Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

Comparison of the intratumor DNA ploidy distribution pattern between differentiated and undifferentiated gastric carcinoma

OBJECTIVE: To compare the differences in intratumor DNA ploidy distribution pattern between differentiated and undifferentiated carcinoma of the stomach. STUDY DESIGN: Two hundred five cases of surgically resected gastric carcinoma were studied, comprising 125 differentiated and 80 undifferentiated carcinomas. DNA ploidy was determined by means of flow cytometry in stepwise sections of the entire tumor. The intratumor DNA ploidy distribution pattern was compared between these two histologic types. RESULTS: Differentiated carcinomas comprised 35 cases of predominantly diploid (28%) and 90 cases of predominantly aneuploid (72%) tumors, while undifferentiated carcinomas comprised 46 cases of predominantly diploid (57.5%) and 34 cases of predominantly aneuploid (42.5%) (P < .01). The frequency of cases showing predominantly aneuploidy among the differentiated carcinomas was significantly higher than among the undifferentiated carcinomas at stages IA and IIIA. The rate of cases showing heterogeneity was lower among the early-stage cases than among the advanced-stage cases in both histologic types. Among the differentiated carcinomas, there were 22 cases that had more than six DNA stemlines in each tumor, whereas among undifferentiated carcinomas, there were six such cases (P < .05). CONCLUSION: There were more cases with high malignant potential among the differentiated carcinoma cases than among the undifferentiated carcinoma cases in the present series.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffinembedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p<0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffin-embedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p less than 0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

DNA ploidy of human breast cancer

Ploidy was determined on 663 resectable primary tumors from untreated patients. Nuclei obtained by mechanical disaggregation of frozen tissue were stained with propidium iodide and analysed in a FACS IV. Aneuploidy was detected in 73% of cases. It was not significantly related to nodal involvement or tumor size, although the highest frequencies were observed in large tumors (88%) or with more than 10 positive nodes (77%). Aneuploidy was more frequently observed in ductal infiltrating (81%) than in lobular histology and in tumors lacking both progesterone and estrogen receptors (85%). Analysis of ploidy in primary and synchronous lymph node metastases from the same patient showed a high agreement rate (90%) of DNA patterns simply defined as diploid or aneuploid. However, differences in DNA stemlines and DNA indices between the two synchronous lesions from the same patient were a rather frequent event.     

Flow cytometric analysis of DNA stemline heterogeneity in primary and metastatic breast cancer.

Flow cytometric DNA-ploidy analysis was used to investigate intratumor DNA stemline heterogeneity in primary breast carcinomas and lymph node metastases (LNM). The study was done in tumor specimens from 44 patients 35 of whom had LNM. In all, measurements were done in 214 different samples of primary tumors and 211 lymph nodes. Sixty-one percent (27/44) of the primary tumors were found to have multiple DNA aneuploid stemlines when the data of the separate samples per tumor (mean 4.9) were compared. Only five of 44 (11%) primary tumors were DNA diploid; two of these had DNA aneuploid metastases. Statistical analysis of these results indicated that, on average, four samples are needed for reliable determination of the DNA ploidy status of primary tumors by flow cytometry. In the majority of the cases (26/35), distinct tumor DNA stemlines found in LNM were also present in the primary tumor, which suggests that the generation of DNA ploidy diversity may have taken place prior to metastasis. Multiploidy was not related to tumor size but, particularly for LNM, was significantly correlated with age (r = 0.40, P = 0.02). The results of this study support the view that breast cancer is an extremely heterogeneous disease and that underestimation of this factor might account for the disagreement in literature about the prognostic value of DNA ploidy determinations.     

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.     

The prognostic significance of the DNA distribution in squamous-cell carcinomas of the uterine cervix

To establish the prognostic parameters of DNA distributions of squamous-cell carcinomas of the uterine cervix, cervical smears from 30 patients were examined cytomorphometrically ten years after the diagnosis had been made. Most of the patients had stage II cervical carcinomas that had been treated with primary combined irradiation. In the DNA histograms, the position of the stemline proved to be the prognostically significant factor. All of the patients with diploid or euploid-polyploid stemlines were still alive after ten years while only 17.6% of those with aneuploid stemlines have survived. No correlation between the ten-year survival rate and the proliferation rate of the tumors could be established.     

