High S-phase Fraction Research Articles

Loss of heterozygosity at chromosome 11 in breast cancer: association of prognostic factors with genetic alterations.

We examined DNA from 116 female and four male breast cancer patients for loss of heterozygosity (LOH). DNA was analysed by polymerase chain reaction using ten microsatellite markers on chromosome 11. Three distinct regions of LOH were identified: 11p15.5, 11q13 and 11q22-qter with a LOH frequency of 19, 23 and 37-43% respectively. The marker D11S969 showing the highest frequency of LOH (43%) is located at the 11q24.1-q25 region. No previous molecular genetic studies have shown frequent LOH at the region telomeric to q23 on chromosome 11. Southern analysis revealed that LOH at 11q13 was due to amplification, whereas LOH at 11q22qter was due to deletion. LOH at 11p15.5 was associated with paucity of hormone receptor proteins, high S-phase and positive node status. An association was found between LOH at 11q13 and positive node status. LOH at the 11q22-qter region correlated with a high S-phase fraction. A significant association was found between LOH at 11p15 and chromosome regions 17q21 (the BRCA1 region) and 3p.

Reproducibility in DNA flow cytometric analysis of breast cancer: Comparison of 12 laboratories' results for 67 sample homogenates

Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S-phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra- and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non-diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid+tetraploid) vs. aneuploid (the remaining non-diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54-0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non-diploid; overall kappa = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall kappa = 0.47 (for low SPF vs. high SPF vs. "no SPF reported")]. Discrepancies were chiefly attributable to differences in the categorization of the S-phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near-diploid and small non-diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S-phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S-phase calculation and tetraploid definition.

Do DNA ploidy and S-phase fraction in primary tumour predict the response to chemotherapy in metastatic breast cancer?

The relationship between the response to chemotherapy with cyclophosphamide, epirubicin and fluorouracil as well as the time to progression of metastasised breast cancer and DNA ploidy and S-phase fraction (SPF) of primary tumours was examined using paraffin-embedded tumour tissue from 81 patients. The response to chemotherapy was significantly better in patients with tumours with a high SPF, and in addition the time to progression was longer in the high-SPF group. There was no significant difference when the DNA ploidy and response to treatment were compared.

DNA-FLOW CYTOMETRY (PLOIDY AND S-PHASE FRACTION) AS PROGNOSTIC FACTOR IN A RETROSPECTIVE SERIES OF 515 PRIMARY BREAST-CANCER

Paraffin-embedded tissues are used in retrospective studies to evaluate the prognostic significance of DNA-flow cytometry (DNA-FCM) in human breast cancer. Although paraffin-embedded samples yield information on disease-free survival (DFS) and overall survival (GAS) of homogeneously selected patients, the resulting DNA-histograms have a lower resolution of aneuploid subpopulations and higher debris levels than those of fresh tumor samples. The aim of this study was to evaluate, retrospectively, the prognostic value of ploidy and the S-phase fraction (SPF) using 515 samples of paraffin-embedded consecutive primary breast cancer tissue (median follow-up: 75.4 months). Ploidy was detectable in 89% cases (34% diploid and 66% aneuploid) and SPF in 77%. The optimal cut-off for SPF was 6%. High SPF values were significantly correlated with shorter DFS (p=0.028) and OAS (p=0.018); aneuploidy was significantly correlated only with a shorter OAS (p=0.0058). Using the Cox proportional hazards regression model to evaluate the independence of DNA-FCM derived parameters, only high SPF was able to predict both a shorter DFS (p=0.02) and OAS (p=0.002). Furthermore, high SPF values were found correlated to aneuploidy (p<0.00001), tumor necrosis (p<0.015) and high histopathological grade (p<0.03). The data reported confirm that SPF is a valuable single independent prognostic factor in human breast, cancer and strongly support the use of archival tumor specimens to study the prognostic role of DNA-FCM in human cancer.

Debris compensation of DNA histograms and its effect on S-phase analysis.

