Immunocytochemical Staining of Serous Effusions With the Monoclonal Antibody Ber-Ep4

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.     

Comparison of cytologic and acridine-orange flow-cytometric detection of malignant cells in human body cavity fluids

Flow cytometrically (FCM) derived DNA and RNA profiles were studied in acridine orange (AO)-stained body cavity fluid (BCF) specimens obtained from 78 patients with various solid tissue and hematologic malignancies. The ploidy (DNA index), RNA content (RNA index), proliferative activity (% S + G2M) and DNA and RNA scattergram patterns were tested "double-blind" against the cytologic scoring of specimens as malignant, benign or reactive. It was determined that expression of an "abnormal" RNA index (greater than or equal to 2.8) and an elevated proliferative activity (% S + G2M greater than or equal to 7.4) was dependent on the presence of malignancy; 21 of 22 specimens having those abnormal indices had DNA aneuploidy and were cytologically scored as positive. The AO FCM sensitivity and specificity for detecting malignant cells (when measured against cytology scoring) were 61% and 90%, respectively, using the "abnormal" RNA index and % S + G2M cut-offs together with the cellular DNA aneuploidy marker. By supplementing the cytologic scoring with AO FCM DNA and RNA features, the sensitivity for detecting malignant cells was 94%, as compared to 72% for cytology alone. Two specimens gave false-positive FCM results: a tuberculous effusion with a tetraploid subpopulation and a reactive mesothelial proliferation that was diploid and negative cytologically. Scoring for malignancy based on the visual pattern of the DNA and RNA FCM scattergrams, while showing good correlation for aneuploid specimens, in some cases failed to identify diploid disease. The results demonstrate the usefulness of FCM DNA and RNA analysis for supplementing cytologic examination of BCF specimens for the purpose of detecting malignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytologic findings in vitreous fluids. Analysis of 74 specimens

OBJECTIVE: To review the cytologic findings of vitreous specimens and propose a simplified approach to them. STUDY DESIGN: Seventy-four vitreous specimens from 60 patients obtained either during a pars plana vitrectomy or by vitreous aspiration were reviewed. Clinical correlation was obtained on all patients. RESULTS: Findings suggestive of a specific disorder were present in 30 specimens (41%); cytologic examination of the remaining 44 showed nonspecific changes. A lymphoproliferative disorder, the intraocular malignancy suspected most often in this series, was identified in eight specimens (11%). Large cell lymphomas were evident in 5 specimens, 2 specimens were suspicious for lymphoma, and 1 specimen was consistent with plasmacytoma. Twelve specimens (16%) contained hemorrhage. In rare instances, specific infectious agents, such as parasites (5%), bacteria (1%) and fungi (3%), could be identified. The diagnosis of viral infections required ancillary studies. Lens fragments were identified in four cases (5%), and a diagnosis of lens-induced endophthalmitis could be rendered in one case (1%). Changes consistent with sarcoidosis were present in 3% of cases. CONCLUSION: Based on this experience with vitreous specimens submitted for clinical reasons, we found that they could be divided into three broad diagnostic categories: inflammation/infection (54 specimens/41 patients), hemorrhage (12 specimens/12 patients) and malignancy (8 specimens/7 patients).     

Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens

Cytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin-embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin-Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High-resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.     

Cytomorphological and immunocytochemical study of non-Hodgkin's lymphoma in pleural effusion and ascitic fluid

INTRODUCTION: Non-Hodgkin's lymphoma (NHL) is often complicated by pleural effusion and ascites. The present study is an attempt to categorize the lymphomatous effusions according to the WHO classification, using archival material. METHODS: May-Grünwald-Giemsa and Papanicolaou-stained smears of 31 lymphomatous effusion specimens were reviewed. Of these, detailed cytological assessment was done on 12 pleural effusions and ten ascitic fluid specimens from 22 patients using the WHO lymphoma classification system. Immunocytochemical studies were performed in 21 specimens. RESULTS: Based on cytomorphological features, the 22 lymphomatous effusion specimens were categorized into lymphoplasmacytoid lymphoma (1), follicle centre cell (FCC) grade-1 (centrocytic) lymphoma (3), FCC grade-2 (centrocytic-centroblastic) lymphoma (3), FCC grade-3 (centroblastic) lymphoma (4), large cell immunoblastic lymphoma (4), lymphoblastic lymphoma (2), anaplastic large cell lymphoma (3) and miscellaneous types (2). Immunocytochemically, the lymphoma cells were T-cell (positive for CD3) and B-cell type (CD20 positive) in five and six cases respectively. CONCLUSION: Cytological examination of pleural effusion and ascitic fluid samples, supported by immunocytochemical studies, may be useful for the classification of lymphomas under the WHO system.     

