High S-phase Fraction Research Articles

S-phase fraction of 155 soft tissue sarcomas: Correlation with clinical outcome

Traditionally, grade is considered the most important prognostic factor for soft tissue sarcomas (STS). However, because of the alleged difficulties in reproducibility of grading, new, objectively determined prognostic factors would be of value. The aim of our study was to establish if S-phase fraction (SPF) measured with flow cytometry was of prognostic significance for STS. In this study, we included all 193 adult STS patients with superficial trunk or limb tumors who were treated by the Helsinki University Central Hospital (HUCH) STS group between January 1987 and May 1993. One hundred and seventy-two formalin fixed paraffin embedded tumor samples were available. SPF measurement was successful in 155 cases. Eighty-six cases were diploid. Ploidy was found to have no effect on overall survival. The median SPF was 6.8% (diploid tumors, 4% and nondiploid tumors, 12.9%). A high SPF predicted a shorter survival in patients with diploid tumors (P=0.003). The prognostic value was even stronger when we studied disease specific survival and excluded from analysis samples that contained less than 50% tumor cells (P=0.011). However, no prognostic value could be detected in nondiploid tumors or in the material as a whole. Our results suggest that high SPF is an adverse prognostic factor for survival of patients with diploid STS. However, further studies are needed to confirm these results.

Mutant p53 protein as a predictor of survival in endometrial carcinoma

The expression of mutated p53 protein was studied in paraffin-embedded, formalin-fixed tumour specimens from 183 women with endometrial carcinoma. Fifty-five per cent of the specimens were negative, whereas the staining intensity was weak, moderate or strong in 15, 2 and 28% of cases, respectively. Strong p53 expression (> 75% of the cells stained) was more common in uterine papillary serous cancers and clear cell cancers than in other tumour subtypes (P < 0.001), as well as in poorly differentiated tumours (P < 0.01) and in tumours with nuclear grade 3 (P < 0.0001). Strong p53 expression was also more frequently found in aneuploid tumours (P < 0.0001) and in tumours with a high S-phase fraction (P < 0.001). Strong p53 expression was highly predictive of poor survival in the univariate analysis (P = 0.006) and in the Cox multivariate analysis which included age, stage and grade. However, it lost most of its impact when the strongly prognostic nuclear grade and ploidy were added to the multivariate models.

Low biological aggressiveness of screen-detected lung cancers may indicate over-diagnosis.

All cases of lung cancer diagnosed in the Tampere University Hospital catchment area in 1983-1987 were identified, analyzed for DNA flow cytometry and followed up to 1992. The patients were classified into 3 groups: screen-detected, symptom-detected, and detected by chance. The biological aggressiveness as indicated by DNA flow cytometry was not related to the survival of the symptom-detected patients. Also the screen-detected patients with an aggressive tumour (aneuploid or high S-phase fraction, SPF) had the same survival as the symptom-detected patients. The survival of screen-detected patients with a diploid or low SPF tumour was significantly better than that in the other groups. It is concluded that some of the previously known discrepancy of no effect on mortality and effect on survival of lung-cancer screening may be due to over-diagnosis, i.e., detection of morphologically malignant but biologically indolent lesions by screening.

Metaphase cytogenetics and DNA flow cytometry with analysis of S-phase fraction in prostate cancer: Influence on prognosis

Objectives To compare the prognostic significance of chromosome aberrations, DNA ploidy, and S-phase fraction (SPF) in prostate adenocarcinomas and to compare the sensitivity of metaphase cytogenetics with flow cytometry (FCM) in detecting abnormal tumor clones. Methods Prostate adenocarcinomas from 57 men were previously successfully analyzed with metaphase cytogenetics. Archival material from these tumors were further analyzed with FCM for DNA content and SPF. Results The patients were followed for 4.5 to 7.7 years. DNA ploidy was analyzed in 51, and SPF in 45 of the 57 tumors. Clonal chromosomal aberrations, DNA aneuploidy, and high SPF were all significantly associated with poor survival. Of these three variables, SPF was the best predictor of survival, but compared with tumor stage and grade in multivariate analysis, SPF was not an independent prognostic factor. Patients with locally advanced tumors or metastatic disease with SPF less than 8% had a median survival of 5.9 years, compared with only 1.3 years for those with SPF more than 8%. Twenty-eight abnormal clones were detected with FCM and 20 with cytogenetic analysis, but only for two of these clones could the results from the two different methods be regarded as concordant. Conclusions SPF was superior to karyotype and ploidy in predicting death in prostate cancer, but it remains to be shown whether SPF analysis adds prognostic information to tumor stage and grade. The cytogenetic analyses correlated poorly with results of FCM, indicating low sensitivity of both methods.