Debris compensation is an important variable affecting S-phase fraction (SPF) analysis in flow cytometric DNA histograms. The SPF was estimated in fresh frozen breast carcinomas using the following four debris subtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moel (EXP-SC); the random cutting of nuclei into multiple pieces of varying sizes (MC); and a combination of both nuclear cutting models (SC-MC). Comparison of SPF estimates indicated that the various debris subtraction models yielded differences in SPF, with SPF values obtained using the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms based on nuclear slicing and fragmentation. However, SPF estimates could be affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G0/1 peak, and the relative amount of debris. Using the ratio of the height of the G0/1 peak to the height of the debris between the chicken red blood cells (CRBC) and G0/1 peaks as an objective measurement of nuclear debris, debris compensation was necessary in diploid DNA histograms where this ratio was as low as 1.5%. Taken in the context of SPF prognostic cutoff levels, variation in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF categories, thereby making the comparison of SPF values derived from different studies difficult.

CD5-Expressing B-Cell Non-Hodgkin's Lymphomas With bcl-1 Gene Rearrangement Have a Relatively Homogeneous Immunophenotype and Are Associated With an Overall Poor Prognosis

Mantle cell lymphomas (MCLs) are typically CD5-expressing B-cell non-Hodgkin's lymphomas (NHLs) that frequently harbor the chromosomal translocation t(11;14) or bcl-1 gene rearrangements. Insufficient data are available on the biologic features and clinical behavior of rigorously characterized MCL. As these NHLs have been reported to exhibit various histologic and cytologic expressions, and in order to avoid using somewhat arbitrary and subjective morphologic definitions, we chose to study cases of MCL selected on more objective grounds. Specifically, 15 samples (from 14 patients) of CD5-expressing B-cell NHLs with detectable bcl-1 gene rearrangement were included. Overall, these patients had relatively uniform clinical manifestations. Most were older men (mean age, 67 years) who presented with lymphadenopathy, high-stage disease, and bone marrow involvement. All but two patients relapsed, demonstrated residual tumor, or had disease progression after an initial response to various therapies. Nine patients have died; these patients had a median survival of only 19 months. All cases could be classified within the broad morphologic spectrum previously described for MCL, and no predominant histologic subtype was observed. However, cases could be segregated into two major groups according to tissue architecture: one with a purely diffuse pattern and the other with at least a focal nodular component. Patients with purely diffuse tumors had a lower survival rate (0%) than those with tumors having a nodular component (62% survival rate). In contrast to the morphologic variability, these NHL exhibited a rather homogeneous immunophenotypic pattern. All cases demonstrated intense CD20 expression, with typically intense IgM and light chain expression, and relatively weak IgD expression. In no case was CD10 detected on the neoplastic cells. DNA content analysis showed aneuploidy only in three instances, and two groups of cases could be arbitrarily defined on the basis of their S-phase fraction. A relationship between a purely diffuse growth pattern and a high S-phase fraction (greater than 5%) was observed. As expected from this association, patients with tumors having high S-phase fractions fared worse (14% survival rate) than those patients with tumors showing lower S-phase fractions (57% survival rate). Thirteen NHLs from 12 patients had amplifiable bcl-1 gene rearrangements at the major translocation cluster (MTC). The bcl-1 breakpoints aggregated within a 63-bp region of the MTC, and the amplified tumor DNA from each patient had unique N-nucleotide junctional sequences and Ig joining region breakpoint sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between low estrogen receptor values and other prognostic factors in primary breast tumors

The current study compared the immunocytochemical expression of estrogen (ER) and progesterone (PgR) receptors by malignant breast cells to the hormone receptor concentrations reported from radioligand assays. These values were examined in relation to DNA ploidy and the fraction of cells in S phase. ER and PgR concentrations, DNA ploidy, and S-phase fractions were measured by standard techniques with 124 samples of invasive ductal carcinoma. Suspensions of tumor cells were examined by immunocytochemical assay (ICA) for the percentages of ER and PgR positive cells. Twenty-six of the 38 tumors from patients 50 years of age or younger were classified as high S-phase fraction, and 28 tumors had aneuploid levels of DNA. The 20 ER positive tumors each contained less than 100 fmol/mg. Thirty-nine of the 86 tumors from patients older than 50 years were classified as high S phase, and 41 were aneuploid. Sixty-five samples were considered ER positive by radioligand assay. ICA showed that tumors in either age group with less than 40 fmol/mg did not contain ER positive cells. The proportion of samples with PgR levels between 10 and 100 fmol/mg was small, and fewer PgR positive tumors were categorized as negative when examined by ICA for receptor containing cells. The reclassification of the hormone receptor status of a tumor based on ICA appeared to be independent of S-phase and ploidy values. Tumors that are classified as ER or PgR positive based on accepted cutoff values for radioligand assays may actually be receptor negative because the tumors do not appear to contain receptor positive cells.

