Cytometric DNA Analysis Research Articles

Abstract 2151: Gingerol, paradol and shogaol overcome colorectal cancer cell resistance to sorafenib via enhancing its cellular uptake, entrapment and intracellular metabolism

Abstract Sorafenib is tyrosine kinase inhibitor used for the treatment of hepatocellular and renal carcinomas but suffers from resistance in some tumors such as colorectal cancer. Colorectal cancer multi-drug resistance might be attributed to over-expression/over-activation of P-glycoprotein (P-gp) efflux pump and/or intracellular metabolism. Gingerol, paradol and shogaol are naturally occurring major compounds within the family Zingiberaceae. Gingerol and its related hydroxyphenylalkanones are known for their potential interference with P-gp function as well as intracellular drug metabolism. In the present study, we investigated the influence of gingerol, paradol and shogaol on the cytotoxic profile of sorafenib in colorectal cancer cell lines (HT-29, HCT-116, LS-174T and CaCo-2) via their influence on sorafenib cellular accumulation and intracellular metabolism. Sorafenib alone showed considerable cytotoxic activity against all testes colorectal cell lines with IC50 ranging from 2.0±0.3 to 11.10±0.8 μM. Gingerol, paradol and shogaol significantly enhanced the cytotoxicity of sorafenib against HT-29 cells and decreased its IC50 from 9.2±0.4 μM to 6.6±0.7, 3.8±0.4 and 4.7± 0.4 μM, respectively. Similarly in HCT-116, gingerol, paradol and shogaol significantly decreased sorafenib IC50 from 11.3±0.8 μM to 5.7±0.7, 5.9±0.4 and 7.7± 0.5 μM, respectively. Using DNA cytometric analysis, combination of gingerol, paradol or shogaol with sorafenib significantly decreased the proliferating cell fractions from 16.9±1.1% to 11.6±1.1%, 14.7±1.1% and 9.16±1.8%, respectively. In parallel, combination of gingerol, paradol or shogaol with sorafenib significantly increased the c-PARP concentration from 60.0±20.0 pg/cell to 256.5±25.8, 244±61.4, and 202.9±37.2 pg/cell, respectively. Gingerol and paradol, but not shogaol increased the intracellular accumulation of the P-gp probe, rhodamine at concentration range (3-100 μM). However, using recombinant ATPase attached p-gp molecules, shogaol and paradol significantly inhibited the ATPase activity. Gingerol, paradol and shogaol increased sorafenib uptake and decreased its extracellular concentration from 1491.7±132.0 ng/ml to 511.0±16.0, 216.0±39.0 and 256.4±15.6 ng/ml, respectively. Only gingerol and paradol increased sorafenib intracellular accumulation and increased its level from 239.0±16.0 pg/cell to 297.0±32.6 and 327.0±12.0 pg/cell, respectively. Both paradol and shogaol enhanced the intracellular metabolism of sorafenib to its N-oxide active metabolite and entrapped it within cellular compartment. In conclusion, gingerol, paradol and shogaol significantly enhanced the anti-cancer profile of sorafenib via influencing its cellular pharmacokinetics. Citation Format: Mohamed G. Mehanna, Fahad A. Al-Abbasi, Tarek Elawady, Ali M. El-Halawany, Ahmed M. Al-Abd. Gingerol, paradol and shogaol overcome colorectal cancer cell resistance to sorafenib via enhancing its cellular uptake, entrapment and intracellular metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2151.

Expression of so-called adhesion proteins and DNA cytometric analysis in malignant parotid tumours as predictors of clinical outcome

Tumours of the salivary glands are rare, and account for only 0.5–1% of all tumours. We have analysed the cytoarchitectural structure of such tumours by studying 3 binding proteins that act on different parts of the glandular epithelial architecture: e-cadherin, laminin, and CD44. We analysed the DNA using image cytometry to evaluate ploidy, S-phase, and 5c exceeding rate, and to compare the biological aggressiveness of the proteins. Our goal was to correlate the degree of structural integrity and the histological grade of the injury, and to try to find new biological factors that would help to predict the evolution of disease in the salivary glands. The immunoexpression pattern of the so-called adhesion proteins of the salivary glands, when combined, yields important data about the aggressiveness of malignant neoplasms, and provides useful tools with which to predict the biological evolution of malignant lesions.

Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions

To study the DNA ploidy pattern of uterine cervical squamous intraepithelial lesions (SILs) and its diagnostic and prognostic significance. The study included 31 cases of SIL: 11 low-grade (LSIL) and 20 high-grade (HSIL). Feulgen-pararosaniline staining was performed on previously Papanicolaou-stained smears and a DNA image cytometric study was performed. An internal reference was used to calibrate the samples. All 31 cases of SIL, either LSIL or HSIL, were non-diploid. Of the 11 cases of LSIL, four were tetraploid and seven were aneuploid, whereas, of the 20 cases of HSIL, four were tetraploid and 16 were aneuploid. Stemline aneuploidy was not a significant discriminator between LSIL and HSIL (P=0.32). Based on single-cell analysis, HSIL cases had significantly higher DNA content than LSIL cases (P<0.01). When a mean of 30% or more was used for the 6c-exceeding event (6cEE) value, the sensitivity and specificity to indicate HSIL were 83% and 64%, respectively, with a positive predictive value (PPV) of 81% and negative predictive value (NPV) of 65%. All HSIL cases were cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on biopsy. In addition, cases which showed recurrence had more DNA content by single-cell analysis than those with an indolent clinical behaviour: P=0.04 and P=0.03 for LSIL and HSIL, respectively. Image cytometric DNA analysis is a useful technique for diagnostic and prognostic purposes in uterine cervical SIL when appropriate 'c' values are used in single-cell analysis. We propose that a >6c DNA content of 30% is useful as a cut-off level for predicting cases with CIN2+ in DNA image cytometry of cervical smears.

Bryonia dioica aqueous extract induces apoptosis through mitochondrial intrinsic pathway in BL41 Burkitt's lymphoma cells

Ethnopharmacological relevanceBryonia dioica Jacq. is a climbing perennial herb with tuberous roots which is widely used in traditional medicine in Algeria for the treatment of cancers; it belongs to the genus Bryonia (Cucurbitaceae). Aim of the studyTo investigate the cytotoxic and apoptogenic activities, the phytochemical composition and acute toxicity of the aqueous extract of Bryonia dioica roots growing in Algeria. Materials and methodsDried roots of Bryonia dioica were extracted with water (decoction). The cytotoxic effects of the aqueous extract in the Burkitt's lymphoma BL41 cell lines were evaluated by flow cytometry. Apoptosis induction was assessed by two corroborative assays; propidium iodide (PI) staining of cell DNA and flow cytometric light scatter analysis. The mitochondria membrane potential was investigated using a fluorescent dye DIOC6. The expression of caspases-3, -8, -9 and PARP was assessed by Western blot. The phytochemical screening of the roots of Bryonia dioica was performed using qualitative phytochemical standard procedures. ResultsThe Bryonia dioica aqueous extract induced cell death in a dose-dependent manner. The IC50 of Bryonia dioica aqueous extract was estimated to be approximately 15, 63μg/ml. This was accompanied by induction of apoptosis, activation of caspase-3 and -9, cleavage of PARP and loss of mitochondria membrane potential. Furthermore, the phytochemical screening of roots of Bryonia dioica showed the presence of various bioactive such as polyphenols, sterols and triterpenes, alkaloids, c-heterosides, carbohydrates and saponins. ConclusionThe aqueous extract of Bryonia dioica induces apoptosis in the Burkitt's lymphoma BL41 cell lines via the mitochondrial pathway. The flavonoids, sterols and triterpens detected could be responsible for the cytotoxic and apoptogenic activities of the aqueous extract of Bryonia dioica. These findings suggest that Bryonia dioica could be considered as a promising source for developing novel therapeutics against Burkitt's lymphoma.

Effects of deoxynivalenol on apoptosis of human gastric carcinoma cell line SNU in vitro.