Correlation of flow cytometry to clinical factors, hormone receptors, and histopathological grade in stage I and II invasive breast carcinoma.

DNA index (ploidy) and S-phase fraction (SPF) were measured by flow cytometry in 131 invasive stage I and II breast carcinomas. Ploidy showed a strong correlation with SPF (p = 0.0001), with aneuploid tumors having a high SPF. Both cytometric parameters correlated with tumor size and hormonal receptor status. Smaller tumors tended to be diploid and have low SPF. Nodal status did not demonstrate an association with cytometric findings. There was a highly significant connection between tumor grade, especially nuclear grade, and SPF (p = 0.0001). The study demonstrates the relationship between conventional prognostic factors, DNA content, and proliferative activity of breast tumors.

Prognostic significance of plasminogen activator inhibitor-1 (PAI-1) in primary breast carcinoma

The plasminogen activator system plays a key role in the proteolysis of malignant tumours. In 155 patients with primary breast cancer levels from PAI-1 and uPA were measured in cytosol with monoclonal antibodies and by an enzyme-linked immunosorbent assay. 35 tumour tissue samples form benign breast were also examined. Malignant tumours contain higher levels of PAI-1 (8.6 ng/mg) than benign tumours (1.28 ng/mg) (p < 0.01). Also the median level of uPA was significantly higher (p < 0.01) in malignant tissue (2.38 ng/mg) in comparison to benign disease (0.54 ng/mg). No correlation was found between the proteases and the classical prognostic parameters like axillary lymph node involvement, tumour size and menopausal status. However, a significant correlation (p < 0.01) was found in tumours with lymphangiosis carcinomatosa, negative hormone receptors, grade III tumour cells and high S-phase fractions (> 5%). After a median follow-up of 46 months we found that high levels of PAI-1 correlated with short DFS (p = 0.0005) and OAS (p = 0.003). However, in the Cox multivariate regression analysis only PAI-1 was significantly independent for OAS and could therefore give additional information. We conclude that levels from PAI-1 antigen measured in cytosol of primary breast cancer is an independent prognostic parameter to identify patients with high and low risk for relapse and for individualised treatment.

Classification of duct carcinoma in situ (DCIS) with a characterization of high grade lesions: Defining cohorts for chemoprevention trials

In the last 6 years a number of non-randomized, predominantly single institutional trials of breast conservation therapy (BCT) with DCIS, have demonstrated that it constitutes a very heterogeneous group of diseases with markedly different risks of local recurrence and invasive transformation. There has been a consensus that DCIS, which exhibits a "comedo" morphology, generally defines a high risk group. Most studies, moreover, have identified the same two features, nuclear grade and necrosis, as contributing most significantly to prognosis. Nuclear grade and necrosis have been identified as independent prognostic variables in several studies. High nuclear grade DCIS which exhibits comedo necrosis defines the majority of all DCIS which will result in local recurrence and invasive transformation after BCT. Studies utilizing image cytometry, to determine ploidy and S-phase fraction and immunohistochemical studies of proliferation and oncogene distribution have shown a significant association with morphologically identified high nuclear grade and aneuploidy, high S-phase fraction or proliferation rate, presence of HER-2/neu and P53 oncogenes and absence of estrogen receptors. Generally the inverse of this association is seen with low nuclear grade DCIS. However, initial hopes that these adjunctive studies would identify subsets within the high nuclear grade group which might be more likely to recur have not been fulfilled.

Review of known prognostic variables.

