Cytokinetic analysis of lung cancer by in vivo bromodeoxyuridine labelling.

Cytokinetic parameters of various types of lung cancer were determined in bronchoscopy specimens after in vivo labelling with the thymidine analogue bromodeoxyuridine (BrdU). The S-phase fraction and BrdU labelling index were measured flow cytometrically, allowing calculation of the S-phase transit time and potential tumour doubling time. The methodology used was found to be feasible for obtaining cytokinetic data from 76% of the bronchial biopsy samples. Despite the difference in clinical behaviour and growth pattern between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), no significant differences were observed between the mean values of the cytokinetic parameters of SCLC and NSCLC. The estimated cell loss factor was higher in NSCLC than in SCLC. It appears that the growth of a tumour, as clinically observed, is to a considerable extent influenced by cell loss. In accord with this assumption is the fact that we have observed non-BrdU labelled S-phase cells, both in tumour biopsies and in apparently normal tissue. The presence of these so-called unlabelled S-phase cells in relation to cell loss is discussed.     

Late relapse of testicular cancer presenting as differentiated teratoma - report of a case and discussion of the clinical implications

This report describes the unusual case of a patient with late relapse of testicular cancer, histologically defined as differentiated teratoma occurring 76 months after primary therapy for metastatic non-seminomatous germ cell cancer. During initial treatment the patient received 4 cycles of cisplatin-based chemotherapy followed by secondary resection of residual retroperitoneal and pulmonary metastases which had histologically revealed necrotic tissue. The patient had been without evidence of disease during the follow-up until May 1994 when a cystic mass was noted in the left fossa obturatoria. There was no concomitant elevation of serum tumour markers, while alpha-fetoprotein (AFP) could be detected in fluid gained by fine-needle aspiration from inside the cystic structure. Histological resection showed a differentiated teratoma. Late relapses defined as tumour recurrences later than 24 months after primary chemotherapy for metastatic testicular cancer are rather rare and affect only 2-5% of patients. However, the prognosis of patients with late relapse is poor with only 20-30% of patients achieving a second remission after chemotherapy +/- surgery. Patients presenting with only differentiated teratoma at late relapse and in whom the tumour can be completely surgically resected have the best prognosis among the subgroup of patients with late relapses with a long-term survival in approximately 50% of patients. The incidence and clinical implications of late relapses after chemotherapy for testicular cancer are discussed in the current report.     

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.     

Cytophotometric Estimation of Cell Proliferation in Breast Cancer: Correlation to the clinical course during long-term follow-up

Tumours from 117 patients with breast cancer operated during a 5-month period 1975-1976 were investigated by absorbance scanning cytophotometry. the relations of these data to breast cancer recurrence and death during a follow-up period of 8.5 years were analysed using Cox's proportional hazards model. When individually tested, nodal status, grade of malignancy and high rates of proliferation, as indicated by cells in S-phase, were statistically significant predictors of the clinical outcome. the risk ratio associated with S-phase decreased significantly over time from primary treatment. No significant relation was found between DNA-ploidy or tumour size and the clinical course. in the multivariate analysis, based on 76 patients, nodal status alone was a significant prognostic factor.     

Clinicopathological features and survival of testicular tumours in a Southeast Asian university hospital: a ten-year review

Introduction: Testicular cancer mainly affects young men worldwide. There is lack of published data on patients with this malignant condition from the Southeast Asian region. The aim of this study was therefore to determine the clinicopathologic features of testicular cancer patients treated in a Southeast Asian university hospital and their overall survival rate. Materials and methods: This was a retrospective study of testicular cancer patients treated between January 2001 and February 2011. Their epidemiological data, clinical presentation, pathologic diagnosis, stage of disease and treatment were gathered and the overall survival rate of this cohort was analyzed. Results: Thirty-one patients were included in this study. The majority of them were of Malay ethnicity. The average age at presentation was 33.7 years. The commonest testicular cancer was non-seminomatous germ cell tumour, followed by seminoma, lymphoma and rhabdomyosarcoma. More than half of all testicular germ cell tumour (GCT) patients had some form of metastasis at diagnosis. All the patients were treated with radical orchidectomy. Adjuvant chemotherapy was given to those with metastatic disease. Four seminoma patients received radiotherapy to the para-aortic lymph nodes. The 5-year survival rate for all testicular cancers in this cohort was 83.9%. The survival rate was 88.9% in 5 years when GCT were analyzed separately. Conclusion: GCT affects patients in their third and fourth decades of life while lymphoma patients are generally older. Most of the patients treated for GCT are of Malay ethnicity. The majority have late presentation for treatment. The survival rate of GCT patients treated here is comparable to other published series in other parts of the world.     