Contextual analysis and intermediate cell markers enhance high-resolution cell image analysis for automated cervical smear diagnosis.

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.     

Nuclear texture features for classifying benign vs. dysplastic or malignant squamous epithelium of the larynx.

OBJECTIVE: To search for nuclear features and feature combinations able to assess malignancy and premalignant changes on tissue sections of laryngeal squamous epithelium. STUDY DESIGN: A total of 139 lesions of benign changes (BC) (n = 44), epithelial dysplasias (ED) (n = 50) and invasive laryngeal cancer (LC) (n = 45) were retrieved from archival pathology specimens. The goal of this study was to identify the best features or feature combinations that discriminate BC from LC and also reflect the degree of ED. In order to verify the results on independent data, the groups were split into two separate subgroups, one for training and one for testing. RESULTS: On the test set of slides, the overall correct classification of BC vs. LC cases was 82% using only one feature, fractal2_area. This classification rate could be increased to 91% when a discriminant function based on 10 features was used. However, this gain was not significant. CONCLUSION: Fractal texture features can be used to assess malignancy on tissue sections as an alternative to DNA measurement. In this study feature combinations did not significantly improve classification rates.     

Proffered papers 14.00–15.00 Tuesday 16 September 2003

Background  ERCP-directed brush cytology is used to sample lesions of the pancreatic and biliary ducts and the ampulla of Vater. With conventional preparations, the sensitivity and specificity range from 44% to 63% and 80% to 98%, respectively, and increased N : C ratio, nuclear molding and loss of honeycombing are reliable features of malignancy. The performance and morphology of specimens prepared by ThinPrep, a liquid-based cytology technique is mostly unknown.
Methods  The laboratory information system was searched for all cases prepared by ThinPrep. Patient disease classification of benign or malignant was determined by linkage with the provincial cancer registry and was the gold standard against which sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. True positives and negatives were reviewed to identify predictive cytomorphologic features.
Results  Between 1996 and 2001, there were 149 ThinPrep specimens; 55 (37%) were reported as positive for malignancy and 94 (63%) as negative. Disease was classified as malignant in 86 (58%) patients and benign in 63 (42%). There were 42 false negative, 11 false positive, 52 true negative, and 44 true positive cytology results. Sensitivity was 51.2% (CI; 40.2 : 62.0), specificity 82.5% (CI; 70.5 : 90.6), and PPV and NPV 80.0% (CI; 66.6 : 89) and 55.3% (CI; 44.7 : 65.5), respectively. Cell groups with crowded, enlarged, irregular nuclei and nuclear features of vesicular chromatin and large, multiple irregular nucleoli correlated with malignant disease, while monolayered sheets of uniform columnar cells, regular nuclei and a finely granular chromatin correlated with benign disease.
Conclusions  The performance of ThinPrep brushings from this anatomic site equals conventional preparations. Cytomorphologic features of malignancy are more frequent and pronounced with ThinPrep.     

Proffered papers 14.00–15.00 Tuesday 16 September 2003 8 Ampullary,pancreatic and biliary duct exfoliative brush cytology prepared by the thinprep method: performance and morphology

Background ERCP‐directed brush cytology is used to sample lesions of the pancreatic and biliary ducts and the ampulla of Vater. With conventional preparations, the sensitivity and specificity range from 44% to 63% and 80% to 98%, respectively, and increased N : C ratio, nuclear molding and loss of honeycombing are reliable features of malignancy. The performance and morphology of specimens prepared by ThinPrep, a liquid‐based cytology technique is mostly unknown. Methods The laboratory information system was searched for all cases prepared by ThinPrep. Patient disease classification of benign or malignant was determined by linkage with the provincial cancer registry and was the gold standard against which sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. True positives and negatives were reviewed to identify predictive cytomorphologic features. Results Between 1996 and 2001, there were 149 ThinPrep specimens; 55 (37%) were reported as positive for malignancy and 94 (63%) as negative. Disease was classified as malignant in 86 (58%) patients and benign in 63 (42%). There were 42 false negative, 11 false positive, 52 true negative, and 44 true positive cytology results. Sensitivity was 51.2% (CI; 40.2 : 62.0), specificity 82.5% (CI; 70.5 : 90.6), and PPV and NPV 80.0% (CI; 66.6 : 89) and 55.3% (CI; 44.7 : 65.5), respectively. Cell groups with crowded, enlarged, irregular nuclei and nuclear features of vesicular chromatin and large, multiple irregular nucleoli correlated with malignant disease, while monolayered sheets of uniform columnar cells, regular nuclei and a finely granular chromatin correlated with benign disease. Conclusions The performance of ThinPrep brushings from this anatomic site equals conventional preparations. Cytomorphologic features of malignancy are more frequent and pronounced with ThinPrep.     