DNA flow cytometric analysis in renal neoplasms associated with acquired renal cystic disease.

In order to better understand the potential malignancy of renal neoplasms arising in patients with acquired renal cystic disease and to try and establish differences from other renal tumours we analysed DNA ploidy as well as the level of S-phase fraction in 11 neoplasms associated with acquired cystic disease by means of flow cytometry. The results were correlated with known prognostic factors such as nuclear grade, size and stage, as well as the clinical behaviour of the tumours. We found a close relationship between DNA aneuploidy and high S-phase fraction and a poor clinical outcome. We also found some differences in the DNA ploidy profile of these tumours when compared with those reported in other renal neoplasms.

Enhanced expression of catalytic subunit isoform PP1γ1 of protein phosphatase type 1 associated with malignancy of osteogenic tumor

The expressions of the three catalytic subunits of protein phosphatase (PP) type 1 and 2A, PP1α, PP1γ1, and PP2AC, were examined in 14 cases of three types of osteogenic tumor using immunohistochemical analysis. The percentage of tumor cells stained positively with antiserum against PP1 catalytic subunit isoform PP1γ1 was significantly higher in malignant osteogenic tumors than in benign osteogenic tumors. Furthermore, malignant osteogenic tumor showed markedly high S-phase fraction in the cell cycle of tumor cells, as compared to benign osteogenic tumors. These results suggest that PP1γ1 is involved in the accelerated growth of malignant cells in osteogenic tumors.

Prognosis following locally recurrent soft‐tissue sarcoma. A staging system based on primary and recurrent tumour characteristics

We have shown that the clinical growth rate of local recurrence from soft-tissue sarcoma could be expressed as a growth-rate index (GRI) which was predictive for metastasis, and which was able to identify 2 equal populations of good (80% 2-year MFS) and poor survivors (33%). We now report the associations between characteristics of the primary and GRI, and combine primary and locally recurrent tumour characteristics in a staging system. We studied 460 adult patients with soft-tissue sarcomas of the extremities and trunk wall who were diagnosed and treated between 1964 and 1990, of whom 134 developed local recurrences and 151 metastases. The association of primary tumour size, histologic malignancy grade, depth, spontaneous necrosis, intratumoral vascular invasion and S-phase fraction with local recurrence, GRI and metastasis were examined. High GRI was associated with primary tumours that were larger, deeper, more malignant, underwent spontaneous tumour necrosis, demonstrated intravascular invasion and had a higher S-phase fraction. The same factors were also strongly associated with the incidence of metastasis. A multivariate analysis found GRI and primary tumour necrosis to be the strongest and most significant prognostic factors. GRI and tumour necrosis were combined in a staging system that identified groups with good survival (79 to 94% 2-year MFS), intermediate survival (61% 2-year MFS) and exceptionally poor survival (6% 2-year MFS). These findings validate our earlier assertion that high GRI reflects highly malignant tumours. A staging system composed of primary tumour necrosis and GRI can identify patients who may be suitable candidates for trials of adjuvant chemotherapy.

High Cell Proliferation Activity Determined by DNA Flow Cytometry and Prognosis in Epidermoid Lung Carcinoma