Prognostic value of DNA flow cytometry in stomach cancer: a 5-year prospective study

The role of DNA flow cytometry in the prediction of prognosis for patients with stomach cancer remains to be defined. Thus we studied prospectively the role of DNA flow cytometry as a prognosis indicator in stomach cancer patients in a high-incidence area. Between November 1990 and December 1992, primary stomach cancer tissues were obtained from the surgical specimens from 217 patients (148 male, 69 female). DNA flow cytometric analyses of DNA ploidy and S-phase fraction were performed and the results were correlated with patient survival. The median age of the patients was 55 years (range 24-78). Aneuploid cell population was found in 114 of 217 samples (53%). Tumour S-phase fraction was obtained in 96 of 103 diploid tumours (93%) and 61 of 114 aneuploid tumours (54%). After median follow-up of 66.1 months, the patients with tumours with an S-phase fraction over 17% had significantly worse survival rates than patients with tumours with S-phase fractions of lower than 8% or 8-17% (45% vs 59% and 63% of patients surviving, P = 0.007). Tumour ploidy status did not correlate with patient survival. Multivariate analyses showed that the TNM stage remained the most important prognostic indicator. The tumour S-phase fraction was also an independent prognostic indicator (relative risk 2.300, 95% CI, 1.252-4.223). Tumour S-phase fraction obtained by DNA flow cytometry is an independent prognostic indicator for the survival of the patients with stomach cancer.     

No longer any role for routine follow-up chest x-rays in men with stage I germ cell cancer

Following radical orchidectomy for testicular cancer, most patients undergo protocolled surveillance to detect tumour recurrences rather than receive adjuvant chemotherapy. Current United Kingdom national and most international guidelines recommend that patients require a chest x-ray (CXR) and serum tumour markers at each follow-up visit as well as regular CT scans; there is however, variation among cancer centres with follow-up protocols. Seminomas often do not cause tumour marker elevation; therefore, CT scans are the main diagnostic tool for detecting relapse. For non-seminomatous tumours, serum beta-HCG (HCG) and AFP levels are a very sensitive harbinger of relapse, but this only occurs in 50% of patients [1], and therefore, imaging remains as important. CXRs are meant to aid in the detection of lung recurrences and before the introduction of modern cross-sectional imaging in the early 1980s, CXRs would have been the only method of identifying lung metastasis. We examined the Thames Valley and Mount Vernon Cancer Centre databases to evaluate the role of CXRs in the 21st century for the follow-up of men with stage I testicular cancer between 2003 and 2015 to assess its value in diagnosing relapsed germ cell tumours. From a total of 1447 patients, we identified 159 relapses. All relapses were detected either by rising tumour markers or planned follow-up CT scans. Not a single relapse was identified on CXR. We conclude that with timely and appropriate modern cross-sectional imaging and tumour marker assays, the CXR no longer has any value in the routine surveillance of stage I testicular cancer and should be removed from follow-up guidelines and clinical practice. Omitting routine CXR from follow-up schedules will reduce anxiety as well as time that patients spend at hospitals and result in significant cost savings.     

Proliferation and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas.

