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DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA-indices measured after staining with propidium iodide and with 4',-6-diamidino-2-phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stcmlincs, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1.67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA-indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and welldifferentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNAindices of >2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prnstat,e mnst, tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.Key terms: Aneuploidy, DNA, solid tumors, metastases, flow cytometry A significant number of human solid tumors are characterized by abnormalities in cellular DNA content (DNA-aneuploidy) (1-3,lO-12,14-17,19-21). The percentage of tumors with aneuploid cells varies for different types of tumor and also varies for the the same type of tumor when reported by different investigators. This variability indicates that more data should be accumulated before a final classification of tumors according to abnormalities of DNA content can be made, and so that a relationship between this parameter and clinical prognosis may be established.Flow cytometry (FCM) provides a fast and precise method for determination of DNA-aneuploidy index. Using mechanical disaggregation and staining of nonfixed cells, results may be obtained in less than 1 h. However, the usefulness of this parameter for clinical prognosis, selection of treatment, and prediction of response has not been conclusively demonstrated. Although some reports suggest a correlation between DNA-aneuploidy and clinical characteristics of the tumor (17,21), detailed and prolonged observations with larger numbers of patients are necessary to firmly establish such correlations. The relationship between DNA-aneuploidy and metastatic capability is a topic of significant interest and also needs to be studied in a large number of patients....

Fifty-three patients with T1 squamous cell cancer of the floor of mouth and ventral surface of the tongue with a known clinical outcome were retrospectively analyzed and arbitrarily divided into "aggressive" and "nonaggressive" groups based on their clinical behavior. Various host and tumor factors were then evaluated in an attempt to determine whether the tumor behavior could have been predicted. The paraffin-embedded tumor specimens were evaluated for tumor differentiation, tumor thickness and tumor invasion, microvessel density, and p53 expression. In addition, a composite morphologic grading score was obtained by combining cell differentiation, nuclear polymorphism, mitosis activity, depth of infiltration, type of infiltration, and lymphatic infiltration. No single technique appeared capable of identifying "aggressive" behavior, although possibly an evaluation of composite factors might show promise in the future.

Local and regional recurrence is the principal reason for treatment failure in squamous cell carcinoma (SCC) of the head and neck. The conventional method of evaluating surgical margins for cellular atypia does not always predict risk of local recurrence accurately. Immunostaining of surgical margins for tumor markers may provide a more precise evaluation of risk of local recurrence. Paraffin-embedded tissue blocks of surgical margins from 24 patients with oral cavity and oropharyngeal squamous cell carcinoma were immunostained for p53 protein. Fifty-eight percent of the patients had at least one margin stain positive for p53, including eight often patients whose SCC recurred locally. The sample odds ratio test predicted a 5.333 times higher chance of local recurrence with at least one p53 positive surgical margin. The implications of these results for patient management and further investigations will be discussed.

The expression of H-ras, K-ras and N-ras oncogenes was analyzed on frozen sections of squamous cell carcinoma of the head and neck (SCCHN) by immunohistochemistry using anti-ras monoclonal antibodies. Of 22 primary SCCHN, 15 (68%) stained positive for H-ras, 10 (45%) for K-ras and seven (32%) for N-ras. Thirteen specimens (59%) stained positive for at least two anti-ras monoclonal antibodies. The presence of immunohistochemically detectable H-ras, K-ras and N-ras proteins was most frequently associated with an increase in tumor size and later stages of disease (T3 and T4), with no apparent correlation with lymph node involvement, site of occurrence, degree of differentiation, age, sex, or race. Thus, overexpression of members of the ras gene family occurs as a relatively common even in SCCHN and may be an important event in the later stages of tumorigenesis.

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Inactivation of tumor suppressor genes, including p53 and retinoblastoma (Rb), are commonly found in all cancers, including head and neck squamous cell carcinoma. Alterations at either p53 or Rb, however, are only weakly associated with tumor aggressiveness. In many cancers loss of heterozygosity (LOH) at multiple loci is associated with decreased survival. The polymerase chain reaction and highly informative microsatellite markers were used to compare DNA from matched sets of 63 head and neck squamous cell cancers and normal tissue for LOH at the p53 and Rb loci. At p53, 50 were informative, with LOH occurring in 19 (38%). Of the 57 that were informative at Rb, LOH occurred in 21 (37%). Of the 46 that were informative at both p53 and Rb, LOH occurred in 10 (22%) at both loci. When LOH for p53 and Rb individually was compared to stage, differentiation, and survival, there was no correlation. However, the patients with LOH at both loci had a significantly poorer survival (P = .009). This strongly supports the contention that simultaneous alterations of these two tumor suppressor genes favor tumor aggressiveness and can be used as a prognostic indicator.

OBJECTIVE: To compare the frequency of gene expression of matrix metalloproteinases (MMP) stromelysins -1, -2 and -3 (MMP-3, -10, and -11), matrilysin (MMP-7), MT1-MMP (MMP-14), and of TIMPs (Tissue Inhibitors of MMPs) -1, -2, -3 and -4 in head and neck squamous cell carcinomas with those of matched adjacent normal tissues. MATERIALS AND METHODS: The present study included 20 surgically removed head and neck squamous cell carcinomas, seven of which were accompanied by matched adjacent oral mucosa excised from the border of the specimens outside the tumor area. RNA isolated from tumors and control samples was subjected to RT-PCR using primers specific for MMP-3, -7, -10, -11 and -14 and for TIMPs -1, -2, -3, and -4. RESULTS: Our findings demonstrate that each of the five MMP genes studied were expressed in essentially all the tumors, while the adjacent marginal tissue samples showed a more varied picture: while stromelysin-3 was located to a majority of the marginal samples, matrilysin was expressed in four of seven adjacent samples, stromelysin-1 and MT1-MMP genes were each expressed in three of these samples, and stromelysin-2 transcript was only expressed in two marginal tissue samples. Whereas TIMP-1 and TIMP-2 transcripts were identified in all tumor and adjacent tissue samples studied, TIMP-3 was expressed, albeit often at low levels, in 17 of 20 tumor samples but only in three of seven adjacent tissues. The novel TIMP-4 gene was not expressed at all. CONCLUSIONS: Specific MMP (MMP-3, -7, -10, -14) and TIMP-3 transcripts observed in head and neck squamous cell carcinomas compared to their frequency in specimens of matching tissues provide important information about expression of extracellular matrix degrading