Objective To explore the effects of deoxynivalenol (DON) on apoptosis of human gastric carcinoma cell line SNU in vitro. Methods SNU cells were treated with DON at different concentrations (50, 100, 1000, 2000 μg/L) for 12 hours, and then cells were harvested for cell apoptosis by flow cytometric (FCM) DNA analysis and the expression of Bax, Bcl-2 and Caspase-3 at protein level with FCM and Western blotting. Results FCM results showed that the apoptosis rates of SNU cells in DON treatment groups were all higher than that in control, especially in DON 1000 μg/L and 2000 μg/L groups (P<0.05). In the concentration range from 50 to 2000 μg/L, a significant concentration-depended response correlation could be found between apoptosis rate and DON concentration (r =0.940, P <0.01). FCM and Western blotting showed Bax and Caspase-3 expression in often SNU cells DON treatment for 12 hours were up-regulated while that of Bcl-2 was down-regulated. Conclusion DON can induce apoptosis of SNU cells in vitro in dose-dependent manner, and possible mechanisms of apoptosis induction effects may be up-regulation of the expression of Bax and down-regulation of that of Bcl-2 and activation of the key enzyme of apoptosis Caspase-3. Key words: Stomach neoplasms; Deoxynivalenol (DON); SNU cells; Apoptosis

Reactive nitrogen intermediates and monokines induce caspase-3 mediated macrophage apoptosis by anaerobically stressed Salmonella typhi

A successful pathogen manipulates its host for its own benefit. After ingestion, on reaching the intestine Salmonella encounters the resident tissue macrophages. Rather than being destroyed by these professional phagocytes after internalization, Salmonella survives intracellularly. Invasive Salmonella has been reported to induce apoptosis of macrophages as a part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under different host environments, including the anaerobic stress encountered by the pathogen in the gut, remains to be examined. The present study is aimed at investigating the apoptotic potential of S. enterica serovar Typhi (S. typhi) grown under anaerobic conditions simulating the in vivo situation encountered by the pathogen. Apoptotic cell death was determined by assessment of nucleosomal DNA and flow cytometric analysis. Evaluation of the data revealed that anaerobically grown S. typhi could induce apoptosis in significantly more number of macrophages compared to the bacterial cells grown under aerobic conditions. A significantly enhanced generation of reactive nitrogen intermediates and caspase-3 activity during macrophage apoptosis induced by anaerobic S. typhi correlated with the increased generation of tumour necrosis factor-alpha, interleukin (IL)-1alpha and IL-6. The results indicate that reactive nitrogen intermediates and monokines induce caspase-3 mediated apoptosis of macrophages by S. typhi under anaerobic conditions. These findings may be relevant for clearer understanding of the Salmonella-macrophage interactions and may be of clinical importance in the development of preventive intervention against the infection.

The Mechanism of Action of the Tumor Suppressor HRSL3

Loss of tumor suppressor function is a critical step in the development of tumors. Two classes of tumor suppressors are known. Loss of function of class I suppressors, such as p53, is achieved through genetic alterations, whereas inhibition of class II suppressors is reversible and leaves their genomic structures unchanged. Nazarenko et al . investigated the mechanism of action of the class II suppressor HRSL3. HRSL3 protein is found in normal tissues but is undetectable in human tumors. The authors performed a yeast two-hybrid screen for binding partners for HRSL3 and detected PR65α, the regulatory subunit Aα of protein phosphatase 2A (PP2A), a serine/threonine phosphatase. Using HRSL3 mutants, the authors determined that the N-terminal region of HRSL3 was necessary for binding to PR65α. Coimmunoprecipitation studies in a transfected human ovarian carcinoma cell line (OVCAR-3) demonstrated that HRSL3 did not bind to the catalytic subunit of PP2A, PR36α. The catalytic activity of PP2A isolated from cells that contained HRSL3 was much reduced compared with that of PP2A isolated from cells that contained a noninteracting mutant of HRSL3. Through flow cytometric analysis of nuclear DNA and Western blotting analysis, the authors demonstrated that either increased amounts of HRSL3 or knockdown of PP2A with a PR65-specific siRNA induced apoptosis of OVCAR-3 cells. The presence of HRSL3 in OVCAR-3 cells resulted in increased amounts of phosphorylated PKCζ, a target of PP2A. Blocking PKCζ activity with a pseudosubstrate peptide abrogated HRSL3-induced apoptosis of the OVCAR-3 cells, suggesting a role for PKCζ in regulating cell death in ovarian cancer. I. Nazarenko, R. Schäfer, C. Sers, Mechanisms of the HRSL3 tumor suppressor function in ovarian carcinoma cells. J. Cell. Sci. 120 , 1393-1404 (2007). [Abstract] [Full Text]

