Diagnostic and prognostic implications of DNA ploidy and S-phase evaluation in the assessment of malignancy in biliary strictures.

BACKGROUND AND STUDY AIMS: Brush cytology of biliary strictures has a low sensitivity for diagnosing malignancy, and additional diagnostic tools are needed. The aim of the present study was to assess the diagnostic and prognostic importance of DNA measurements as an adjunct to brush cytology in patients with biliary strictures. PATIENTS AND METHODS: All patients (n = 225) with bile duct strictures who underwent endoscopic retrograde cholangiopancreatography (ERCP) between January 1997 and October 2003 at the Department of Radiology at Karolinska University Hospital, Huddinge, Sweden, were included in the study. While 66 patients had an unclear final diagnosis and were therefore excluded, the remaining 159 patients were assessed with brush cytology and DNA flow cytometry. RESULTS: Sensitivity and specificity of DNA aneuploidy for tumor detection were 43 % and 96 %. Using DNA analysis in addition to brush cytology, the sensitivity was 62 % compared with 57 % for brush cytology alone (not significant). Patients with diploid DNA tumors had a significantly better survival than patients with aneuploid DNA tumors ( P = 0.02). The mean survival was 10 months for diploid cancers and 6 months for aneuploid cancers. CONCLUSION: DNA ploidy measurement may be a diagnostic method that could supplement brush cytology in the identification of malignancy in biliary strictures. DNA aneuploidy is a marker of poor prognosis in patients with malignant biliary strictures.

Colorectal cancer in colonic Crohn's disease--high frequency of DNA-aneuploidy.

BACKGROUND: The risk of colorectal cancer (CRC) in colonic Crohn's disease (CCD) seems to be of the same magnitude as in extensive, longstanding ulcerative colitis (UC) and colonoscopic surveillance has been advocated. Mucosal dysplasia and DNA-aneuploidy are early warning markers of malignant transformation in UC. Data concerning the occurrence of such premalignant lesions in CCD are scarce. AIMS: The objective of this study was to investigate the DNA ploidy pattern in CCD-patients with manifest CRC, both in the tumour, as well as in the adjacent and distant colorectal mucosa. The results from DNA-flow cytometry analyses (FCM) prior to the development of a CRC in CCD were also investigated. MATERIALS AND METHODS: Biopsies obtained at colonoscopy and surgical specimens from 43 patients with colonic or ileocolonic CD developing CRC between 1988 and 1998 were reviewed. The CRC histological phenotype, and the occurrence of dysplasia were registered. CRC-tissue and tissue from areas with dysplasia adjacent to and/or distant from the tumour were obtained from paraffin-embedded blocks and were analysed by FCM after preparation. RESULTS: Twenty-four CRCs in 21 patients (14 men) were suitable for FCM-analyses. The median age at CRC-diagnosis was 53 years (21-73) and the median CCD-duration was 14.5 years (1-50). A predominance of CRC was found either in the cecum (9124) or in the rectum (7/24). DNA-aneuploidy was found in 62.5% (15/24) of the tumours, in 25% (2/8) in adjacent and/or distant mucosa, and in 50% (2/4) of the patients that had been subjected to colonoscopic surveillance prior to the CRC-diagnosis. In 7patients (29%), definite dysplasia was detected adjacent to andlor distant from the tumour. Of the 6 patients undergoing colonoscopic surveillance, 3 (50%) displayed definite dysplasia prior to the colectomy. CONCLUSION: Since DNA- aneuploidy is a' common feature in CRCs in CCD and precede the development of invasive carcinoma, inclusion of FCM-analyses of colorectal biopsies may enhance the sensitivity of identifying high-risk CCD-patients prone to develop CRC within the frame of colonoscopic surveillance programs.

Low IL-1alpha expression in bladder cancer tissue and survival.