DNA aneuploidy and high cell proliferation activity determined by DNA flow cytometry are known to be associated with poor prognosis in many human malignancies. The aim of the present study was to determine the long-term prognostic value of DNA ploidy and cell proliferation activity in lung cancer. The material consisted of samples from 290 paraffin-embedded lung cancers (171 epidermoid carcinomas, 62 small-cell carcinomas, 26 adenocarcinomas, and 31 others), of which DNA ploidy and cell proliferation activity as defined by S-phase fraction (SPF) was determined by DNA flow cytometry. DNA aneuploidy was found in 61% of the tumours and the mean (+/- SD) SPF was 13.6 +/- 9.5%. DNA aneuploidy was associated with a high T-stage (p < 0.05) in epidermoid carcinoma, but not in small-cell carcinoma or adenocarcinoma. DNA aneuploidy and high SPF were associated with poor survival in epidermoid carcinoma. However, only SPF (relative risk (RR) = 1.80), N-(RR = 2.46) and M-stages (RR = 2.17) were statistically significant predictors of survival according to multivariate analysis. Regression-tree analysis showed that the prognostic value of SPF was restricted to localized (M0-stage) epidermoid carcinoma. Neither DNA aneuploidy nor SPF was associated with survival in small-cell lung carcinoma. In conclusion, SPF is an independent prognostic factor in epidermoid lung carcinoma.

S-phase fraction and survival benefit from adjuvant chemotherapy or radiotherapy of breast cancer

Cancer chemotherapy interacts with cell proliferation, but data on the relationship between cancer cell replication and the effect of adjuvant chemotherapy are scarce. We have investigated the S-phase fractions of the primary tumour from premenopausal breast cancer patients who participated in a randomised trial comparing 12 cycles of polychemotherapy (CMF) with post-operative radiotherapy. DNA flow cytometry was performed on frozen tissues from 208 primary breast carcinomas, of which the S-phase fraction was estimated in 176 cases. There was a significantly higher benefit from CMF among patients with a high S-phase fraction (P = 0.0033). The relative risk of distant recurrence or death in the chemotherapy group as compared with the radiotherapy group was 0.19 for patients whose tumours had an S-phase fraction of 10% or over (95% CI 0.07-0.51) and 1.55 (0.88-2.73) for patients whose tumours showed lower S-phase levels. The interaction was still significant in multivariate analysis (P = 0.0057), including lymph node metastases, tumour size and oestrogen receptor content. We conclude that the benefit from adjuvant chemotherapy compared with radiotherapy is largely confined to patients with highly proliferative tumours.

Tumour vascularity and basement membrane structure in breast cancer as related to tumour histology and prognosis.

A series of 202 breast cancer biopsy specimens were analysed immunohistochemically for collagen IV to demonstrate basement membrane (BM) structures and blood vessels within tumour tissue. Integrity of the BM was graded into four categories and the number of vascular channels per square millimetre of tumour tissue were counted. Defective BM structures were significantly related to high grade, lack of tubule formation, invasive disease, high S-phase fraction and variability in nuclear size and shape. High vascular channel density was related to poor tumour differentiation and a high proliferation rate of cancer cells as well as to the absence of tubule formation, inconspicuous intraductal growth and low progesterone receptor content. High vascular density and defective BM structures were signs of poor prognosis and short recurrence-free survival in the entire cohort and also in local tumours. In multivariate analysis, the vascular density had independent prognostic value, as did the diameter, axillary lymph node status and mitotic rate. The counting of vascular channels within the tumour provides additional prognostic information in breast cancer, in contrast to analysis of the BM integrity which shows hardly any prognostic information additional to that provided by the special histological features, e.g. tubule formation and intraductal growth pattern.

FLOW CYTOMETRIC DNA ANALYSIS IN PREDICTION OF INTRAVESICAL RECURRENCE FROM HUMAN BLADDER-CANCER

A series of 128 patients undergoing diagnostic and/or therapeutic transurethral resection for primary urothelial bladder carcinoma was prospectively studied in order to evaluate the relative importance of DNA-ploidy and S-phase fraction (SPF) by flow cytometry and of several clinicopathological features in predicting disease-free survival. Only frozen tissue was used. The mean follow-up was 22 months (range: 3-61 months). DNA-aneuploidy was present in 64% of the cases (82/128), while multiclonality was found in 29% of the DNA-aneuploid cases (24/82). In the univariate statistical analysis, high stage, DNA-aneuploidy (mono- and multiclonal patterns) and high SPF value (>11.6%) proved to be significantly related to the risk of intravesical recurrence, while a significant trend was present for histological grade. In the multivariate analysis only high SPF value (>11.6%) and DNA-aneuploidy were independently related to risk of relapse (RR of 2.39 and 2.40 respectively).