Improved identification of malignant cells in serous effusions using a small, robust panel of antibodies on paraffin-embedded cell suspensions

OBJECTIVE: To evaluate the expression of these markers individually and to find out which markers would be the most effective in a diagnostic panel to reliably discriminate these lesions. STUDY DESIGN: Sections from cell blocks of these fluids were stained with antibodies against calretinin, EMA, HMFG-2, BerEp4, B72.3 and CEA. A preliminary diagnosis was formulated based on cytomorphologic criteria. Subsequently, results of all 6 immunocytochemical stainings were evaluated, the most effective diagnostic pane of antibodies was proposed and staining results using this panel were compared to results obtained by solely cytomorphologic evaluation and to the ultimate diagnosis. RESULTS: Additional immunocytochemical staining with the proposed panel of calretinin, EMA, HMFG-2 and CEA improved sensitivity for malignancy in general from 78% to 96% and specificity from 73% to 91%. Sensitivity for malignant mesothelioma increased from 45% to 91%, with an increase in specificity from 87% to 96%. Sensitivity for adenocarcinoma decreased slightly from 100% to 92%, but specificity increased from 86% to 100%. Overall, diagnostic accuracy increased from 76% to 94%. CONCLUSION: Immunocytochemical staining of standardized cell block reparations of serous fluid cells with a small panel of 4 antibodies significantly improves diagnostic results compared to cytomorphologic evaluation alone.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

Fine needle aspiration of the thyroid: can an image processing system improve differentiation?

OBJECTIVE: To differentiate thyroid nodules by means of fine needle aspiration with subsequent computer-aided diagnosis. STUDY DESIGN: Fine needle aspiration biopsies obtained from 137 patients for whom histopathologic diagnosis was available were investigated: 16 hyperplastic nodules (12%), 39 adenomas (28%), 25 follicular thyroid carcinomas (18%), 19 follicular variants of papillary thyroid carcinoma (14%) and 38 papillary thyroid carcinomas (28%). From each stained specimen 100 cell scenes were scanned and analyzed, constituting a total of 62,325 cell images. RESULTS: All the entities described could be well discriminated from each other by automated image analysis methods. Both the diagnosis of tumor type and the differentiation between benign and malignant could be achieved with a sensitivity of .98. CONCLUSION: With only 7-10 calculated cell features (texture line analysis) and classification with decision trees, a tool for high-quality cell image diagnosis is available. Subtypes of cells characterized by the calculated features could be found in all the specimens and could be assigned to the malignancies with high statistical significance. The method increases the relevance of image processing as an additional diagnostic tool for cytologic examination of thyroid nodules.     