OBJECTIVE: To investigate whether the previously reported association between IL-1alpha mRNA levels and survival in urinary bladder cancer remains in an extended patient material and to search a mechanism behind a possible antitumoral activity of IL-1alpha. PATIENTS AND METHODS: IL-1alpha mRNA levels were determined in 164 tumors with quantitative TaqMan PCR. RESULTS: A large variation was found in mRNA levels of IL-1alpha. We found, by immunohistochemistry, that IL-1alpha is expressed by tumor rather than stromal cells. In a univariate Cox proportional hazards model, low levels (median split) of IL-1alpha mRNA were associated with a relative hazard ratio (RHR) of 1.7 (95% CI: 1.0-2.9) for cancer-specific death (n=157); a restriction to muscle invasive tumors (n=63) resulted in an RHR of 1.8 (0.9-3.3). In bivariate analyses, adjustment for age, stage and grade respectively, decreased the RHR and the association between IL-1alpha expression and cancer-specific survival was not statistically significant. Which factors to regard as confounders remains unclear. CONCLUSIONS: Low levels of IL-1alpha mRNA expression are associated with an increased risk for cancer-specific death in the investigated material. However, confounding is an issue and to determine whether or not the observed association is causal, we need a defined mechanism and data from other studies.

Diagnosis of biliary strictures in conjunction with endoscopic retrograde cholangiopancreaticography, with special reference to patients with primary sclerosing cholangitis.

BACKGROUND AND STUDY AIMS: Strictures of the bile ducts due to malignant changes are difficult to distinguish from benign changes, particularly in patients with primary sclerosing cholangitis (PSC). The aim of this study was to evaluate diagnostic methods for malignancy in biliary strictures in conjunction with endoscopic retrograde cholangiopancreaticography (ERCP). PATIENTS AND METHODS: Bile duct strictures were identified during ERCP in 57 patients, who were thus included in the present study. Brush samples from the strictures were taken for cytology and for evaluation of DNA content by flow cytometry. The tumor markers CA 19-9 and CEA were determined both in serum and bile fluid. Two independent radiologists evaluated all cholangiograms. The diagnostic sensitivity, specificity, and accuracy of each diagnostic method were evaluated separately and in combination. RESULTS: 32 patients were found to have malignant strictures and when the four methods: brush cytology, DNA analysis, serum CA 19-9 and serum CEA were combined, a diagnostic sensitivity of 88 % and specificity of 80 % were reached. Seven of the 20 patients with PSC were found also to suffer from cholangiocarcinoma, yielding a sensitivity and specificity of 100 % and 85 %, respectively. Analyses of CA 19-9 and CEA in bile fluid had no diagnostic significance. CONCLUSION: An ERCP procedure with brush cytology, a DNA analysis, combined with serum analysis of CA 19-9 and CEA, can increase the possibility of distinguishing between malignant and benign biliary strictures, especially in PSC patients.

X-irradiation effects on thymidine kinase (TK): I. TK1 and 2 in normal and malignant cells.

The effect of radiation on TK is more complicated than would be expected from earlier results on bone marrow cells (Feinendegen et al. 1984, Int. J. Radiat. Biol. 45, 205). TK activity increased at 0.01 Gy and then decreased up to 1 Gy in mouse spleen. In contrast to the results for the spleen, an increase in activity at 0.1 Gy was seen in mouse thymus. The activity of dephosphorylated TK1 (TK1a) in both spleen and thymus was reduced to 50% after irradiation at 0.5-1 Gy. The degree of phosphorylation (TK1b/TK1a ratio) changed in spleen, but not in thymus. The activity of TK2 in mouse liver increased at 3 h after 5 Gy by about 60%. In mouse ascites tumour, a dose-independent (1-5 Gy) oscillating TK1 activity was found up to 24 h, especially for TK1a and TK1b. The amount of TK1 was unchanged up to 12 h, but decreased at 24 h. This suggests that the differences in the changes in the degree of phosphorylation of TK1 after irradiation among spleen, thymus and ascites tumour further underline the complexity of the response of TK1 activity to irradiation. The dramatic change in the activities of TK1a and TK1b may illustrate that both of them are more radiosensitive than TK-h, a variant with mixed TK1 and TK2 properties.

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity.

The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1-24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5 cent-amino-5 cent-deoxythymidine (5 cent-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.

Age- and site-dependent cell cycle composition of the normal human colonic mucosa.

