2)-terminal part of the protein as well as its SH2 domains.

Mutations of the epidermal growth factor receptor in non-small cell lung cancer -- search and destroy.

The targeting of the ATP binding pocket of the epidermal growth factor receptor (EGFR) tyrosine kinase, by the small molecule drugs gefitinib and erlotinib, represents a promising new therapeutic strategy in non-small cell lung cancer. However, it is now apparent that only a subset of patients responds to such treatment. Two publications in early 2004 reported the presence of activating mutations in the EGFR tyrosine kinase gene conferring exquisite sensitivity to these drugs. Several publications have since reported prospective data consistent with this finding. This brief review summarises the mutation data from 15 such studies in terms of mutation frequency by clinicopathological features and correlation with response to tyrosine kinase inhibition. A new paradigm for the routine detection of such mutations is needed to facilitate patient selection for treatment and further studies.

Nuclear expression of the c-erbB-4/HER-4 growth factor receptor in invasive breast cancers.

The prevalence and sites of expression of the c-erbB-4 receptor have been determined by immunocytochemical staining in a series of 178 human breast cancers. Most tumors displayed cytoplasmic staining of variable intensity. When compared with adjacent normal tissue, 32 cases (18%) showed lower than normal expression, and 13 (7%) showed greater than normal expression. Nuclear immunoreactivity, confirmed by two different antibodies, was present in 87 cancers (49%) but was found in normal adjacent breast epithelial cells in <5% of cases. There were no significant associations with cytoplasmic or membrane immunoreactivity, but cases showing nuclear expression in >25% of cells were associated with good histological grade, epidermal growth factor receptor expression, c-erbB-3 positivity, cripto, amphiregulin, and transforming growth factor-alpha overexpression.

Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas.

We describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of the epidermal growth factor (EGF) receptor. We have examined a group of ten human tumors including cervical, ovarian, and vulval carcinomas for expression of EGF receptors by immunohistological staining using one of these antibodies and another monoclonal antibody to the extracellular domain of the molecule. The tumors were examined using a sensitive amplified enzyme system and a less sensitive indirect staining method. There was generally a good correlation in staining intensity with the two monoclonal antibody reagents. Both antibodies showed strong staining of squamous cell carcinomas and usually weak or heterogeneous patterns with the adenocarcinomas. Samples of each tumor were solubilized in detergent and analyzed for the presence of functional EGF receptors by immunoprecipitation and autophosphorylation. Three of the squamous cell tumors gave labeled bands, Mr 170,000, on sodium dodecyl sulfate:polyacrylamide gels. DNA was extracted from seven of the tumors and digested with two restriction endonucleases, and the fragments were analyzed on Southern blots using probes representing the extracellular and cytoplasmic domains of the molecule. The tumor DNA showed no apparent rearrangements or amplifications when compared to the EGF receptor gene in human placental DNA. These results suggest that there is a high level of EGF receptors on some squamous cell tumors.

Differential isolation of normal luminal mammary epithelial cells and breast cancer cells from primary and metastatic sites using selective media.

The present studies were aimed at determining if the use of a cell culture medium that supports proliferation of human mammary epithelial cells of the luminal lineage would allow routine isolation of breast cancer cells from primary and metastatic tumor specimens. Results obtained with mammary epithelial cells derived from reduction mammoplasty specimens and primary breast carcinomas indicated that growth of cells on type I collagen-coated dishes in Ham's F-12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, cholera toxin, and 5% fetal bovine serum resulted in the growth and serial passage of cells that stained positively for the luminal cell marker cytokeratin 19. By contrast, growth of mammary epithelial cells in a growth factor-supplemented serum-free medium resulted in the emergence of mammary epithelial cell colonies that were uniformly negative for keratin 19. Filter isolation methods were used to isolate individual keratin-19-positive colonies from primary cultures derived from breast cancer specimens. All of the luminal mammary epithelial cells isolated from breast cancer tissues expressed characteristics of normal cells. Keratin-19-positive colonies isolated from several different tumors all grew rapidly for 30 to 60 days in culture and then senesced. Cells were isolated from one tumor that was known to have undergone a loss of heterozygosity at a specific locus in the p53 gene. All colonies isolated from this specimen contained both p53 alleles, which was consistent with their origin from normal luminal cells. Cells were also isolated from one tumor in which the c-erbB2 protein was drastically overexpressed in the neoplastic cells. Once again, keratin-19-positive colonies isolated from this tumor did not overexpress the c-erbB-2 protein. Experiments were then performed with cells derived from pleural effusions and metastatic lymph nodes. Results obtained with these specimens indicated that the growth conditions that support the growth of normal luminal mammary epithelial cells do not support the growth of neoplastic cells. However, the omission of cholera toxin, epidermal growth factor, and type I collagen substratum resulted in the isolation of two long-term cell lines. Both cell lines have population doubling times of approximately 100 h, are hyperdiploid, and stain positively for cytokeratin 19. Thus, culture conditions that support the growth of normal luminal mammary epithelial cells do not, in general, support the growth of breast cancer cells.