Diagnostic accuracy of cytology and immunocytology in carcinomatous effusions

Background: Immunocytology substantially improves the diagnostic accuracy of conventional cytology in the diagnosis of carcinomatous effusions. Due to the unequivocal characterization of the various cell populations, a sensitivity of 92% and specificity of 100% was achieved by immunocytology, examining samples of 1234 serous effusions. Objective: Cytology plays a central role in the aetiological clarification of serous effusions. The sensitivity of this method for the diagnosis of carcinomatous effusions varies between 40% and 80%. The aim of the present study was to investigate whether immunocytology substantially improves the diagnostic quality of the cytological examination in the diagnosis of carcinomatous effusions. Method: Consecutive serous effusions were examined by conventional cytology and by immunocytology. The immunocytological examination was performed on smears, using a standard panel of three antibodies against pancytokeratin, human epithelial antigen 125 and calretinin. Results: Altogether, 1234 effusion samples were examined. A total of 603 effusions were caused by carcinomas, five by malignant mesotheliomas, 11 by malignant lymphomas and 615 by non‐malignant disorders. In conventional cytology, carcinomatous effusions were correctly diagnosed in 314 samples, corresponding to a sensitivity of 52%. In 31 specimens (5%) tumour cells without further specification were described and in 161 samples (27%) the presence of tumour cells was suspected (84% overall sensitivity). A total of 97 carcinomatous effusions (16%) were diagnosed false‐negatively and 50 (8%) of the 615 non‐malignant effusions false‐positively (92% specificity). In immunocytology, 561 carcinomatous samples were correctly diagnosed, representing a sensitivity of 93%. In six cases (1%) the presence of tumour cells was suspected. A total of 36 carcinomatous effusions (6%) were diagnosed false‐negatively (94% over‐all sensitivity). Out of the 615 non‐malignant specimens, there were no false‐positive diagnoses (100% specificity). Conclusion: Immunocytology is a simple, cost‐effective, routinely practicable method which substantially improves the diagnostic accuracy of conventional cytology in the diagnosis of carcinomatous effusions. Therefore, we recommend the use of immunocytology in all those cases where cytology on its own is not completely unequivocal.     

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.     

Brushing cytology of the upper gastrointestinal tract. Obsolete or not?

The merit of brushing cytology of the upper gastrointestinal tract has been questioned since it appears to duplicate biopsy. To determine its value, the reports on all endoscopic biopsy and cytology specimens from a one-year period were reviewed. The 683 procedures included 481 in which only a histologic biopsy sample was obtained, 47 in which only a cytologic brushing sample was obtained and 155 in which both types of samples were obtained ("combined specimens"). Among the 155 combined specimens, 4 of the confirmed malignancies were detected by histology only while 2 were detected by cytology only. A diagnosis of suspicious or positive for malignancy was made for 20% of the combined specimens as compared to less than 5% of the biopsy-only or brushing-only specimens. While 15% of the specimens that included a brushing sample (either alone or with biopsy) showed fungal infections, only 1.2% of the biopsy-only specimens did. It appears that the clinicians at this hospital tend to use brushing in combination with biopsy when either a malignancy or a fungal infection is suspected. Other empiric advantages of endoscopic brushing cytology include its rapid turn-around time, minimal invasiveness and good recognition of lymphoid cells. The selective use of brushing cytology should increase the probability of detecting malignancies and fungal infections without any increased risk or discomfort to the patients.     

Metastatic transitional cell carcinoma causing a unilateral pleural effusion: a case report

BACKGROUND: Transitional cell carcinoma (TCC) is a common neoplasm, but it is only rarely associated with serous effusions. The cytologic features of metastatic TCC in pleural effusions have been described only in occasional studies. One feature that raises the possibility of metastatic TCC in this setting is the presence of eosinophilic cytoplasmic inclusions (ECIs). CASE: Metastatic TCC was diagnosed in a pleural fluid from a 50-year-old man with a unilateral effusion. Two years previously he had been diagnosed with a poorly differentiated TCC of the urinary bladder (WHO grade 3, stage pT2 at least), and more recently he had also been diagnosed with an omental metastasis. Cytologic examination of the pleural fluid sample revealed numerous pleomorphic malignant cells, many of which were vacuolated. Numerous eosinophilic inclusions were identified within the malignant cells in the liquid based cytology (ThinPrep) preparation. Examination of the omental cake biopsy revealed similar appearances. CONCLUSION: ECIs within malignant pleural effusion fluid specimens should, if detected, raise the possibility of metastatic transitional cell carcinoma.     

The distinction of mesothelioma from adenocarcinoma in malignant effusions by electron microscopy

To determine the usefulness of the electron microscopic (EM) differential diagnosis between malignant mesothelioma and metastatic adenocarcinoma in cytologic specimens of serous fluids, we undertook a prospective study of 17 pleural and peritoneal effusions from 14 patients. In the nine effusions identified as malignant by routine cytologic examination, EM correctly diagnosed three mesotheliomas and six adenocarcinomas. EM resolved the differential diagnosis of mesothelioma versus adenocarcinoma in three cases in which routine cytologic examination could not. As with tissue specimens, EM cannot be used to diagnose the malignancy of cytologic specimens; it can, however, reliably identify the origin of cells diagnosed as malignant by routine cytologic examination. We conclude that, when EM is used to evaluate cytologically malignant effusions, it can accurately distinguish mesothelioma from adenocarcinoma. This technique will be diagnostically useful in selected cases and may be helpful in avoiding more invasive procedures as well as delays in diagnosis and therapy.     