BACKGROUND AND AIM: As a reference to studies of DNA-ploidy and S- and G2/M-phase fractions in patients with inflammatory bowel diseases, we describe the mucosa of normal individuals with respect to age and localization in the colon. MATERIALS AND METHODS: One hundred and sixty-five biopsies from the right, transverse and left colon from 44 subjects (20 men, 24 females, median age 55 years (range 21-80)) who were referred for colonoscopy due to rectal bleeding, diarrhoea or suspicion of neoplasia, but with normal macroscopic and microscopic findings, were analysed by DNA-flow cytometry for ploidy and cell cycle composition. The biopsies were immediately fixed in buffered formalin and then analysed by a method for high quality preparations of cell nuclei without any centrifugation steps, resulting in minimal cell damage and low frequencies of aggregates, making the background levels low in the DNA-histograms. RESULTS: The median S-phase fraction of the biopsies, all diploid, was 2.35% (0.1-8.3). The S-phase fraction increased linearly with age (p = 0.001) and decreased from the right colon (median 2.75% (0.5-8.3)) over the transverse colon (median 2.3% (0.1-6.2)) to the left colon (median 1.9% (0.8-6.5), p < 0.02). The fraction of G2-cells (median 1.1%, range 0.2-5.1) increased significantly with increased S-phase fraction (p < 0.0001). CONCLUSION: DNA-FCM analyses of normal colonic tissue demonstrate an age- and site-dependent variation with regard to cell proliferation. This variation has to be taken into consideration when biopsy specimens from chronic colitis mucosa are evaluated.

Investigation on cell proliferation with a new antibody against thymidine kinase 1.

The cytosolic thymidine kinase 1 (TK1) is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti-TK1 antibody against a synthetic peptide from the C-terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti-TK1 antibody works in archival specimens and is a specific marker of cell proliferation.

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.

Can DNA cytometry be used for evaluation of malignancy and premalignancy in bile duct strictures in primary sclerosing cholangitis?

BACKGROUND/AIMS: The significance of DNA ploidy determinations for diagnosing cholangiocarcinoma (CC) in primary sclerosing cholangitis (PSC) has not previously been evaluated. Knowledge of tumour cell ploidy by DNA cytometry may facilitate the evaluation of malignant and premalignant lesions in PSC. METHODS: Twenty-eight patients with CC were studied; 10 of the patients had PSC. Seventeen samples from 15 patients with PSC but without CC were used as controls for benign strictures. Gallbladder tissue from 100 patients with chronic cholecystitis was also analysed. DNA was measured using flow cytometry on cells from paraffin-embedded tissues. RESULTS: Tumours from patients with PSC displayed non-tetraploid DNA aneuploidy significantly more often (80%) than tumours from patients without PSC (39%) (p<0.05). CC from patients with PSC significantly more often displayed DNA aneuploidy: 80% (8/10) compared with 12% (2/17) in bile ducts in PSC without CC (p=0.0007). The frequency of DNA aneuploidy in gallbladder tissue from patients with chronic cholecystasis was 1% (1/100). CONCLUSION: The high prevalence of DNA aneuploidy in PSC-related CC and the low prevalence in benign PSC strictures point to DNA cytometry as a possible future method for detecting malignant and premalignant changes in bile duct strictures in patients with PSC. This method may be useful in selecting PSC patients for liver transplantation.

Analysis of proliferating cell fraction determined by monoclonal antibody to M1-subunit ribonucleotide reductase and Ki-67 in relation to p53 protein expression in fine-needle aspirates from non-Hodgkin's lymphomas.