Expression of erbB-4/HER-4 growth factor receptor isoforms in ovarian cancer.

Immunohistochemical expression of erbB4 protein was identified in 93% (49 of 53) of ovarian cancers using the HFR-1 antibody (targeted to the cytoplasmic domain of the erbB4 receptor) and in 89% (47 of 53) of ovarian cancers using the H4.77.16 antibody (targeted to the extracellular domain). Tumors of serous histology were more likely to express a higher level of erbB4 than endometrioid tumors, and for stage III serous tumors, long-term survival was associated with moderate to high coexpression of erbB4 and erbB2. Within ovarian cancer cell lines, high erbB4 expression was associated with cisplatin resistance. Using reverse transcription-PCR, the presence of multiple isoforms of erbB4 mRNA was identified in both ovarian primary tumors and cell lines. Splice variants in the juxtamembrane (JM-a and JM-d) and cytoplasmic (CT-a and CT-b) regions were identified in mRNA of both cell lines and primary tumors. The use of an anti-erbB4 blocking antibody suggested that erbB4 was not the mediator of the growth stimulatory effects of neuregulin in ovarian cancer cells and indeed could potentially antagonize this effect.

Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer.

Sections of formalin-fixed, paraffin-embedded tissue from 185 primary breast carcinomas were stained immunohistochemically using a polyclonal antibody against the c-erbB-2 oncoprotein. Positive staining, which is known to correlate with gene amplification, was associated with earlier relapse, shorter postrelapse survival, and shorter overall survival. Lymph node, epidermal growth factor receptor, and estrogen receptor status, tumor size, and histological grade also had prognostic significance but, applying multivariate analysis, only lymph node status was a more important predictor of relapse-free and overall survival than staining for the oncoprotein. Positive staining was correlated with negative estrogen receptor status and high histological grade, but there was no association with either lymph node or epidermal growth factor receptor status or tumor size. Expression of the c-erbB-2 oncoprotein appears to be an important independent indicator of prognosis in human breast cancer.

Differential immunohistochemical detection of amphiregulin and cripto in human normal colon and colorectal tumors.

Thirty-six primary human colorectal tumors, 43 noninvolved colon samples that were adjacent to either carcinomas of adenomas, 22 adenomas, and nine normal colon specimens were immunohistochemically examined for the presence and localization of two epidermal growth factor-related peptides, amphiregulin (AR) and cripto. Within the primary tumors, 18 (50%) showed moderate levels of AR expression. Approximately 60% of the tubular and tubulovillous adenomas were positive for AR expression, whereas only 15% of the adjacent, noninvolved colon mucosa expressed AR. A greater proportion of well-differentiated tumors (71%) were positive for AR expression than were poorly differentiated tumors (18%). All of the nine normal colon specimens were positive. Consequently, AR expression appeared to be associated with both normal and malignant epithelial cells that were more differentiated. The distribution of cripto expression was different. Seventy-nine % of the colon tumors expressed cripto with a frequency of expression that was approximately equivalent between well-differentiated and poorly differentiated tumors. Approximately 86% of the tubulovillous adenomas, but only 43% of the tubular adenomas, were positive for cripto expression. In contrast, whereas AR was expressed in normal colon specimens, none of these tissues expressed cripto, and only 12% of the noninvolved normal colon samples adjacent to tumors or adenomas were positive for cripto. Cripto expression therefore appeared related to neoplasia. These data suggest that AR and cripto may be functioning as potential autocrine and/or paracrine growth factors in the colon and that the differential expression of cripto may serve as a potential tumor marker for colonic carcinogenesis.

Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading.