Progression and survival in prostatic adenocarcinoma: a comparison of clinical stage, Gleason grade, S-phase fraction and DNA ploidy

Clinical data were reviewed in 325 patients with prostatic adenocarcinoma followed up for a mean of 13 years. Paraffin-embedded tumour biopsy specimens from the primary tumours were available for flow cytometry (FCM) in 273 cases. Intra-tumour heterogeneity in DNA index (DI) was found in 4% of the tumours (54 cases were analysed). S-phase fraction (SPF) and DNA ploidy were significantly interrelated. Aneuploidy and high SPF were significantly related to both a high T category and high Gleason score. The progression in T1-2M0 tumours was related to Gleason score (P = 0.009), DNA ploidy (P = 0.006) and SPF (P = 0.007), while the Gleason score (P = 0.0013), DNA ploidy (P = 0.002) and SPF (P < 0.001) had prognostic value in univariate survival analysis. In the entire cohort, the T category (P < 0.001), M category (P < 0.001), Gleason score (P < 0.001), DNA ploidy (P < 0.001) and SPF (P < 0.001) were significant prognostic factors. In Cox's analysis, the M category (P < 0.001), Gleason score (P < 0.001), T category (P = 0.003), age (P = 0.001) and SPF (P = 0.087) were independently related to prognosis. In the T1-2M0 tumours, Gleason score (P < 0.001), T category (P = 0.022) and SPF (P = 0.058) were independent predictors. A novel classification system in which the DNA ploidy or SPF and the Gleason score were combined was found to be of significant prognostic value in all M0 tumours (P < 0.001). The results suggest that FCM can be used as an adjunct to conventional histological assessments for determination of the correct prognostic category in prostatic adenocarcinoma.

Overexpression of p53 and long-term survival in colon carcinoma

Survival analysis of 144 histologically confirmed cases of colon carcinoma diagnosed in a 12 year period (1971-82) at the Tampere University Hospital was performed to test the hypothesis that p53 overexpression is associated with a poor clinical outcome. Immunohistochemical staining of paraffin-embedded sections using a polyclonal antibody CM-1 against p53 protein was performed to identify aberrant expression of the p53 tumour-suppressor gene. Sixty-nine per cent of the tumours (100/144) showed overexpression of the p53 protein. The prevalence of p53 overexpression was independent of age and sex of the patient and subsite of the tumour, but was slightly, although not statistically significantly, higher in advanced than in localised tumours. Overexpression was associated with a higher S-phase fraction. Some indication of a larger proportion of aneuploid tumours among those with overexpression was also observed, although this finding did not reach statistical significance. Significantly reduced patient survival for tumours with p53 overexpression was found. Patients with p53-overexpressing tumours had a corrected 5 year survival rate of 37% compared with 58% among patients whose tumours had normal expression of p53. The corresponding 10 year rates were 34% and 54%. In the multivariate analysis using a Cox model, the survival difference was independent of age and sex of the patient, as well as of subsite and stage of the tumour. Furthermore, the effect of p53 overexpression remained after controlling for flow cytometric parameters in an analysis of a subset of tumours. Thus, p53 overexpression appears to be a useful prognostic indicator in colon carcinoma.

Prognostic value of Ki-67 expression in 182 soft tissue sarcomas. Proliferation--a marker of metastasis?

Soft tissue sarcomas (STS) are characterized by deregulated proliferation. Ki-67 is a cell cycle antigen which may be elevated in proliferative states. We analysed Ki-67 expression in fixed and embedded tissues from STS in order to examine associations between proliferation, primary tumour characteristics, and metastasis. One hundred and eighty-two adult patients with trunk wall or extremity STS were treated at our institution between 1980 and 1992 (35 developed local recurrence and 56 developed metastases). Median follow-up time for survivors was 6 years (1-13). We used a semiquantitative score to the assess percentage of Ki-67-positive cells: < or = 10% (n = 86), > 10-25% (n = 57), > 25-50% (n = 30), > 50-75% (n = 7), > 75-100% (n = 2). Increasing Ki-67 expression correlated positively with tumour size, malignancy grade, necrosis, vascular invasion, S-phase fraction, and metastasis. A Ki-67 index Ki-D < or = 10% (n = 86) and > 10% (n = 96) defined two groups who had 84% and 56% 3-year metastasis-free survival (p = 0.0001), respectively. Tumours with Ki-D > 10 were typically large, high grade, necrotic, DNA aneuploid, and had intravascular invasion and a higher S-phase fraction. Ki-67 expression may be helpful in predicting survival of patients with soft tissue sarcomas.

