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Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. Methods and results: Ploidy was measured in 22 paraffin-embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker gamma-H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. Conclusions: The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.

The introduction of new technologies, designed to assess biologically relevant alterations in tumour cells, has the potential to provide additional prognostic and predictive information from biopsies, as well as providing a more objective assessment than conventional histopathology alone. One such technique is the assessment of the ploidy status of tumours. DNA ploidy studies, using various technologies, are abundant in the literature, but questions remain as to how such studies should be translated into evidence-based practice. In this Expert Opinion, we provide two perspectives on this field. Baak and Janssen discuss the technology and set the assessment of ploidy in the context of other quantitative techniques. Clinical applications are then illustrated. In the paper by Peter Hall, aspects of the biology of ploidy are discussed, and the potential relevance of a more detailed understanding of the biology of ploidy to clinical application. Some of the general short-comings of the applied literature are discussed and some approaches suggested for the improvement of the evidence base on which healthcare decisions relating to the application of new technologies are made.

BACKGROUND Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.

lntratumoral heterogeneity of DNA ploidy has been identified in breast carcinomas; however, optimal sampling methods have not been determined. In this study of 28 invasive breast carcinomas measuring more than 1.4 cm in greatest dimension, two different techniques for obtaining cells for flow cytometric DNA ploidy analysis were compared. Two solid pieces of tissue were taken from opposite halves of the tumor. A third sample was obtained by scraping multiple cut surfaces of the tumor. Heterogeneity of DNA ploidy was detected in 43% of cases. Most cases demonstrating heterogeneity contained multiple aneuploid populations. However, in five cases classification of the tumors as either DNA euploid or DNA aneuploild differed among samples. A total of 39 non-diploid populations were detected in 23 of the cases. Thirty-thrlee (85%) were detected by scraping and 35 (90%) were detected in either one or both tissue pieces. lntratunioral DNA heterogeneity emphasizes the need for adequate sampling. The scraping technique was as effective in identifying aneuploid cell populations as the combined results of the two pieces of tissue and better than sampling a single piece of tissue. Scraping also offers the advantage of tissue conservation which may be critical when various analytic studies are performed. o 1995 Wiley-Liss, inc. Key terms: DNA neoplasms-analysis, flow cytometry, DNA ploidy, breast neoplasms, heterogeneity

Background/aims: Image analysis in dermatopathology has been used for DNA ploidy analysis, morphometry, stereology, and quantitative immunohistochemistry. The object is to review image analysis in dermatopathology and evaluate these modalities and their application in pigmented lesion pathology, for elucidation of tumor behaviour and architecture and as an aid in tumor identification and prognostication. Conclusion: Image analysis in dermapathology has a huge potential. The techniques are difficult and at present mainly used in specialized centres.

A 62-year-old man with a mucinous carcinoma of the skin on the left cheek is described. The excised specimen showed a characteristic histopathologic picture. The findings from histochemical, immuno-histochemical, and electron microscopic examinations suggested an eccrine differentiation of the tumor cells. The removed tumor was examined using DNA cytofluorometry in order to analyze the cell kinetics and to estimate its biological behavior and prognosis. The tumor cells showed a diploid mode with a small number of polyploid cells. Only a small number of cells in the S and G2/M phase ranges were detected. After four years, there has been no evidence of tumor recurrence nor metastasis. The results of DNA ploidy analysis were thus compatible with the nature of slow growth with a good prognosis in this case.

The histogenesis of atypical fibroxanthoma (AFX) and its relationship to malignant fibrous histiocytoma (MFH) are a subject of controversy. Many investigators have proposed that AFX may represent a reactive process, while others contend that it is a true fibrohistiocytic neoplasm, closely related to MFH. In an attempt to determine whether biologic differences between AFX and MFH may be accounted for at the cellular DNA level, we performed ploidy analysis on 14 cases of AFX by flow cytometry and compared our results with previously reported DNA analyses of MFH. Thirteen of the 14 lesions demonstrated diploid distribution of nuclear DNA, and only 1 case had an aneuploid population. This contrasts with prior data on MFH, the vast majority of which are aneuploid. Our results suggest that, despite histologic similarities, AFX may be distinguished from MFH on the basis of DNA content. These findings may be significant in understanding the biologic behavior of AFX.

A microfluorometric DNA study was conducted on isolated cells from 64 fragments of ovarian epithelial tumors. Data analysis considered the DNA content of the main stemline and the prevalence of cells in the different compartments of the DNA ploidy histogram in relation to the mitotic activity index and the histologic architecture. Main stemlines were found remarkably stable in different parts of the same tumor. Peridiploid (1.8c to 2.2c) stemlines were found both in well-differentiated and in poorly differentiated tumors. However, low aneuploid (2.2c to 3.0c) stemlines were mostly found in nonsolid tumors and high ploidy (3.2 to 5.04 c) stemlines were prevalent in solid tumors. A comparative analysis between DNA ploidy parameters and mitotic activity was useful to evaluate the expanding modalities of neoplastic cell populations. This analysis revealed that several tumors accumulate an excessive amount of heteroploid cells and others an excessive amount of G2 cells. Yet, most neoplastic cell populations vary from slow to rapidly growing patterns without relevant abnormalities in their ploidy graphic profiles. No relationship was found between stemline ploidy and histologic architecture compared with expanding modalities. These findings indicate that multifaced criteria combining features such as main ploidy, graphic profile, mitotic rate, and histologic architecture measured on one or more microsamples of the same tumor may help to objectively estimate the aggressiveness reached by the neoplastic cells at the time of clinical presentation.