Treatment of branchial cleft cyst with intracystic injection of OK-432

Conclusions. The data suggest that branchial cleft cyst (BCC) can be primarily treated with OK-432 sclerotherapy and only the remaining lesions with excision. Objectives: To evaluate the effectiveness of sclerotherapy using OK-432 in the treatment of BCC. Materials and methods. We treated 12 BCC patients (3 males and 9 females; mean age 25 years) with OK-432 sclerotherapy at an outpatient clinic. The cystic fluids were aspirated and diagnosed by cytomorphology and DNA cytometric analysis to exclude malignancy. The fluid aspirated from the cyst was replaced with an equal volume of OK-432 solution. The sizes of cysts were measured and compared before and after injection. The remaining cysts were excised and histopathologically compared with the excised BCCs that had not been treated with OK-432. Results. Seven of 12 patients (58%) showed a complete response after OK-432 injection, administered one to three times. Three patients (25%) had only partial response and two (17%) were stationary. Five patients with remaining lesions underwent excision. There was no difficulty in dissecting around the cysts and no increased morbidity during operation. None of the patients had evidence of recurrences or malignancies developing during the follow-up period (mean 21 months, range 17–26 months). There were no major side effects except fever after sclerotherapy.

Pathological anatomy and molecular pathology

The incidence of malignant mesotheliomas in Germany has increased since about the mid 1980s, and a further increase is expected until about 2020 due to the peak in asbestos processing in Germany between 1965 and 1980. About 90% of the mesotheliomas recorded in the files of the German Mesothelioma Registry in Bochum are asbestos-related and therefore possibly due to an occupational exposure. In 2003, 717 mesotheliomas were newly diagnosed at the German Mesothelioma Registry. Mesotheliomas are very heterogeneous in terms of histological appearances and of prognosis. At present, the diagnostic gold standard is conventional histology in combination with additional immunohistochemical analysis. We were not able to confirm a promising report that described telomerase reverse transcriptase catalytic subunit (TERT) for the differentiation between reactive and neoplastic mesothelial lesions, which can be extremely difficult. DNA cytometric analysis may also help differentiate between reactive and neoplastic mesothelial lesions. There are some characteristic patterns of chromosomal imbalances as detectable by comparative genomic hybridization (CGH), but at present, specific chromosomal or genetic defects that give rise to a mesothelioma are not known. A reliable pathological diagnosis is the basis for therapeutic, prognostic, and medicolegal consequences. In general, it can be achieved by thoracoscopic inspection with specifically directed biopsy. Furthermore, a description of the peculiarities of each mesothelioma by the pathologist might be the key to a more individual therapy in the future.

Role of concurrent S. mansoni infection in H. pylori-associated gastritis: a flow cytometric DNA-analysis and oxyradicals correlations