Cytophotomeric DNA-analysis of aspirated cells from prostatic carcinoma.

In the present study material obtained from prostatic lesions by transrectal aspiration biopsy was subjected to a comparative morphologic and cytophotometric DNA analysis. Based on the morphologic pattern, the clinical material was divided into benign lesions (prostatic hyperplasia), suspected prostatic malignancy and highly, moderately and poorly differentiated prostatic carcinoma. The cytochemical analysis, based on quantitative cytophotometric measurements of Feulgen stained nuclei, showed that cell nuclei from benign lesions (prostatic hyperplasia) exhibited the normal diploid amount of DNA. Contrary to this, cell populations from prostatic malignancies, were characterized by various degrees of heteroploidy, i.e. the existence of cells with nuclear DNA quantities increased above the normal diploid level. A general correlation between degree of heteroploidy (frequency of heteroploid cells) and degree of clinical malignancy seemed to exist; high grade malignant prostatic carcinoma (poorly differentiated) exhibited a pronounced degree of heteroploidy with more or less distinct aneuploid stemlines, whereas low-grade prostatic carcinomas (highly differentiated) were more similar to benign cell populations, in showing a large proportion of cells with a diploid DNA quantity suggesting the existence of diploid or near diploid stemlines. Cases morphologically classified as moderately differentiated prostatic carcinoma, which previously have been shown to exhibit individual variations in degree of clinical malignancy, also showed large individual variations in degree of hetyroploidy. Approximately half of these cases had cytochemical DNA characteristics similar to that of highly differentiated prostatic carcinoma in showing a modal DNA value in the diploid region, while the other half showed cytochemical characteristics similar to that of poorly differentiated prostatic carcinoma, i.e. aneuploid DNA distributions. However, no morphologiifferentiated prostatic carcinoma. In conclusion it can be stated that the present investigation suggests the possibility that quantitative cytochemical DNA analysis may be used in combination with, and offer additional information to, morphologic analysis in the malignancy grading of prostatic carcinoma. A future clinical follow-up, now in progress, will hopefully give a more definite answer to that idea.     

DNA ploidy pattern and S-phase characteristics of metastatic melanoma

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

Sputum cytology of metastatic carcinoma of the lung.

The cytopathology of 47 cases of metastatic carcinoma of the lung and of 28 cases of recurrent or metastatic bronchogenic carcinoma is reviewed. The diagnostic yield was better for recurrent than for metastatic carcinoma but overall was comparable to that of primary bronchogenic carcinomas. The metastatic tumors were located in all areas of the lung and included single as well as multiple lesions. The positive yield did not differ significantly in relation to any of the pathologic features but was somewhat higher if the metastases were large and centrally located. A definite differentiation of the metastatic tumors, usually adenocarcinomas, from new primary bronchogenic carcinomas is often possible particularly if the cytopathology can be compared with that of the primary lesion. Specific cytologic features include the relative lack of cohesion and the formation of columns in metastatic breast carcinomas, the formation of larger cohesive well circumscribed nodules composed of tall columnar cells in metastatic colon carcinomas, clear cell features in some metastatic adenocarcinomas of the kidney, and the small cell size and uniform, regular nuclear features in the often cytologically well differentiated metastatic carcinomas of the prostate.     

DNA ploidy pattern and S-phase characteristics of metastatic melanoma.

Samples of 130 metastatic melanomas from 92 patients were analyzed by DNA flow cytometry. DNA aneuploidy was observed in 67% of the patients. DNA indices were evenly distributed from 0.6 to 2.6 Tumors originating from primary lesions in the lower extremities were more frequently DNA aneuploid than those of other sites. S-phase fraction (SPF) was evaluable from 73 tumors. DNA aneuploid tumors had a significantly higher SPF than diploid tumors, and females had a higher SPF than males. Furthermore, distant metastases had a higher SPF than metastases in regional lymph nodes and in transit metastases, probably indicating a higher growth potential in metastases spreading to distant sites.     