The prognosis of patients with primary breast cancer depends on prognostic factors per se and on treatment. In certain subgroups of patients, the overall treatment benefits of systemic adjuvant therapy are modest. Therefore, proper selection of patients and treatment modalities is absolutely necessary. Patients with primary breast cancer and a very good prognosis can be classified by known prognostic factors such as tumor size T < or = 1 cm, grade 1, hormone receptor positive tumors, and no other factors indicating poor prognosis. [table: see text] Patients with very poor prognosis are characterized by young age (< 35 years), large tumor size (T > 3 cm), grade 3, hormone receptor-negative tumors, and high S-phase fractions. Tumor size is the best prognostic factor for the decision to treat (breast conservation surgery, primary preoperative or adjuvant systemic therapy) or not to treat. Table 4 shows possible treatment decisions (type and timing of therapy) in primary breast cancer based on known prognostic and predictive variables. The decision to treat or not to treat outside clinical trials should be based on this kind of information. The type and aggressiveness of adjuvant systemic treatment should depend on the receptor status of the tumor and on the age and menopausal status of the patient, as classical predictive factors; however, prognosticators which can describe the metastatic potential of breast cancer cells have yet to be defined.

Classification of duct carcinoma in situ (DCIS) with a characterization of high grade lesions: Defining cohorts for chemoprevention trials

In the last 6 years a number of non-randomized, predominantly single institutional trials of breast conservation therapy (BCT) with DCIS, have demonstrated that it constitutes a very heterogeneous group of diseases with markedly different risks of local recurrence and invasive transformation. There has been a consensus that DCIS, which exhibits a "comedo" morphology, generally defines a high risk group. Most studies, moreover, have identified the same two features, nuclear grade and necrosis, as contributing most significantly to prognosis. Nuclear grade and necrosis have been identified as independent prognostic variables in several studies. High nuclear grade DCIS which exhibits comedo necrosis defines the majority of all DCIS which will result in local recurrence and invasive transformation after BCT. Studies utilizing image cytometry, to determine ploidy and S-phase fraction and immunohistochemical studies of proliferation and oncogene distribution have shown a significant association with morphologically identified high nuclear grade and aneuploidy, high S-phase fraction or proliferation rate, presence of HER-2/neu and P53 oncogenes and absence of estrogen receptors. Generally the inverse of this association is seen with low nuclear grade DCIS. However, initial hopes that these adjunctive studies would identify subsets within the high nuclear grade group which might be more likely to recur have not been fulfilled.

Nm23-H1 protein immunohistochemical expression in human breast cancer

A series of 76 patients undergoing surgery for primary breast carcinoma has been prospectively studied in order to evaluate the relative weight of nm23-H1 protein expression in disease-free survival. Expression of nm23 protein was immunohistochemically assessed. In all, 39% (29/74) of the turners showed positive staining for nm23-H1 protein expression. Negative nm23-H1 expression was found in poorly differentiated, tumors (p<0.02). There was no significant relationship between nm23-H1 and the other clinicopathological and biological features examined. In the univariate statistical analysis, node positivity, G3 histological grade and high flow cytometric S phase fraction (SPF) value proved to be significantly related to risk of relapse. In the multivariate analysis, only histological grade (G3) and high SPF values (>10.6) proved to be independently related to risk of relapse, with a hazard ratio of 9.84 and 7.98 respectively. Our preliminary study suggests that immunohistochemical nm23-H1 expression should not be considered a marker for predicting tumor progression and patient prognosis.

The implication of DNA content and S-phase fraction in oral carcinomas with and without metastasis

Flow cytometry DNA analysis was performed on fresh tissue samples of 90 primary lesions and 32 metastatic lymph nodes of squamous cell carcinomas of the oral cavity and related regions to elucidate the characteristics of tumors with metastatic potency. The incidence of aneuploidy for carcinomas with metastasis was 67%, which was significantly higher than the 44% of carcinomas without metastasis, and aneuploid carcinomas (39%) had a higher tendency to metastasis than diploid carcinomas (20%). The incidence of aneuploidy and metastasis was related to the T classification, the degree of differentiation, and the histologic grade of malignancy. The incidence of aneuploidy and mean DNA index of metastatic lesions were 31% and 1.12, respectively, and the values were significantly lower than the 67% and 1.30 of the corresponding primary lesions. The results indicate that the chance of evolution of metastatic cell lines is higher in aneuploid carcinomas than diploid carcinomas, possibly because the former is more heterogeneous than the latter, but most of the cell lines causing lymph-node metastasis are diploid cell lines. Metastatic lesions had a lower S-phase fraction than primary lesions, indicating that a high S-phase fraction does not always reflect the presence of metastatic cell lines.