Fifty-one primary human breast tumors were analyzed for amplification of the c-erbB-2 protooncogene. Thirteen (25%) of the DNA samples contained multiple gene copies. Paraffin-embedded tumor sections, available from 47 of the cases, were stained with a c-erbB-2 specific antiserum. Eighty-three % (10 of 12) of the tumors containing amplified c-erbB-2 gene copies stained positively with the c-erbB-2 specific antiserum (P = 0.03). Thirteen tumors containing single copy c-erbB-2 sequences also stained positively with the antiserum. This suggests that mechanisms other than gene amplification may lead to elevated levels of c-erbB-2 protein. Finally, there was a statistically significant correlation between c-erbB-2 protein expression and parameters used in breast cancer prognosis. Positive staining was associated with positive nodal status of the patient (P = 0.02) and with tumors showing a poor nuclear grade (P = 0.02). This is the first study showing that a determination of the level of c-erbB-2 protein in paraffin-embedded tumor sections may have prognostic value for the course of human breast cancer.

Prognostic significance of c-erbB-2 and estrogen receptor status in human breast cancer.

Using the 21N polyclonal antibody, we immunohistochemically stained 314 primary breast carcinomas to identify those tumors overexpressing the c-erbB-2 oncoprotein and to ascertain the prognostic significance of this expression on disease-free and overall survival. Positive membrane staining was present in 52 (17%) of these carcinomas of which 7 (13%) were ductal carcinomas in situ. There was no significant relationship between c-erbB-2 positivity and (a) age at diagnosis, (b) menopausal status, (c) tumor size, (d) lymph node status, (e) estrogen receptor status, or (f) whether or not the patient had disseminated disease outside the axillary fields. However, c-erbB-2-positive tumors were significantly associated with poorer grade (P = 0.02). Patients who were positive for this oncoprotein had a shorter disease-free survival (P = 0.002) and reduced overall survival (P = 0.0001). Overexpression of this oncoprotein was predictive of a worse prognosis in lymph node-positive disease (P = 0.003) and in patients presenting with grade II tumors (P = 0.001). Stratifying the patients on the basis of estrogen receptor status suggested that c-erbB-2+/estrogen receptor-negative status was predictive of a poorer prognosis when compared with the other subgroups (P less than 0.001). Primary and recurrent tumor tissues were available from 42 of the 314 patients. Identical patterns of c-erbB-2 expression occurred in 95% of cases, arguing against a direct role for c-erbB-2 expression in the process of tumor dissemination. The high incidence of staining in ductal carcinomas in situ suggests that expression of this oncoprotein is an early event in tumorigenesis. Finally, multivariate analysis indicated that the c-erbB-2 oncoprotein was an independent prognostic indicator for overall survival in breast carcinoma patients.

Specific short transmembrane sequences can inhibit transformation by the mutant neu growth factor receptor in vitro and in vivo.

The neu oncogene is activated by a point mutation within its transmembrane domain that results in the substitution of glutamic acid for valine at position 664, and is associated with constitutive activation of the tyrosine kinase. It has been proposed that the mutation allows for stabilization of homodimers of the receptor that are necessary for transduction of the mitogenic signal. To investigate the role of the alpha-helical transmembrane sequence in the function of neu, we constructed an expression vector to produce a variety of short transmembrane neu proteins, lacking ligand binding or intracellular kinase domains. Such sequences should interact with full-length receptors and prevent receptor dimerization and thus act as specific inhibitors of function. These small proteins all included a pentapeptide from position 661-665, which has been proposed to be necessary for packing. We show that the short transmembrane molecules are expressed at the cell surface and can retard the growth of neu-transformed cells in monolayers, as colonies in soft agar and as tumours in animals. As predicted by molecular modelling, the magnitude of inhibition depended on the nature of the packing surface, suggesting that the neu transmembrane domain is directly involved in neu protein dimerization.

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation.

Expression of a receptor for human macrophage-colony stimulating factor (M-CSF or CSF-1), containing a point mutation which changes an aspartate to a valine at position 802 of the activating loop of the kinase domain, potently transforms the haemopoietic cell line FDC-P1 yet prevents Rat-2 fibroblast transformation. In order to understand this apparent paradox, aspartate 802 was changed by cassette mutagenesis to each of the other 19 amino acids. All hydrophobic amino acid substitutions were transforming when tested in FDC-P1 cells yet inactivating when tested in Rat-2 fibroblasts. These same amino acid substitutions also activated receptor degradation, strongly suggesting a causal relationship between receptor degradation and inactivation in fibroblasts. Point mutations or small deletions of Y708 within the kinase insert region of the mutant D802V receptor partly inhibited receptor degradation. The more stable D802V receptor derivatives were able to transform both FDC-P1 cells and Rat-2 fibroblasts, so establishing that the cell specific effect of the c-fmsD802V activating loop mutation is attributable to receptor degradation which accompanies kinase activation and prevents the transformation of Rat-2 but not of FDC-P1 cells.