We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin''s lymphoma using flow cytometry. A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression. Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin''s lymphoma (NHL) patients revealed a non-normal distribution for both parameters. The median fraction of apoptotic tumour cells was 1.1% (25 percentiles 0.5%, 2.7%). In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells (1.9%; 25 percentiles 0.7%, 4.0%; P = 0.03). The median fraction of tumour cells in S-phase was 1.4% (25 percentiles 0.8%, 4.8%), the median fraction of normal cells in S-phase was significantly lower than for the tumour cells (1.0%; 25 percentiles 0.6%, 1.9%; P = 0.004). When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data. One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7%. These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as ''low'' (< 3%) or ''high'' (> 3%), independent of the median S-phase fraction. The apoptotic fractions were log-normally distributed. The median apoptotic fraction was higher (1.5%) in the ''high'' S-phase group than in the ''low'' S-phase group (0.8%; P = 0.02). However, there was no significant correlation between the two parameters (P > 0.05).     

Carcinoma in situ of the testis: review of biological and clinical features

Carcinoma in situ of the testis (CIS) is the uniform precursor of testicular germ-cell tumours. Morphologically, CIS consists of large, intratubular, gonocyte-like cells with large nuclei and abundant glycogen. CIS cells are probably derived from primordial germ cells and are supposed to be present in the testis of a future testis cancer patient at the time of birth. CIS cells appear to spread inside the seminiferous tubules until CIS progresses to invasive cancer. Diagnosis is best achieved by surgical biopsy of the testis and subsequent immunohistological staining of placental alkaline phosphatase (PlAP). This enzyme is present in embryonal germ cells, CIS and seminoma as well as several other types of germ-cell tumour but usually not in normal germ cells. CIS is found in testicular tissue adjacent to testicular germ-cell tumours in about 90% of cases, and it is observed in all clinical groups known to be at risk for testicular cancer: cryptorchidism (2% to 4%), infertility (0% to 1%), ambiguous genitalia (25%) and contralateral testis of patients with testicular cancer (5%). Conversely, CIS is found in less than 1% of the normal male population, and this prevalence corresponds well to the life-time risk of testicular cancer in males. If CIS is left untreated, there is a 50% probability of progressing to frank germ-cell neoplasm within 5 years. Localised low-dose radiotherapy to the testis eradicates CIS and germ cells, while Leydig cells are preserved. The patient can thus be spared orchiectomy and hormone supplementation. Currently, dose-reduction studies are looking for the optimal radiation dose, which is expected to be around 14 to 16 Gy. After chemotherapy, there is a cumulative risk of 42% for recurrence of CIS within 10 years. The concept of CIS offers the chance of very early detection of testicular cancer and organ-preserving early treatment.     

A comparison between flow cytometric assessment of S-phase fraction and Nottingham histologic grade as prognostic instruments in breast cancer

Flow cytometric DNA analysis with assessment of S-phase fraction and DNA ploidy was compared to Nottingham histologic grade. The study population consisted of 654 patients who presented between 1987 and 1996 with primary operable breast cancer and whose tumours had been analysed for S-phase fraction and DNA ploidy at the time of surgery. Grade, tumour size, node status, steroid receptor status, age, S-phase fraction and DNA ploidy were analysed univariately and multi-variately in a Cox proportional hazard analysis. In the univariate analyses all parameters were statistically significantly associated with breast cancer mortality during the follow-up period of 2–11 years. The most powerful predictor of death from breast cancer in the multiple regression analysis was grade. Patients with grade 1 tumours have excellent prognosis. We conclude that tumour grade is a strong prognostic indicator applicable to all breast cancer patients, regardless of size and nodal status, and advocate its general use.     

Testicular tumours presenting as gynaecomastia.

AIM: Gynaecomastia is the commonest benign breast condition seen in men. It is usually due to age, co-existing disease, drugs or idiopathic factors. Rarely is the cause a testicular tumour. We have assessed those men who were subsequently diagnosed as having a testicular tumour in a population of men referred to a specialist breast surgeon. METHODS: A retrospective review of 175 men who had presented with breast enlargement and/or 'lumps' over seven years (1993-2000) to a specialist breast surgeon was performed. All patients were investigated by a protocol including biochemical assessment. RESULTS: 175 men, median age 44 years (range 18-89) who presented to the breast clinic were assessed. 127 men had gynaecomastia (39 bilateral), 8 had breast cancer and 4 had testicular cancer. Of the men with testicular tumours, two had bilateral gynaecomastia; a testicular mass was palpable in two and the diagnosis confirmed on scrotal ultrasound in all four. CONCLUSION: The possibility of a testicular tumour must be considered in any male presenting with gynaecomastia. Clinical testicular examination is essential and the determination of serum tumour markers useful in the overall assessment of those presenting with 'true gynaecomastia'.     