Squamous cell carcinoma antigen in genitourinary tumors

We have evaluated the serum levels of squamous cell carcinoma (SCC) antigen in patients with adenocarcinoma of the kidney, adenocarcinoma of the prostate, germ cell tumors of the testis, transitional cell carcinoma of the bladder, and SCC of the penis, urethra, and bladder. Serum SCC antigen levels were elevated in 5 of 11 patients (45%) with metastatic SCC of the penis, and in the 3 patients for whom serial determinations were made, the serum levels correlated correctly with the progression of disease or response to treatment. The antigen was elevated in 1 of 3 patients with SCC of the urethra, and 1 apparent false-positive value was observed in a patient with adenocarcinoma of the prostate. Otherwise, no SCC antigen elevations were noted among 10 patients with metastatic adenocarcinoma of the prostate, 8 with metastatic adenocarcinoma of the kidney, 11 with metastatic transitional cell carcinoma of the bladder, 8 with metastatic nonseminomatous germ cell tumors of the testis, and 2 patients with metastatic SCC of the bladder.     

Flow cytometric study of DNA distribution in cytopunctures of benign and malignant breast lesions

One hundred seventy-eight cytopunctures of mammary lesions were obtained for cytologic diagnosis and flow cytometric (FCM) analysis of the nuclear DNA content. All lesions were excised and evaluated histologically; 106 were carcinomas and 72 were benign lesions. The benign lesions showed a diploid DNA content, with one exception. Among the 106 carcinomas, 35 (33%) were diploid, 14 (13%) were tetraploid and 57 (54%) were aneuploid. For 79 carcinomas, the relationship between ploidy and (1) "T" and "N" of TNM staging, (2) the histologic grading of Scarff, Bloom and Richardson, (3) axillary nodal involvement, (4) the presence of estrogen and progesterone receptors, (5) age and (6) menopausal status was investigated. The percentage of aneuploidy was significantly higher (P less than .05) in grade III tumors as compared to grade I tumors. There was no significant relationship between aneuploidy and the other factors. However, a trend was observed between the lack of steroid receptors and a high probability of the tumor being aneuploid. FCM DNA analysis was also carried out on breast carcinomas obtained at surgery in 40 patients for whom FCM DNA analysis had previously been performed on breast cytopuncture specimens. The FCM DNA analyses were found to be best performed on the samples obtained by cytopuncture, which may increase the yield of tumor cells.     

Clinical applications of image cytometry to human tumour analysis

Image cytometry (ICM) is widely applied to the automated screening, the detection, the diagnosis, the classification, the prognosis and the therapeutic follow-up of different types of cancers (breast, bladder, cervix,...). This review describes the analysis methods and the applications of nuclear image analysis, the determination of DNA content and the analysis of morphometry and of nuclear texture. DNA content analysis can contribute to a prognostic information in addition to other prognostic factors for breast, renal and prostate cancers. For ovarian cancer, aneuploidy seems to be related to prognosis. Bladder tumours with DNA aneuploidy were frequently of high malignancy while ploidy was significantly correlated to relapse risk. For digestive cancers, patients presenting DNA diploid tumours show a better survival than patients with aneuploid ones. Morphometry seems to be a more important criterion than other conventional prognostic factors of invasive breast and digestive carcinomas. A differential diagnosis between normal and neoplastic thyroids is more precise when based on a quantitative evaluation of texture associated to morphometry. Textural parameters permit the discrimination of two populations of patients having a different prognosis and could thus be an aid for prognosis in prostatic cancers. Morphonuclear parameters contribute to separate low and high grade bladder carcinomas. Although ICM was frequently reported, results from the reported examples were not always obvious. In conclusion, the measurements obtained with ICM could be helpful for a decision in several cancers but could not be a substitute for the classical approach of the pathologist.     

DNA distribution in non-small-cell lung carcinomas and its relationship to clinical behavior

A study of 187 surgical specimens of tumors of patients with non-small-cell lung carcinomas was carried out by means of flow cytometry. Eighty-four percent of the tumors were classified as tumors with abnormal DNA stemlines (DNA aneuploidy). Patients with tumors demonstrating DNA aneuploidy had significantly shorter survival times than those with tumors demonstrating DNA diploidy (p = .009). Cell cycle analysis was possible in 122 tumors. Patients whose tumors had 0-8% S-phase cells died later than patients whose tumors had 9-16% S-phase cells (p = .018). In addition, patients with tumors with a low fraction of labeled S-phase cells (autoradiography) had a better prognosis than patients with tumors with a high proportion of labeled S-phase cells (p = .041).     