The purpose of this study was to analyse the proliferative fraction with the monoclonal antibody M1-R-R to M1-subunit ribonucleotide reductase and with MIB-1 to Ki-67 antigen in relation to p53 protein expression in fine needle aspirates from B-cell non-Hodgkin's lymphomas. One hundred and thirty-seven cases, previously diagnosed and sub-typed according to the Kiel classification and characterized by immunophenotyping, were included in the study. The M-1 subunit ribonucleotide reductase (M1-R-R), Ki-67 and p53 antigens were detected using monoclonal antibodies on stored cytospin preparations. There was a good correlation (r = 0.72) between Ki-67 and M1-R-R positive cell fraction in both high and low grade lymphomas. High-grade lymphomas had a median percentage of M1-R-R/MIB-1 positive cells of 53.0/73.0 for lymphoblastic, 61.0/52.0 for immunoblastic and 33.5/41.0 for centroblastic lymphomas, respectively. In low grade lymphomas figures of median percentage of M1-R-R/MIB-1 were 9.0/15.0 for centroblastic/centrocytic, 11.0/9.5 for chronic lymphocytic leukaemia, 16.0/27.0 for centrocytic and 12.0/9.0 for immunocytomas, respectively. The median percentages of M1-R-R/MIB-1 for high and low grade lymphomas were 37.0/50.5 and 11.0/12.0, respectively. In the p53 positive cases the proliferation rate as measured by staining for M1-R-R and MIB-1 was higher than in p53 negative cases, but the difference was not statistically significant. The results show that cytospin material obtained by fine needle aspiration and stored at -70 degrees C for years can be used reliably for both peroxidase-avidin-biotin and three-step alkaline phosphatase immunocytochemical staining. In addition, proliferation fraction determined by M1-R-R monoclonal antibody staining correlates well with that measured by an established marker for cell proliferation, the Ki-67 antibody. However, the proliferation fraction as measured by the two antibodies differs in the various subtypes of non-Hodgkin's lymphoma which indicates that they may contribute different prognostic information.

Evaluation of tumor heterogeneity of prostate carcinoma by flow- and image DNA cytometry and histopathological grading.

BACKGROUND: Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. MATERIAL AND METHODS: Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 Böcking's grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow- and fluorescence image cytometry (FCM, FICM). Cell cycle composition was measured by FCM. RESULTS: By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM) was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. CONCLUSION: Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.

Reliability of DNA cytometric S-phase analysis in surgical biopsies: assessment of systematic and sampling errors and comparison between results obtained by image and flow cytometry.

Three major parameters in DNA histograms that contribute to the reliability of S-phase analysis were evaluated. These parameters are (1) the extent of background in relation to the amount of S-phase cells (and the validity of its subtraction), (2) the size of the "free" S-phase range (S(free)), and (3) the sampling error of cell counting. Tests in histograms obtained from surgical biopsies by flow cytometry (FCM) showed that the background subtraction is reliable if the found S-phase fraction is higher than the fraction of background events in the histogram range of the cell population. The size of S(free) was determined in computer-generated test histograms as a function of variables such as the coefficient of variation (CV) and the DNA index (DI). To calculate the sampling error of cell counting above background and in S(free), a model was developed that was validated by experimental data. This error can serve as an indicator of the uncertainty in S-phase analysis. The poor correlation found between %S values measured by image cytometry (ICM) and FCM in surgical biopsies was assigned to high uncertainty by low cell numbers in ICM histograms. A method is proposed to estimate quantitatively the reliability of S-phase analysis that can facilitate the interpretation of results.

Chromosomal numerical aberrations detected by fluorescence in situ hybridization on bladder washings from patients with bladder cancer.

OBJECTIVE: Previous studies on touch biopsy specimens have determined numerical or structural changes involving many different chromosomes in bladder cancer. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) assay in bladder washings as an objective technique to detect chromosomal numerical aberrations in bladder cancer. The main advantages of bladder washings are that they can be easily collected during the clinical follow-up of patients with superficial bladder cancer and they do not contain so many degenerate cells as urine samples. METHODS: We collected specimens from 25 patients who underwent transurethral resection of bladder tumors. Double target FISH assays with centromeric labeled probes for chromosomes 7, 8, 9 and 11 were used on the bladder washings and on the touch biopsy slides. The results were compared to flow cytometry and tumor grade and stage. RESULTS: We found monosomy 9 and trisomy 7, 8, 9 and 11 in 28, 32, 36, 28 and 25% respectively of the patients. FISH analysis of bladder washing versus touch biopsy specimens were concordant in approximately 90% of the slides. Total DNA aneuploidy correlated well with numerical aberrations of chromosomes 7, 8 and 11, but not with chromosome 9. CONCLUSION: Although better hybridization efficiency was obtained on touch biopsy slides, the results in bladder washings were in high concordance. FISH analysis on bladder washing samples may become a simple tool to improve the accuracy of cytology.