Background/aim: Helicobacter pylori infection is associated with the development of atrophic gastritis and increased gastric epithelial proliferation that is important in developing gastric carcinoma. Some countries with a high prevalence of H. pylori infection have high gastric cancer rates, whereas in others these rates are low. Several theories have been advanced to explain this phenomenon. One of these explanations is that the concurrent parasitic infection that is common in the African population might alter the immune response to H. pylori infection and reduce the incidence of atrophic gastritis. The aim of the present study was to assess whether concurrent Schistosoma mansoni infection with H. pylori has an effect on gastric mucosal injury in view of cell proliferation, apoptosis, pathological changes, nitric oxide (NO), oxyradicals and antioxidant capacity status. Patients/methods: Between April 2001 and March 2002, 73 patients were subjected to upper gastrointestinal endoscopy for dyspepsia and liver cirrhosis in the National Liver Institute, Menoufiya University. Biopsies were obtained from any lesion as well as from apparently healthy mucosa. Specimens were preserved in RNA later solution, and then kept at −80 °C until utilized for estimation of DNA-flow cytometric assay, reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), NO and lipid peroxidation (LPO) product—malondialdehyde (MDA). Diagnosis of bilharziasis was done by stool analysis, or by sigmoidoscopy and rectal snip. Results: Of the 73 patients, 48 were H. pylori-positive, 34 of them were positive and 14 were negative for S. mansoni. Of the 25 H. pylori-negative cases, 18 were positive and 7 were negative for S. mansoni. Concurrent infection with S. mansoni occurred in 34 patients and they had reduced DNA S-phase (7.57±4.99 vs. 14.5±3.11, P=0.001), reduced proliferation activity (9.95±3.95 vs. 16.78, P<0.004) and reduced apoptosis (21.83±11.64 vs. 26.0±8.31, P>0.05) compared with H. pylori infected patients alone. Conclusions: The results demonstrate that concurrent helminthes infection may modify the inflammatory response to gastric H. pylori infection manifested by the reduction of oxyradical-induced DNA-damage, apoptosis and cellular proliferation activity, and the increase in antioxidant production. Concurrent S. mansoni infection may have a protective effect against the possible progression of H. pylori-induced gastritis towards gastric carcinoma.

Flow cytometric analysis of cell cycle control by tumor suppressor genes.

Determination of cell cycle distribution is an important tool in cell biology. Flow cytometric analysis of cellular DNA can provide rapid, quantitative information about DNA ploidy and cell cycle distribution. Presented here are three basic protocols for analysis of DNA by flow cytometry. There are many variations of these protocols that may be more appropriate for particular cell types or applications (1).

Clinicopathologic and DNA Cytometric Analysis of Carcinoid Tumors of the Thymus

Twelve cases of carcinoid tumors of the thymus were reviewed in terms of clinicopathologic, histochemical, and immunohistochemical features and DNA ploidy patterns. The collective consisted of nine male and three female patients, aged 34 to 74 years, of whom five (42%) had symptoms. Eleven patients underwent surgical resection, and one with systemic metastases was autopsied. In the 11 resected patients, tumors had invaded surrounding structures in four cases, and mediastinal lymph node metastases were detected in six. Recurrence occurred in two of the resected patients (18%), and the 5-year survival rate was 82%. Histologically, all tumors showed an organoid growth pattern with delicate fibrovascular stroma. In addition, three tumors had unusual morphologic features such as combined features of carcinoid tumor and thymoma and solid growth pattern with occasional large tumor cells. Mitotic counts ranged from 1 to 14 per 10 high-power fields with a mean count of 4.9. Central necrosis within solid nests was observed in nine tumors. Classification of this series using the WHO histologic classification system resulted in categorization of all 12 tumors as atypical carcinoids. All tumors were positive for Grimelius staining and for cytokeratin. Immunohistochemical staining documented the presence of moderately to strongly positive neuroendocrine markers such as neuron-specific enolase, chromogranin A, synaptophysin, and neural cell adhesion molecule. No correlation between proliferative activity based on the Ki67 labeling index and prognosis or lymph node metastasis was found. Concerning DNA ploidy patterns, only one tumor with multiple lymph node metastases was considered to be aneuploid. In conclusion, although all of our cases were histologically classified as atypical carcinoid tumors of the thymus, most were diploid, and the patients enjoyed a relatively good prognosis.