Traitement médical des tumeurs endocrines digestives

Gastroenteropancreatic neuroendocrine tumors constitute a highly heterogeneous group of tumors with very different prognoses. It is important to distinguish between the well-differentiated neuroendocrine tumors (carcinoid tumors affecting the gastrointestinal tract and pancreatic endocrine tumors), which generally progress slowly, and the poorly differentiated endocrine tumors, which are characterized by being aggressive and of rapid progression. The treatment of the poorly differentiated forms is essentially based on chemotherapy, although prognosis remains poor. The well-differentiated forms require a more complex approach – depending on the site of the primary tumor, staging, the resectability of the lesions, and disease spread – and should therefore be defined within a multidisciplinary setting. The treatment of choice remains surgical excision of the primary tumor and metastases. No additional treatment has been demonstrated to be effective. For functional tumors, symptomatic treatment with a somatostatin analog for carcinoid tumors or a protein pump inhibitor for gastrinoma must be started rapidly to reduce the complications related to hormone secretion. For metastatic disease or for tumors that are not amenable to complete resection, several options can be proposed: careful monitoring, chemoembolization of liver metastases, systemic chemotherapy, or enrollment in therapeutic protocols offering targeted therapies. Stepwise introduction of these various therapies prolongs survival, even in metastatic disease.     

Flow cytometric analysis of paraffin-embedded material in human gastric cancer

Flow cytometric DNa analysis was performed on formalin-fixed, paraffin-embedded samples obtained by gastroscopic biopsy from 9 patients with histologically normal gastric mucosa (36 specimens) and by radical gastrectomy from 42 cases of human gastric cancer (120 specimens). Ploidy patterns and the distribution of cells in the different cell cycle phases were estimated, and the results were correlated with the histologic and clinical features. All samples of normal mucosa showed a diploid modal DNA content whereas DNA aneuploidy was encountered in 71.4% of the gastric tumors. The correlation between aneuploidy and histologic malignancy grading was statistically significant: aneuploidy was found in 36.4% of highly differentiated (grade 1 and grade 2) tumors and in 75.0% of poorly differentiated (grade 3) tumors (P less than .05). The percentage of cells in S-phase in normal gastric mucosa (median: 5.0%) was lower than that in the tumors (median: 11.3%) (P less than .05). There was a trend for grade 3 tumors to have higher median values (median: 13.4%) than grade 1 and 2 tumors (median: 9.3%); however, this was not statistically significant. An aneuploid DNA pattern was associated with a poorer prognosis, both in early and in advanced stages of gastric tumors, while proliferative activity did not correlate with postoperative survival.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

Expression of a melanoma-tumor-associated antigen as demonstrated by a monoclonal antibody (D6.1) in cytopathologic preparations of human tumor cells from effusions and needle aspirates

Immunocytopathologic studies were performed on 79 fine needle aspiration biopsies (FNABs) and effusions from 13 melanomas and 57 other human neoplasms with the monoclonal antibody (MAb) D6.1 raised against a partially purified melanoma-tumor-associated antigen (MTAA). The purposes of these studies were (1) to evaluate the ability of MAb 6.1 to react with melanoma cells in cytopathologic preparations and (2) to define the spectrum of reactivity of MAb D6.1 in cytopathologic preparations of non-melanomas. Cytocentrifuge preparations of the cytopathologic specimens were permitted to react with the primary antibody and were then stained by the avidin-biotin-immunoperoxidase method. Thirteen of 13 FNABs of malignant melanomas exhibited staining reactivity with MAb D6.1. Among the nonmelanoma tumors tested, staining reactivity was observed in 30 of 57 specimens. Among specific neoplasms, staining was present in 5 of 11 adenocarcinomas of the breast, 2 of 7 ovarian adenocarcinomas and 5 of 6 metastatic adenocarcinomas from the colon. Among 17 lung cancers examined, staining was noted in 4 of 7 adenocarcinomas, 3 of 4 large-cell undifferentiated carcinomas and 2 of 3 poorly differentiated squamous-cell carcinomas. Two small-cell undifferentiated carcinomas and one carcinoid failed to stain. Three of three adenocarcinomas of the pancreas showed staining. Among the remaining neoplasms examined, one specimen each of carcinoma of the prostate and the cervix and one carcinoma of undetermined primary exhibited staining. Two malignant lymphomas did not stain. Staining of mesothelial cells was observed in three of nine benign effusions.(ABSTRACT TRUNCATED AT 250 WORDS)