Prognostic significance of tissue prostate-specific antigen in endocrine-treated prostate carcinomas.

Fine-needle aspiration biopsy is a minimally invasive technique for obtaining sample material suitable not only for cytological grading but also for flow cytometry and for biochemical analyses. The prognostic value of tissue prostate-specific antigen (T-PSA) from fine-needle aspiration biopsies was compared with serum total and free prostate-specific antigen, the ratio of free:total serum prostate-specific antigen, tumor stage, cytological grade, and DNA ploidy in 179 patients with stage T2-T4 prostate cancer (CAP). The patients, who were free from bone metastases at the time of diagnosis, were treated by either orchidectomy or medical castration with GnRH analogues or high-dose parenteral depot estrogens. They were followed for at least for 71 months or until death, and the different variables were correlated to time to progression and time to death from CAP. Using Cox univariate analysis, T-PSA was shown to be the most important factor in predicting time to progression and time to death. When the patients were divided into three groups with respect to T-PSA, 56 of 60 (93%) of the patients with low T-PSA levels developed progressive disease, and 52 of 60 (87%) died of CAP. For patients with intermediate T-PSA levels, the corresponding figures were 9 of 60 (15%) and 6 of 60 (10%). None of the 59 patients with high T-PSA values developed progressive disease. Similar but less pronounced relationships were found between tumor progress and CAP-specific death on the one hand and clinical stage, cytological grade, and DNA ploidy on the other. In a Cox multivariate stepwise analysis, T-PSA was the only important factor for time to progression and death. This was also true for the subgroup of patients with stages T2 and T3 disease only. The study shows that T-PSA is superior to other hitherto routinely used markers for the prediction of outcome of hormone-treated patients with newly diagnosed CAP.

Use of markers in defining urothelial premalignant and malignant conditions.

Markers have revealed the presence of phenotypically abnormal areas in histologically benign urothelium in bladders containing transitional cell carcinomas. This finding strongly suggests that at least some bladder cancers are associated with changes in the field and that markers can detect these lesions before they reach a grossly malignant stage. Markers have been used clinically for the detection of cancer in patients who are under regular surveillance for recurrence of bladder cancer. Much less information is available regarding the use of markers to detect bladder cancer without a prior history of the disease and for the prediction of which tumors are biologically more aggressive. However, ongoing clinical trials are addressing the latter issue. The type of specimen and its preparation will determine what type of markers can be analyzed. Although marker performance is based upon sensitivity and specificity, the prevalence of bladder cancer in the population being tested will dramatically affect the positive predictive value of an assay. Markers with high positive predictive value are indicators for interventions, such as biopsy, while markers with high negative specific values are useful for avoiding interventions. Cytology is used to detect occult high-grade neoplasms such as carcinoma in situ. While not yet clinically validated, tests with high negative predictive value could be used to decrease the frequency of cystoscopic evaluation. Markers must be validated by testing them prospectively using previously defined cut-off values. Furthermore, markers that will be used to alter treatment should be tested prospectively to determine the safety and cost-effectiveness of this strategy. Recommendations for future work include: (1) evaluation of markers in patients with dysplasia defined by the current pathologic classification; (2) evaluation of markers as indicators of tumor recurrence; (3) evaluation of markers as indicators of tumor progression; and (4) evaluation of markers in chemoprevention studies.

Correlations between serum progesterone and smoking, and the growth fraction of cervical squamous cell carcinoma.