Expression of the c-erbB-3 protein in normal human adult and fetal tissues.

c-erbB-3 is a member of the type I family of growth factor receptors which includes the epidermal growth factor (EGF) receptor and c-erbB-2. Whereas for EGF receptor and c-erbB-2 the expression patterns in normal tissues are well documented, there is currently little information available about the sites of c-erbB-3 expression. In order to examine the normal tissue distribution of c-erbB-3, polyclonal antibodies were raised against eight synthetic peptides corresponding to distinct sites on the intracellular domain of c-erbB-3. Of these, three produced antibodies which reacted with a 160-kDa protein on immunoblots of human embryonal cells (293 cells) transfected with the cDNA encoding c-erbB-3, and two of the three antibodies immunoprecipitated a protein of similar size from the same cells. These antibodies were used for immunochemical staining of a wide variety of normal human adult and fetal tissues employing formalin-fixed, paraffin-embedded material. The c-erbB-3 protein was identified in cells of the gastrointestinal, reproductive, respiratory and urinary tracts as well as the skin, endocrine and nervous system in a distribution distinctly different from that observed for EGF receptor and c-erbB-2. The level of expression of the mRNA for c-erbB-3 was also examined in extracts of a selection of fetal tissues. In general the sites of mRNA and protein expression were concordant.

Intracellular expression of a single-chain antibody directed to the EGFR leads to growth inhibition of tumor cells.

A gene encoding a single-chain antibody (scFv) which specifically binds the epidermal growth factor receptor (EGFR) has been constructed from hybridoma cells producing the R1 monoclonal antibody. The gene, designated scFv-R1R, was introduced into EGFR transformed NIH3T3 cells via retroviral infection. scFv-R1R was directed to the lumen of the endoplasmic reticulum (ER) where it bound the extracellular domain of the receptor inhibiting its appearance on the plasma membrane. In these cells, EGF induced tyrosine phosphorylation of the EGFR and several substrates was greatly reduced. Furthermore, intracellular retention of EGFR caused a partial inhibition in the transformed growth of the cells. Intracellular expression of receptor tyrosine kinase directed scFvs is a novel approach for affecting tumor cell growth. We have recently shown that scFv-5R directed to ErbB2, another member of the ErbB family, blocks the anchorage independent growth of ErbB2 transformed cells. In order to examine the effects of scFv-R1R and scFv-5R on the long-term growth of tumor cells overexpressing either EGFR or ErbB2, retroviruses encoding the two scFvs were used to infect various human tumor cell lines. Intracellular expression of the scFvs resulted in a marked inhibition of stable colony formation in some of the cell lines. In general, inhibition was observed when overexpressed receptor was targeted. However, in some cases expression of both scFvs was incompatible with long term cell growth suggesting that heterodimers of ErbB2 and EGFR are essential for the growth of some human tumor cell lines.

Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer.

The rat neu proto-oncogene, which is a putative growth factor receptor closely related to the epidermal growth factor receptor, can be activated in vivo by a single point mutation in the sequence encoding its transmembrane region. The human homologue of neu, c-erbB-2, can be activated in vitro to an oncogenic form by point mutations in the same relative position in the gene. We have sought the presence of such activating mutations in a series of 100 cases of human breast cancer by polymerase chain reaction amplification and sequence-specific oligonucleotide hybridization, and also by a designed restriction fragment length polymorphism strategy in cases with Southern blot evidence of c-erbB-2 amplification. No evidence of activating point mutations in the c-erbB-2 protooncogene was found in any of these cases.

c-erbB3 and c-erbB4 expression is a feature of the endocrine responsive phenotype in clinical breast cancer.

We examined c-erbB3 and c-erbB4 mRNA expression in 47 primary breast cancer samples by simultaneous RT-PCR and have investigated correlations between these parameters and the expression of both ER and EGFR mRNA and protein as measured by RT-PCR and ICA and with Ki67 immunostaining. A direct association was found between c-erbB3 and c-erbB4 mRNA and ER marker status measured by either RT-PCR (c-erbB3 P = 0.0003; c-erbB4 P = 0.02) or ICA (c-erbB-3 P = 0.002; c-erbB4 P = 0.01). Inverse associations were seen between c-erbB3 and c-erbB4 mRNA marker status and EGFR membrane protein (c-erbB3: P = 0.003; cerbB4: P = 0.003) and mRNA (c-erbB4: P = 0.009) status. These associations were reinforced by Spearman Rank Correlation Tests. A significant relationship was seen between Ki67 and c-erbB4 mRNA status and level. Measurements of c-erbB3 protein levels in tumour samples removed from a further 89 patients of known response to endocrine therapy: (i) confirmed the relationship between c-erbB3 and ER and (ii) identified that patients whose ER positive tumours expressed high levels of c-erbB3 were most likely to benefit from endocrine measures. A non-significant trend was recorded between c-erbB3 levels and Ki67 immunostaining. These results clearly demonstrate that increased c-erbB3 and c-erbB4 expression appears to be associated with the prognostically-favourable ER phenotype.