High proliferative rates demonstrated by bromodeoxyuridine labeling index in breast carcinomas with p53 overexpression.

The protein p53 is a product of a suppressor oncogene. Mutations occurring in 13-15% of breast carcinomas are associated with p53 stainability within nuclei and progression of the tumor. We determined the extent to which p53 abnormality was associated with proliferation by measuring p53 immunohistochemically with a polyclonal antibody and monoclonal PAb1801 in invasive carcinomas of known S-phase fraction (SPF) assessed histologically by bromodeoxyuridine incorporation. Results with the two antibodies always agreed. One of 20 low, 2/18 midrange, and 9/17 high SPF carcinomas were positive for p53. P53 positivity was also related to other indicators of aggressiveness including size of primary tumor, nuclear and nucleolar size, and estrogen and progesterone receptor content, but relationships between p53 and vascular invasion and lymph node metastasis were not found. We conclude that nuclear p53 accumulation is more closely related to proliferation than to invasion and metastasis, and that it identifies some but not all breast carcinomas with high proliferative indices.

Flow cytometry detected S-phase fraction and ploidy as prognostic parameters in primary, node-negative breast cancer

100 specimens of patients with primary node-negative breast cancer were examined by flow cytometry. 64 tumours were shown to be diploid and 36 tumours were aneuploid. The median was the cut-off level for tumours with low S-phase (< or = 10.9%) and high S-phase values (> 10.9%). Aneuploid tumours were associated with high S-phase fraction. The average length of follow-up was 42 months. In univariate analysis for disease-free survival, diploid tumours were shown to have a slightly better prognosis than aneuploid tumours (p = 0.07). This trend could not be shown in multivariate analysis. The S-phase fraction proved to be an independent prognostic factor. Patients, whose tumours had S-phase fractions < or = 10.9%, had significantly longer disease-free survival in univariate (long-rank-test: p = 0.021), as well as in multivariate analysis according to the Cox regression model (p = 0.033). Since, as already stated, the S-phase fraction is an independent prognostic factor for node-negative breast cancer, further adjuvant therapy studies should take this factor into consideration.

Evaluation of thymidylate synthase RNA expression by polymerase chain reaction

5-Fluorouracil (5FU), an antimetabolite often used for the treatment of breast cancer, binds to and inactivates the enzyme thymidylate synthase (TS). Measurement of TS levels may be useful in examining resistance to 5FU, but current methods involving ligand binding assays present considerable problems due to the low basal level of this enzyme in many tissues. Therefore, a semiquantitative polymerase chain reaction (PCR) technique to measure TS RNA concentration in breast tumour cell lines has been developed with potential for measuring TS RNA levels in individual breast tumours. Using less than 1 μg of total RNA, we determined that the T47D breast cancer cell line expressed TS levels 1·3 to 4·4 times lower than those of four other breast cancer cell lines, MCF-7, MDA-MB-468, MDA-MB-231, and MDA-A1 R. Using the MCF-7 cell line as a representative of relatively high TS expression, the relationship between the S-phase fraction (SPF), and TS levels in MCF-7 and T47D cells was next examined because cell cycle kinetics can also influence drug response. TS expression was directly related to SPF. MCF-7 cells had a higher SPF, corresponding to their higher TS level relative to T47D cells. Lastly, 5FU sensitivity was assayed for and it was found that MCF-7 cells were more sensitive to 5FU than T47D, despite having higher TS levels. A semiquantitative technique for assessing TS RNA expression by PCR in breast cancer cell lines has been demonstrated. In the cell lines tested, there was no direct relationship between TS level and 5FU sensitivity, though there was a direct relationship between cell proliferation and sensitivity to 5FU.