The use of fine‐needle aspiration cytology in the molecular characterization of neuroblastoma in children

BACKGROUND The utility of fine-needle aspiration (FNA) material in the molecular characterization of a group of neuroblastic tumors (NT) in children is presented. METHODS A recent study showed a high accuracy rate for FNA cytology (FNAC) in combination with immunostaining as a diagnostic method in 26 children with NT. In the current study FNA smears from 18 children were analyzed, either at the time of diagnosis or retrospectively, on available stored smears for cellular DNA content (DNA ploidy) by means of image cytometry (ICM) and 1p deletion and N-myc amplification by interphase fluorescent in situ hybridization (FISH). RESULTS A total 62 analyses (DNA ploidy, 20 analyses; chromosome 1 or 2 number, 11 analyses; 1p deletion, 16 analyses; and N-myc, 15 analyses) resulted in clear information in 60 cases, whereas 2 tests for N-myc amplification failed. The results were compared with each other and with cytometric DNA analyses on tumor touch imprints (n = 12) and N-myc analyses on material from surgical specimens (Southern blot analysis, n = 12). The results were concordant in all but seven analyses (all with clear informative results) in six children. These discrepancies may be explained as an effect of either chemotherapy or tumor heterogeneity. CONCLUSIONS FNA is a fast and noninvasive diagnostic technique that yields sufficient material for the molecular characterization of neuroblastic tumors by means of FISH and ICM. Such analyses are of prognostic significance because they predict tumor behavior and response to therapy according to International Neuroblastoma Staging System/International Neuroblastoma Risk Groups criteria. In the majority of cases it also is possible to obtain material for storage and future investigations. Cancer (Cancer Cytopathol) 1999;87:60–8. © 1999 American Cancer Society.

The use of fine-needle aspiration cytology in the molecular characterization of neuroblastoma in children

The utility of fine-needle aspiration (FNA) material in the molecular characterization of a group of neuroblastic tumors (NT) in children is presented. A recent study showed a high accuracy rate for FNA cytology (FNAC) in combination with immunostaining as a diagnostic method in 26 children with NT. In the current study FNA smears from 18 children were analyzed, either at the time of diagnosis or retrospectively, on available stored smears for cellular DNA content (DNA ploidy) by means of image cytometry (ICM) and 1p deletion and N-myc amplification by interphase fluorescent in situ hybridization (FISH). A total 62 analyses (DNA ploidy, 20 analyses; chromosome 1 or 2 number, 11 analyses; 1p deletion, 16 analyses; and N-myc, 15 analyses) resulted in clear information in 60 cases, whereas 2 tests for N-myc amplification failed. The results were compared with each other and with cytometric DNA analyses on tumor touch imprints (n = 12) and N-myc analyses on material from surgical specimens (Southern blot analysis, n = 12). The results were concordant in all but seven analyses (all with clear informative results) in six children. These discrepancies may be explained as an effect of either chemotherapy or tumor heterogeneity. FNA is a fast and noninvasive diagnostic technique that yields sufficient material for the molecular characterization of neuroblastic tumors by means of FISH and ICM. Such analyses are of prognostic significance because they predict tumor behavior and response to therapy according to International Neuroblastoma Staging System/International Neuroblastoma Risk Groups criteria. In the majority of cases it also is possible to obtain material for storage and future investigations.

Breast tumors with myofibroblastic differentiation: clinico-pathological observations in myofibroblastoma and myofibrosarcoma