Cell cycle phase influences tumour cell sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro.

Photodynamic therapy (PDT) is a form of cancer treatment based on the destruction of cells by the interaction of light, oxygen and a photosensitizer. Aminolaevulinic acid (ALA) is the prodrug of the photosensitizer protoporphyrin IX (PpIX). ALA-induced PDT depends on the rate of cellular synthesis of PpIX, which may vary with cell cycle phase. This study has investigated the relationship between cell cycle phase, PpIX generation and phototoxicity in synchronized and unsynchronized bladder cancer cells (HT1197). In unsynchronized cells, relative PpIX fluorescence values (arbitrary units) were significantly different between cell cycle phases after a 1-h ALA incubation (G1 24.8 +/- 0.7; S-phase, 32.7 +/- 0.8, P < 0.05; G2 35.4 +/- 0.8, P < 0.05). In synchronized cells after a 1-h ALA incubation, cells in G1 produced less PpIX than those in S-phase or G2 [6.65 +/- 1.1 ng per 10(5) cells compared with 15.5 +/- 2.1 (P < 0.05), and 8.1 +/- 1.8 ng per 10(5) cells (not significant) respectively] and were significantly less sensitive to ALA-induced PDT (% survival, G1 76.2 +/- 8.3; S-phase 49.7 +/- 4.6, P < 0.05; G2 44.2 +/- 2.4, P < 0.05). This differential response in tumour cells may have implications for clinical PDT, resulting in treatment resistance and possible failure in complete tumour response.     

MIB-1 labelling index is an independent prognostic marker in primary breast cancer.

The proliferative activity of a tumour is considered to be an important prognostic factor in primary breast cancer. We have investigated the prognostic value of the MIB-1 labelling index in 341 patients with primary breast cancer and compared the results with the S-phase fraction in 220 patients of the same cohort. All patients were treated in one hospital and had a median follow-up of 128 months. No correlation between MIB-1 labelling and S-phase fraction could be demonstrated. MIB-1 had prognostic value for disease-free survival in the whole group of patients (P < 0.001) and in the node-negative subgroup (P < 0.001). In multivariate analysis, MIB-1 was an independent prognostic factor (P = 0.004) besides axillary lymph node status (P = 0.001). In univariate analysis high S-phase fraction was associated with decreased overall survival (P = 0.04); however, not in multivariate analysis. Moreover, S-phase fraction had a borderline prognostic significance for post-relapse survival in multivariate analysis (P= 0.08). Thus, in conclusion, the growth fraction of a tumour as determined by the MIB-1 labelling index is an important prognostic factor in patients with primary breast cancer.     

Hypermethylation of the 5' CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis

The Testisin gene (PRSS21) encodes a glycosylphosphatidylinositol (GPI)-linked serine protease that exhibits testis tissue-specific expression. Loss of Testisin has been implicated in testicular tumorigenesis, but its role in testis biology and tumorigenesis is not known. Here we have investigated the role of CpG methylation in Testisin gene inactivation and tested the hypothesis that Testisin may act as a tumour suppressor for testicular tumorigenesis. Using sequence analysis of bisulphite-treated genomic DNA, we find a strong relationship between hypermethylation of a 385 bp 5' CpG rich island of the Testisin gene, and silencing of the Testisin gene in a range of human tumour cell lines and in 100% (eight/eight) of testicular germ cell tumours. We show that treatment of Testisin-negative cell lines with demethylating agents and/or a histone deacetylase inhibitor results in reactivation of Testisin gene expression, implicating hypermethylation in Testisin gene silencing. Stable expression of Testisin in the Testisin-negative Tera-2 testicular cancer line suppressed tumorigenicity as revealed by inhibition of both anchorage-dependent cell growth and tumour formation in an SCID mouse model of testicular tumorigenesis. Together, these data show that loss of Testisin is caused, at least in part, by DNA hypermethylation and histone deacetylation, and suggest a tumour suppressor role for Testisin in testicular tumorigenesis.     