The epidermal growth factor receptor family.

The epidermal growth factor receptor family consists of four receptor genes and at least 11 ligands, several of which are produced in different protein forms. They create an interacting system that has the ability to receive and process information that results in multiple outputs. The family has an important role in directing and coordinating many normal processes, including growth and development, normal tissue turnover and wound healing. Its members are also aberrantly activated by overexpression or mutation in many common human tumour types and as such have been the target for anticancer drug development.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

Planorbis corneus haemoglobin. Circular dichroism and susceptibility to proteases.

The purified haemoglobin of Planorbis corneus was subjected to protease digestion and the resulting products characterised by gel filtration and detergent-gel electrophoresis. Small functional subunits of molecular weights approximately 20,000 were obtained corresponding to a single haem group, but multiples of this unit were also always obtained even at high proteolytic enzyme: haemoglobin ratios. This suggested that the subunits of the native molecule (one-tenth containing perhaps ten O2-binding sites) were made up of single-binding site domains linked by regions of polypeptide chains having different susceptibilities to proteases. The far ultraviolet CD of the native haemoglobin indicated the presence of a high helix content (75--80%) in the protein. The near ultraviolet and visible CD spectra of oxy- deoxy-, and CO-haemoglobin were reported. Planorbis haemoglobin CD was more like that of vertebrate haemoglobins than that of haemoblobins. Nevertheless the Soret CD of Planorbis oxyhaemoglobin had only about half the rotational strength of that of human haemoglobin A, and was halved again upon removal of the ligand. Also in contrast to Lumbricus and human haemoglobins there was only a small decrease in rotational strength in the 260 nm band when Planorbis oxyhaemoglobin was deoxygenated.

Human breast cancer: prognostic significance of the c-erbB-2 oncoprotein compared with epidermal growth factor receptor, DNA ploidy, and conventional pathologic features.

PURPOSE: A study was undertaken to define the prognostic value of the expression of the c-erbB-2 oncoprotein in a series of breast cancer patients when compared by multivariate analysis with expression of the epidermal growth factor receptor (EGFR), DNA ploidy, and conventional clinicopathologic features. PATIENTS AND METHODS: Prognostic indicators were analyzed in 165 primary breast cancers. The c-erbB-2 oncoprotein was recognized by the polyclonal antibody 21N using an immunocytochemical method. Expression of the EGFR was stated immunocytochemically using the monoclonal antibody EGFR1. DNA ploidy was assessed in paraffin-embedded sections using a standard flow-cytometric method. RESULTS: Overall, 27% of carcinomas had membrane 21N-staining and were classified as c-erbB-2-positive. Overexpression of the c-erbB-2 oncoprotein was poorly associated with EGFR expression and the conventional pathologic features, and it was weakly associated with DNA ploidy and nodal status. Univariate analysis showed that c-erbB-2 expression, nodal status, DNA ploidy, and EGFR provided significant prognostic information concerning 4-year relapse-free survival (RFS) with the odds ratios (ORs) of not relapsing of 2.94, 2.83, 2.34, and 2.20, respectively. Regarding overall survival (OS) at 4 years, only nodal status and DNA ploidy had prognostic significance, with the ORs of not dying of 2.68 and 2.80, respectively. Applying multivariate analysis to RFS, 21N when adjusted for nodal status, EGFR, and DNA ploidy (full model) failed to retain prognostic value (P = .202), whereas nodal status was the most significant indicator of relapse (P = .027) followed by DNA ploidy (P = .056) and EGFR (P = .093). CONCLUSIONS: This study suggests that overexpression of the c-erbB-2 oncoprotein appears to be an important indicator of relapse in stage I-II breast cancer when singly evaluated. Multivariate analysis shows that the determination both of nodal status and DNA ploidy improves our ability to identify subsets of patients with different prognoses, and allows for a better selection of patients for systemic adjuvant treatments.