This report describes the clinico-pathological features of myofibroblastic tumors of the breast in six patients. Four women and one man presented with a benign myofibroblastoma. The sixth patient was a woman with myofibrosarcoma. All myofibroblastomas were composed of a fascicular arrangement of spindle cells embedded in dense bundles of collagen. Tumors differed with respect to their proportion of neoplastic cells and collagenous stroma as well as cellular pleomorphism. Based on this variation, the tumors could be subclassified as classic, collagenized, epithelioid and cellular myofibroblastoma. Immunohistological staining confirmed myofibroblastic differentiation by strong expression of either desmin or smooth muscle actin with coexpression of vimentin. In addition, numerous cells reacted with antibodies to CD68. Proliferative activity was rather low in the myofibroblastoma with an average of 0-2 mitotic figures per 10 HPF. DNA cytometric analysis was performed in two cases and showed diploid stem lines with minor S-phase fractions (1% and 3%). In the myofibrosarcoma, cells contained pleomorphic nuclei with some giant cells and numerous mitotic figures (6-7/10 HPF) and had infiltrating margins that were apparent even grossly. Immunohistochemically, tumor cells strongly expressed vimentin, smooth muscle actin and fibronectin. Ultrastructurally, neoplastic cells met the criteria of myofibroblasts, i.e. contained abundant intermediate filaments and myofilament bundles with focal densities as well as fibronexus junctions. DNA cytometric analysis exhibited again a diploid stemline but marked proliferative activity was present as indicated by an S-phase fraction of 20%. In conclusion, in benign myofibroblastoma there may be some cellular pleomorphism but mitotic activity is always low. The malignant counterpart, myofibrosarcoma, is characterized by marked cellular pleomorphism, infiltrating margins and high mitotic rate.

Cytometric DNA analysis and prognostic biomarkers in breast carcinoma. Expression of P53 product in the different ploidy classes.

The aim of the study was to correlate the DNA Index (DI) and S‐phase fraction (SPF) values determined by multiparametric flow cytometry in breast cancer (mainly T1 and T2 stages) with several clinico‐pathologic variables and other biological parameters. For this purpose, a total of 136 breast cancers were submitted to flow cytometry and to several types of immunohistochemical analyses. Among clinico‐pathologic data we considered pT, pN and grade and among immunohistochemical markers, hormonal receptors, P53, c‐erbB‐2 and MIB‐1. We found that DNA aneuploidy was strongly correlated with high tumoral grade, absence of hormonal receptors, high proliferation, as shown by high MIB‐1 (≥36%) and SPF values (≥13.3%), and P53 positivity. Grouping the tumours according to their DI values, we observed a relative significantly higher correlation of the near‐triploid range carcinomas (DI 1.40–1.60) with immunohistochemical expression of P53 (p=0.0001). Near‐triploid DI was also associated with a high proliferative activity, expressed both by SPF (p=0.0001) and MIB‐1 reactivity (p=0.0001), high tumoral grade (p=0.0001) and presence of axillary metastases (p=0.03). These data suggest that DNA near‐triploid tumours in breast cancer may have a more aggressive behaviour in comparison with other DI classes.

The suitability of DNA cytometry for the prediction of the histological diagnosis in women with abnormal cervical smears.

To analyse the suitability of DNA cytometry for predicting the histological diagnosis in women with cervical dyskaryosis. Survey with the use of diagnostic information to revise disease probability. Colposcopy clinic of a university hospital. One hundred and ten women with two mildly or moderately dyskaryotic cervical smears and 98 women with one severely dyskaryotic smear. DNA cytometric analysis using cytocentrifuge preparations of single cell suspensions from a cervical scrape. The main DNA cytometric parameter was N5C (i.e. the absolute number of cells with a DNA content of more than 5C on a given surface with a predefined cell density). The probability of finding CIN II or worse. On arbitrary grounds, a positive test should point to a probability of 85% or higher. In the patients with cervical neoplasia, the value of N5C increased significantly with an increasing CIN grade (P < 0.001). In the patients with one severely dyskaryotic smear and in those with two mildly or moderately dyskaryotic smears, the prior probability of finding CIN II or worse was 94% and 53%, respectively. Therefore, DNA cytometric analysis might be particularly useful in women with mild or moderate dyskaryosis; further analysis was restricted to this group. All of the women in whom the N5C value was higher than 52 were diagnosed as having CIN II or worse. Only 16 (14.5%) of the 110 women had an N5C value of 52 or higher. When the N5C value was 27, the probability of finding CIN II or worse was estimated to be 85%. Only 28 (25%) patients had an N5C value of 27 or higher. DNA cytometry produced significant diagnostic information, as was shown by the relation between N5C and the histological diagnosis. However, the N5C value could not discriminate sufficiently between women with CIN II or worse and CIN I or better. Therefore, the management of individual patients with cytological abnormalities cannot be based on the results of DNA cytometric analysis.