Flow cytometric DNA analysis and lysosomal cathepsins b and l in locally advanced laryngeal cancer. Relationship with clinicopathologic parameters and prognostic significance

The traditional factors of locally advanced laryngeal squamous cell carcinoma (LSCC) have limited predictive value for the identification of high risk patients. Therefore, it is extremely important to define prognostic factors that identify the more aggressive types. Reliable and reproducible prognostic indicators are being investigated to help clinicians identify high risk groups and address more rational treatment. Flow cytometric DNA ploidy and S-phase fraction (SPF) measurements were performed on frozen tumor tissues from a consecutive series of 71 patients with Stage III and IV LSCC: Lysosomal cathepsin B and L activity levels were determined biochemically in matched paired sets of tumor tissue and normal mucosa samples. By univariate analysis, lymph node positivity, poor histologic differentiation, DNA aneuploidy, high SPF, and high tumor/mucosa ratio of cathepsin B activity were significantly related to risk of relapse, whereas only DNA aneuploidy and high SPF proved to be significantly related to risk of death. Multivariate analysis showed that high histologic grade and high SPF values (> 15.1%) were independent prognostic factors related to risk of relapse (relative risk [RR] = 3.54; 95% confidence limits [CL] = 1.05-12.0; and RR = 4.22; CL = 1.54-11.6, respectively), whereas only high SPF was related to risk of death (RR = 3.63; CL = 1.17-11.3). S-phase fraction is an independent predictor of relapse free and overall survival in patients with locally advanced LSCC. On the basis of these findings, SPF should be used in addition to other established prognostic factors to refine the prognostic assessment of these patients further. More studies are needed for a better evaluation of the prognostic significance of DNA ploidy and that of lysosomal cysteine proteinases in these tumors.

Increased Glucose Metabolism in Untreated Non-Hodgkin's Lymphoma: A Study With Positron Emission Tomography and Fluorine-18-Fluorodeoxyglucose

Glucose metabolism has been shown to be increased in neoplastic tissue. It has been suggested that high activity of glucose metabolism is associated with a high grade of malignancy of human cancer. We studied in vivo glucose metabolism in 22 patients with untreated non-Hodgkin's lymphoma with fluorine-18-fluorodeoxyglucose (FDG) and positron emission tomography (PET). FDG uptake in lymphoma deposits was measured blinded to clinical data, and compared with histologic classification and proliferative activity. Tracer uptake was measured by using two indices of FDG accumulation: the standardized uptake value (SUV) and the regional metabolic rate (rMR) for the tracer. The median SUV of the lymphomas was 8.5 (range, 3.5 to 31.0), and the median rMR 22.7 mumol/100 g/min (range, 9.0 to 124.3 mumol/100 g/min). A high FDG uptake in tumors was associated with high histologic degree of malignancy as defined by the Working Formulation (P = .005 for the SUV, and P = .04 for the rMR) or by the Kiel classification (P = .003 for the SUV, and P = .02 for the rMR). A high FDG accumulation was also associated with a high S-phase fraction (r = .786 for the SUV, P = .0002; and r = .774 for the rMR, P = .02). We conclude that in untreated non-Hodgkin's lymphomas high FDG uptake is associated with high histologic grade of malignancy and a high proliferation rate. This minimally invasive method may find application in assessing lymphoma lesions in patients who are poor candidates for surgery, and it may provide further information in cases where the grade of aggressiveness of lymphoma is not settled based on clinical or histologic data.