Nuclear deoxyribonucleic acid (DNA) ploidy studies with paraffin embedded archival material from 14 pancreatic insulinomas were performed by flow cytometry. Clinical follow-up (2 to 17 years; mean, 8 years) was obtained for all patients. Half of the tumors had a normal DNA histogram and half exhibited an abnormal DNA profile consistent with DNA aneuploidy. Six of the seven patients with aneuploid tumors are alive and disease free (2 to 5 years postresection), and one is alive with metastatic disease. Of the seven patients with tumors showing normal DNA profiles, five are alive and disease free, one is dead of disease, and one is alive with metastatic disease. These data suggest that DNA ploidy analysis is unlikely to provide useful prognostic information for patients with insulinomas.

In this study the authors applied flow cytometric DNA-ploidy analysis to multiple female genital tract malignant tumors in 43 patients, most of whom (n = 37) had bilateral ovarian cancer. An algorithm was developed for calculation of the likelihood ratio of the probabilities that measured DNA index differences between multiple tumor localizations within the same patient could be attributed to measurement variation or to true biologic DNA content differences. The results of this statistical analysis show that in 72% of the cases (31 of 43) this probability ratio exceeded 1. Because the probability that two independent tumors will have a near-identical aneuploid DNA content is very low, this finding supports a metastatic process rather than the occurrence of multiple primary tumors in these patients. Thus, flow cytometric DNA-ploidy analysis can be helpful in the identification of metastatic disease in patients with multiple female genital tract malignant tumors.

The relationship between DNA content, clinicopathologic findings, and patient survival in synovial sarcoma was investigated. Patient age at diagnosis (P less than 0.001), tumor size (P less than 0.001), and ploidy status (P less than 0.003) correlated significantly with patient survival. A marginally significant correlation between mitotic count and patient survival was also observed (P = 0.04). Histologic subtypes (monophasic versus biphasic), mitotic count, and S-phase by flow cytometry had no significant influence on the clinical outcome of patients with synovial sarcoma in this study. The authors conclude that DNA ploidy analysis is a significant objective probe in the prognostication of patients with synovial sarcoma.

The Sixth Annual Clinical Applications of Cytometry Meeting was held September 11-14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.

Renal cell carcinoma is unpredictable in outcome, although the best predictor is tumor stage, followed by histologic grade. The authors retrospectively assessed the clinicopathologic features and DNA ploidy of 103 cases of renal cell carcinoma, the latter determined by flow cytometry of formalin-fixed, paraffin-embedded tissue. The study group comprised 63 men and 40 women (age, 28-80 years; mean, 57 years). Robson stage at diagnosis was Stage I in 52 patients, Stage II in 21, and Stage III in 30. Statistically significant variables in predicting outcome were Robson stage (P less than 0.0001), DNA ploidy (P = 0.0008), mitotic rate (MR, P less than 0.0001), worst nuclear grade (WNG, P = 0.00009), predominant nuclear grade (P = 0.019), and sex (P = 0.044). Tumor size, cell type, and architectural pattern were also assessed but did not prove to be significant. Statistically significant associations occurred between DNA ploidy and WNG (P less than 0.0001), stage (P = 0.0037), and MR (P = 0.015); between WNG and MR (P less than 0.0001) and stage (P = 0.0007); and between stage and MR (P = 0.002). Cox proportional hazards regression analysis of all significant variables showed Robson stage, tumor ploidy, and MR to be independent, significant predictors of outcome. If ploidy data had not been available, WNG would have been independently significant. The authors conclude that DNA ploidy analysis provides significant predictive information on renal cell carcinoma.

Adrenal cortical carcinoma is an uncommon neoplasm in children. Only a handful of congenital adrenal cortical carcinoma cases have been described. A newborn who had metastatic adrenal cortical carcinoma (skin metastases and cerebral lesions) is described. This patient underwent surgical resection of the right adrenal primary, but no further treatment was given. Hemihypertrophy developed in this patient by 2 months of age, and at 4 months of age spontaneous regression of all skin nodules and central nervous system (CNS) lesions was observed. Follow-up at 1 year shows the patient to be alive, well, and disease-free. Evaluation of the tumor included DNA ploidy analysis that showed the tumor to be polyploid, a pattern recently associated with nonmetastasizing adrenal cortical neoplasm. The observation of apparent metastatic disease that regressed spontaneously highlights the prognostic value of DNA ploidy analysis and raises the possibility of an adrenal tumor with properties similar to those of Stage IV-S neuroblastoma.