DNA aneuploidy in early breast cancer

High-resolution flow cytometric (FCM) DNA analysis was performed on 148 unfixed, frozen tissue samples from four groups of early breast cancers: invasive carcinomas (ICs) with predominance of carcinoma in situ (DCIS) (group I), small clinical cancers < or = 15 mm (group II), node-negative, clinical cancers (group III) and small screening-detected cancers < or = 15 mm (group IV). The median tumour size was 12 mm. The aim of the study was to support, with a larger sample, our recent findings with respect to DNA ploidy pattern in the selected group of ICs with predominance of DCIS (group I). Similar results to this group were found for both the small clinical cancers and the node-negative cancers, with respect to frequency of DNA aneuploidy (79% and 90%), DNA index (DI) distribution, intratumoral DNA heterogeneity and S-phase fraction. A high frequency of DNA hyperdiploid clones was found, in particular related to highly differentiated tumours. A significant difference was found compared with the screening-detected cancers, which were characterised by a much lower frequency of DNA aneuploid samples (49%) and may represent a biologically specific group of low-malignant, slowly growing tumours. Associations were shown between histological grade and DI subclasses, and between lymph node status and DNA diploidy/aneuploidy, whereas DI was not correlated with tumour size. The DNA ploidy findings in this series of early cancers are concordant to our own results from preinvasive lesions as well as those reported from series of more advanced cancers.

JUVENILE GRANULOSA-CELL TUMOR OF THE OVARY - INTERPHASE CYTOGENETICS FOR CHROMOSOME-12 AND CHROMOSOME-X AND FLOW CYTOMETRIC DNA-PLOIDY STUDY

We performed interphase in situ hybridization (ISH) for chromosomes 12 and X and now cytometric DNA analysis on seven juvenile granulosa cell tumors to verify this observation and correlate the results with clinicopathologic factors. Five cases were primary ovarian tumors and two were metastatic lesions. Our results show that four tumors exhibited polysomy 12 and four had monosomy X; only two tumors displayed concurrent aberrations of both chromosomes. Of the six tumors with interpretable flow cytometric histograms three showed DNA aneuploidy and three were DNA diploid. All three aneuploid tumors manifested polysomy 12. Of the three diploid DNA neoplasms two showed monosomy X and one displayed disomy for chromosomes 12 and X. No apparent correlation between numerical chromosomal abnormalities and the biological course was observed in this small cohort. Our results indicate that chromosomes 12 and X are frequently altered in these neoplasms and thus could be targeted for further molecular studies in order to identify genetic aberrations which might be associated with JGCT tumorigenesis.

An evaluation of the flow cytometric nuclear DNA analysis of intrahepatic multinodular hepatocellular carcinoma for a diagnosis of their multicentricity

This study aims to evaluate the clinical significance of the nuclear DNA index (DI) for identification of multicentrically occurring (MC) hepatocellular carcinoma (HCC). In 14 multinodular HCC patients, the DI of 30 HCC specimens and 14 non-cancerous liver tissues were analyzed by flow cytometry. Histological studies of the 30 HCCs revealed MC in 6 cases and intrahepatic metastasis (IM) in 7 cases except for a histologically undetermined case who was found to be a hepatitis B virus (HBV) carrier, and the MC of this case was determined by a clonal study using the HBV integration pattern. In four of the seven specimens with MC HCC, the DI of all the intrahepatic tumors was 1.0 (diploid pattern), while the remaining three were different. On the other hand, five of the seven IM cases were identical (3 diploid and 2 aneuploid), one similar level (DI = 1.17-1.18) and one different (1.0 and 1.24). Moreover, in one IM case, the possibility of an alteration of the DI during the course of HCC development was investigated. Although the DI of the recurrent main tumor (DI = 1.17) of this case, which was identified as metastasis of the primary tumor by a clonal study, was also similar to that of subsequent metastatic lesions (DI = 1.18), the DI of the primary tumor was 1.0. These results indicate that DI analysis was not enough to make a differential diagnosis of the multicentric occurrence of HCC.