792 Increased glucose metabolism in non-Hodgkin's lymphoma as studied with FDG pet

Glucose metabolism has been demonstrated to be increased in neoplastic tissue, and to reflect the grade of malignancy of human cancer. We studied <i>in vivo</i> glucose metabolism in 22 pts with untreated non-Hodgkin's lymphoma with fluorine-18-fluorodeoxyglucose (FDG) and positron emission tomography (PET). FDG uptake in lymphoma deposits was measured as standardized uptake values (SUVs) of the tracer, and compared with histological classification and proliferative activity. <h3>Results</h3> The median SUV of the lymphomas was 8.5. A high FDG uptake in tumors was associated with high histological degree of malignancy by the Working Formulation (<i>P</i>=0.005) or by the Kiel classification (<i>P</i>=0.003). A high FDG accumulation was also associated with a high S-phase fraction (r=0.786, <i>P</i>=0.002). <h3>Conclusion</h3> FDG PET may find application in assessing the grade of aggressiveness of lymphoma in clinically problematic cases.

1173 P53 protein as a predictor of survival in endometrial carcinomas

In an immunohistochemical study tumor specimens from 183 women with endometrial carcinoma were analysed as regards p53 expression, using the monoclonal antibody DO-7. Negative specimens were 101 (55%), whereas the staining intensity was weak, moderate or strong in 27 (15%),4 (2%) and 51 (28%), respectively. DNA content was analysed in 159 (87%) tumors. Moderate or strong expression of p53 were significantly more common in aneuploid tumors (<i>P</i><0.0001). S-phase fraction was evaluable in 165 cases. High S-phase fractions were more frequent in with strong expression of p53 (<i>P</i><0.0001). p53 expression was correlated to grade (<i>P</i><0.05) but not to clinical stage. In histological subgroups such as UPSC (Uterine Papillary Serous Carcinoma), clear cell carcinoma and undifferentiated carcinoma of the endometrium, strong expression of p53 was more frequent. Moderate or strong expression of p53 was highly predictive of a poor survival (<i>P</i><0.001).

664 Flow cytometrically determined S-phase fraction is a stronger prognostic factor in node-negative breast cancer than immunohistochemically detected MIB1 (KI-67)

The proliferation markers, S-phase fraction (SPF) and MIB1, were evaluated in 90 primary node-negative breast carcinomas. SPF was determined flow cytometrically using an improved Hedley protocol for release of pure nuclei from formalin-fixed, paraffin-embedded sections. SPF analysis was performed using the ModFit software program (Verity, Maine, U.S.A.). MIB1 immunostaining (Dianova, Hamburg, Germany) was performed on adjacent paraffin sections using APAAP. The percentage of MIB1 positive cells was calculated in 500 randomly chosen tumor cells. Median follow-up was 34 (9–72) months. An optimal cutoff of 8% SPF was found using isotonic regression analysis: 61 (68%) tumors had low (≤ 8%) and 29 (32%) high (> 8%) SPF. The optimal cutoff for MIB1 was 25%: 75 (83%) tumors had a low (≤ 25%) and 15 (17%) a high (> 25%) MIB1 staining percentage. MIB1 was significantly correlated to grading (P = 0.018) and medullary histological tumor type (P

Image analysis evaluation of ploidy, S-phase fraction and nuclear area in canine mammary tumours

Image analysis was used on cytocentrifuge preparations of Feulgen-stained nuclei extracted from formalin-fixed, paraffin wax-embedded tissues to determine ploidy (assessed by integrated optical density), S-phase fraction, and nuclear area of 90 canine mammary tumours (30 benign and 60 malignant). Only two lesions identified histologically as benign were aneuploid, suggesting potential malignancy. Of malignant tumours, 37% were aneuploid. No distinction based on ploidy could be made within benign or malignant groups, except for tubular adenocarcinomas of complex type, all of which were diploid. Mean S-phase fraction of malignant lesions (15.60%) was twice that of benign lesions (7.87%; P < 0.0001). The mean nuclear surface was significantly smaller in malignant lesions (70.62 microns2) than in benign counterparts (77.45 microns2; P < 0.05). No relation was found between a given tumour type and S-phase fraction or nuclear area within histologically malignant or benign tumour groups. Infiltration of lymphatics by neoplastic cells, known to indicate a poor prognosis, was associated with aneuploid lesions (mean DNA index > 1.25) and high S-phase fraction. The presence of necrosis and of a high number of mitoses was significantly associated with high S-phase fraction lesions.

Epithelioid gastric stromal tumors (leiomyoblastomas): a study of fifty-five cases.