The usefulness of a panel of immunostains in the diagnosis and differentiation of metastatic malignancies in pericardial effusions

Pericardial effusions are not uncommon in patients with an advanced malignancy Rarely malignancies may present initially with a pericardial effusion. Cytological examination of pericardial fluid may be valuable in differentiation of these cases. However, a metastatic tumour in serous effusion may not always show the functional differentiation of the primary tumour. In such a situation, although a wide range of special studies have been suggested for the diagnosis of malignancy we have found the use of a panel of a few common immunostains to be useful in confirming or suggesting the site of a primary tumour. The material for this study consisted of 76 pericardial fluids obtained between January 1991 and October 1998 from 46 males (mean age 59 years) and 30 females (mean age 52 years). Metastatic malignancy was diagnosed in 22 of the 76 patients and in 7/22 cases pericardial effusions were the initial presentation. The subsequent follow-up in the seven cases revealed adenocarcinoma of lung (n = 2), small cell anaplastic carcinoma of lung (n = 1), squamous cell carcinoma lung (n = 1), melanoma leg (n = 1), non-Hodgkin's lymphoma retroperitoneal lymph nodes (n = 1) and carcinoma of the breast (n = 1). Of the remaining 15 cases with a known history of malignancy, eight had cancers (three adeno; two small cell; one poorly differentiated, and two squamous cell types) of the lung; breast (n = 3); colon (n = 1); melanoma (n = 2) and non Hodgkin's lymphoma (n = 1). Immunostains which were useful in the diagnosis were EMA, CEA, cytokeratin, B72.3, HMB45, vimentin, S100, LCA, L26 and kappa and lambda light chains.     

Biliary tract brush cytology

OBJECTIVE: To prospectively review brush smears obtained during endoscopic retrograde cholangiopancreatography (ERCP) primarily from the biliary tree. STUDY DESIGN: A total of 175 specimens from 147 patients were included in the study. The smears, prepared directly from the endoscopic brush, were stained by the Papanicolaou technique and analyzed for standard cytologic features. RESULTS: The smears were categorized into benign/reactive, significant atypia and suspicious/positive. The consistent features seen in suspicious or positive smears were tightly cohesive, small, three-dimensional cell clusters that formed cell balls. The cells in the clusters displayed features of malignant cells. CONCLUSION: ERCP-guided brushing is a safe diagnostic procedure for the evaluation of biliary tree lesions. Small, three-dimensional epithelial clusters with marked atypia signify malignancy and warrant the diagnosis of a malignant neoplasm even when only one or two such clusters are seen in the smears. Single cells, cytoplasmic vacuoles and prominent nucleoli are not essential for a diagnosis of malignancy.     

Flow cytometric analysis of head and neck carcinoma DNA index and S-fraction from paraffin-embedded sections: comparison with malignancy grading

Archival, paraffin-embedded, pathology specimens representing pretreatment tissue biopsies from 73 patients with epidermoid carcinoma of the head and neck were analyzed for DNA Index and %S-phase cells by flow cytometry and were scored for quantitative histomorphology. The DNA fluorescence/light scatter size patterns derived from paraffin-embedded specimens were shown to be essentially the same as those from mechanically disaggregated, ethanol-fixed cells obtained from the same tissue specimen. Patterns ranged from lymphocyte-like to highly abnormal DNA Index cytokinetic patterns. The DNA Index values ranged from 0.70 to 3.50 (median 1.42), with an aneuploidy frequency of 63/73 (86%). DNA distribution %S ranged from 4% to 45% (mean 19), with the microscopic malignancy grading showing broad heterogeneity (mean 2.1, range 1.0-3.0, where 1.0-1.7 = well differentiated, 1.8-2.3 = moderately differentiated, 2.4-3.0 = poorly differentiated). Cross-comparison of these data showed that (1) the tumor %S was dependent on DNA Index (higher %S at higher ploidy), (2) low to high malignancy tumors were randomly distributed between diploid/near diploid tumors and high-degree DNA abnormality tumors, and (3) proliferative activity values broadly overlapped between low to high malignancy scored tumors. However, those carcinomas characterized by high DNA Index (greater than or equal to 1.50) and high %S-phase fractions (greater than or equal to 20) had a five fold higher incidence of high-degree malignancy, invasive tumors than diploid/near diploid (%S less than or equal to 19) tumors.