BACKGROUND: Possible correlations between growth fraction of squamous cervical carcinomas and serum progesterone (se-P) concentrations, smoking habits and DNA ploidy were studied. MATERIALS AND METHODS: The DNA S-phase fraction (SPF), measured by flow cytometry was used as a marker of tumour growth in 103 cases of squamous cervical cancer stage IB-IV. DNA-ploidy (peridiploidy vs. aneuploidy), Se-P, se-Estradiol, smoking habits, parity, menopausal status, clinical stage and histopathological grading were compared to SPF < 14% vs. SPF > or = 14%. RESULTS: Aneuploidy, (odds ratio (OR) 10.0), se-P > or = 2.6 nmol/l (OR 7.5) and smoking (OR 3.0) were significantly associated with SPF > or = 14%, after adjustments for all factors included in the study. The association with se-P and smoking was attributed to an increased risk for the premenopausal women in the study. DISCUSSION: In this study an increased tumour growth was associated with increased leves of se-P, smoking and aneuploidy in women with invasive squamous cervical carcinoma. This study seems to experimentally confirm epidemiological studies, where smoking and long-term use of oral contraceptives have been linked to cervical neoplasms.

A high proliferation rate measured by cyclin A predicts a favourable chemotherapy response in soft tissue sarcoma patients.

A small but not insignificant number of patients experience a prolonged survival after treatment of metastatic soft tissue sarcoma. This must be weighed against the majority of the patients who benefit little from the therapy, but nevertheless experience its side-effects. It would therefore be of utmost importance to be able to screen for those patients who respond to the treatment. Since proliferating cells are more sensitive to chemotherapy than non-proliferative cells, we measured the proliferation rate of the primary tumour of 55 soft tissue sarcoma patients with locally advanced or metastatic disease by determining the flow cytometric S phase fraction and immunohistochemical Ki-67 and cyclin A scores. S phase fraction or Ki-67 score did not predict chemotherapy response or progression-free survival. A high cyclin A score, however, correlated with a better chemotherapy response (P = 0.02) and longer progression-free survival time (P = 0.04). Our results suggest that a high cyclin A score predicts chemotherapy sensitivity.

Expression of cyclin A in soft tissue sarcomas correlates with tumor aggressiveness.

Cyclins and cyclin-dependent kinases regulate the cell cycle. Cyclin A has a dual role in cell proliferation. It is essential in the S phase for DNA replication, and it is also involved in G2-M-phase transition, signifying actively dividing cells. The expression of cyclin A was determined by immunohistochemistry in paraffin sections of 126 soft tissue sarcomas. The median cyclin A score was 10.8% (range, 1-54%). Cyclin A expression correlated with the S-phase fraction, Ki-67 score, G2-M phase, and grade. It did not correlate with the size of the tumor. A high cyclin A score predicted a poor metastasis-free survival (P < 0.01) and a poor disease-specific overall survival (P = 0.01). We concluded that the expression of cyclin A is a powerful prognostic factor in soft tissue sarcoma. Moreover, the cyclin A score determines the fraction of tumor cells in the S phase and the G2 phase, which are the most sensitive cell cycle phases for current modalities of cancer treatment.

Assessment of cell proliferation by Ki-67 staining and flow cytometry in fine needle aspirates (FNAs) of reactive lymphadenitis and non-Hodgkin's lymphomas.

This study was undertaken to assess cell proliferation in FNAs from a series of 57 non-Hodgkin's lymphomas (NHL) and 11 cases of reactive lymphadenitis using Ki-67 staining and flow cytometry (FCM). The results were compared and correlated to the cytomorphological subgrouping according to Kiel classification. The mean percentages of Ki-67 positivity were 16.6% and 61.1% for low and high grade lymphomas, respectively (P < 0.001). The mean S-phase fraction (SPF) determined by FCM was 4.61% for low grade and 12.9% for high grade lymphomas (P < 0.001). The figures for Ki-67 positivity and S-phase fraction in reactive lymphadenitis were 16.8% and 40%, respectively. We observed a strong correlation in low grade lymphomas between Ki-67 and SPF. A good correlation was also found in reactive lymphadenitis. In high grade lymphomas, however, with highly scattered Ki-67 and S-phase values, this correlation was lost. In some cases this discrepancy can be explained by a rich admixture of non-neoplastic, non-proliferating cells in aspirates from diploid tumours. In addition, the existence of a minor aneuploid tumour cell population of high proliferation such as that in Ki-1 lymphomas will not be accurately analysed by FCM but is easily assessed by Ki-67 staining. However, the main reason seems to be a high variability between the fraction of cells in S-phase and the total number of cells in G1, S and G2 in individual tumours.