Epithelioid gastric stromal tumors form a distinct histologic subset of gastric tumors whose malignant potential and prognosis are controversial. Fifty-five patients with epithelioid gastric stromal tumors accounted for 11.5% of patients undergoing definitive operations for gastric stromal tumors from 1960 to 1986. Medical records and pathology slides were reviewed, and immunohistochemical staining and flow cytometry were performed. The Kaplan-Meier method was used to estimate survival. Survival curves were compared with log-rank tests and Cox proportional hazards model. Of the 55 tumors, 40 were benign and 15 (27%) were malignant. Mean follow-up was 10.5 years. Ten patients died of their disease. No patient with a benign tumor had recurrence of metastasis, but all patients with high-grade malignancy had died of disease within 3 years after diagnosis. Seventy-five percent of proximal tumors were malignant. Extent of resection had no impact on survival (p = 0.5). The best determinant of tumor behavior was histologic grade. Twenty-seven percent of patients had malignant tumors, and 67% of these died of disease. Other significant prognostic factors included a mitotic count greater than 5/10 high-power fields, size larger than 6 cm, aneuploidy, and higher S-phase fraction (p < 0.01). Proximal lesions were more likely to be malignant. Extent of surgical treatment had no effect on survival.

Expression of insulin-like growth factor I (IGF-I) in female breast cancer as related to established prognostic factors and long-term prognosis.

The expression of insulin-like growth factor I (IGF-I) was analysed immunohistochemically in a series of 211 breast cancers with special emphasis on its relationship to conventional prognostic factors and long-term prognosis. Altogether, IGF-I was expressed by the tumour cells in 91% of the breast carcinomas, and by stromal cells in 29%. The expression of IGF-I in cancer cells was weakly related to a high proportion of intraductal growth (P = 0.032), distinct tumour margins (P = 0.048) and high S-phase fraction (P = 0.074). In a univariate analysis, IGF-I expression in cancer cells was significantly related to a high survival probability in the entire cohort (P = 0.0144) as well as in the axillary lymph node positive tumours (P = 0.0286). Alternatively, expression of IGF-I in the stromal cells was related to metastasis at the time of diagnosis (P = 0.05), tumour diameter (P = 0.04), DNA ploidy (P = 0.07) and nuclear pleomorphism (P = 0.025), but it was without prognostic significance in a univariate analysis (P > 0.1). In a multivariate analysis, the conventional prognostic factors were superior to IGF-I expression in predicting the disease outcome, albeit expression of IGF-I in tumour stroma showed some independent prognostic significance in axillary lymph node negative tumours. The results suggest that IGF-I expression is related to malignant histopathological features in breast cancer, and expression of IGF-I has independent prognostic significance in the early phases of the disease.

Apoptosis suppressing protein bcl-2 is expressed in well-differentiated breast carcinomas with favourable prognosis.

The expression of bcl-2 protein was analysed immunohistochemically in 202 female breast carcinomas. The intensity of bcl-2 expression was inversely related to tumour grade (P < 0.0001), tumor necrosis (P < 0.0001), mitotic index (P < 0.0001), oestrogen receptor content (P < 0.0001), progesterone receptor content (P = 0.0007), S-phase fraction (P = 0.00047), and apoptotic index (P = 0.087). A high fraction of bcl-2-positive cells was related to ductal or lobular type (P = 0.03) and slight nuclear pleomorphism (P = 0.03). Heterogeneous expression of bcl-2 protein was associated with high grade (P = 0.02), severe nuclear pleomorphism (P = 0.02), DNA aneuploidy (P = 0.018), high S-phase fraction (P = 0.05), and early metastasis (P = 0.03). Intense expression of bcl-2 protein was significantly related to favourable outcome in the entire cohort (P = 0.0013), as well as in axillary lymph node-negative (ANN) tumours (P = 0.0124). Long recurrence-free periods in the entire cohort (P = 0.037) and in ANN tumours (P = 0.08) were confined to cases with intense expression of bcl-2 protein. In multivariate analysis, bcl-2 expression had no independent prognostic value in the entire cohort or in axillary lymph node-negative breast carcinomas, whereas it was a weak independent prognostic factor in axillary lymph node-positive breast carcinomas.