Relationship of DNA Ploidy and S-Phase Fraction to Survival After First Recurrence of Breast Cancer

Flow cytometry was performed on frozen specimens from the primary tumour of 184 women with recurrent breast cancer. No significant association was seen between DNA ploidy and the other prognostic factors investigated. Patients with a high S-phase fraction had more often a negative estrogen receptor (ER) status and a short disease-free interval. A shorter survival after disease recurrence was seen both in patients with DNA aneuploid tumours and among those with a high S-phase fraction. Patients with DNA tetraploid tumours showed the longest survival after recurrence. In this subgroup, half of the patients survived more than 3 years after recurrence and the estimated survival rate at 10 years was 17%. In a Cox's regression analysis including 116 patients, site of recurrence, number of positive nodes at time of primary operation, size and ER content of the primary tumour as well as DNA ploidy showed additional prognostic value.     

Significance of S-phase fraction and hormone receptor content in the management of young breast cancer patients.

Tumours from 336 breast cancer patients under the age of 50 were analysed for hormone receptor content and by DNA flow cytometry. Sixty-six percent of the tumours were positive for estrogen receptors (ER), 60% were progesterone receptor (PR) positive and 42% showed DNA diploid profiles. DNA hypodiploid tumours were relatively frequent (7%), especially in patients aged 40 years or less (11%). S-phase fraction (SPF), with a mean of 10%, correlated significantly with receptor status, DNA ploidy, lymph node status, tumour size and age. With a median follow-up period of 34 months, the distant recurrence-free interval was independently predicted by lymph node status, tumour size, SPF and PR content. Amongst the 212 patients who had not received adjuvant systemic treatment, receptor status was, in addition to lymph node status and SPF, independently related to distant recurrence rate. A high SPF identified a subgroup with high recurrence rate, comprising approximately one third of the node-negative patients. Similarly, the one third of node-positive patients who had PR-positive tumours with a low S-phase fraction formed a subgroup with low recurrence rate. We conclude that hormone receptor assays and DNA flow cytometry should be useful tools in the management of breast cancer patients less than 50 years of age.     

Plasminogen activator inhibitor-1 (PAI-1) is not related to response to neoadjuvant chemotherapy in breast cancer.

There is no information available on the relation between response to chemotherapy and the high-risk phenotype assessed by uPA and/or PAI-1. The clinical situation of neoadjuvant chemotherapy provides a means of rapidly assessing the sensitivity of the primary tumour to cytotoxic drug regimens. The goal of the study was to assess prospectively the predictive value of PAI-1 for response to first-line chemotherapy. PAI-1 concentration was measured on hypertonic cytosolic extracts (0.4 M potassium chloride) by ELISA before chemotherapy on a drill biopsy sample of the tumour in 69 T2 and T3 breast cancer patients (median age 46 years). Oestrogen receptor (ER) (51% ER+), progesterone receptor (PR) (58% PR+), S-phase (median 4.0%) and ploidy were also assessed in the majority of cases. The clinical response to treatment was evaluated after four cycles of FAC or FEC regimen (5-fluorouracil, epidoxorubicin or doxorubicin and cyclophosphamide) (one cycle every 4th week). PAI-1 could be assayed in 29 post-chemotherapy surgical samples. The objective response rate (complete response plus partial response) was 59% (41 out of 69). PAI-1 expressed as gram of tissue (range 19-2370 ng g(-1) tissue) was highly correlated (r = 0.98) to PAI-1 expressed as mg protein (range 0.5-68 ng mg(-1) protein). No correlation between PAI-1 level and response could be observed, with any cut-off. The post- and pre-chemotherapy PAI-1 levels were correlated (r = 0.66). Of all biological parameters, only high S-phase (cut-off 5%) was slightly correlated (chi2 = 3.91, P = 0.05) to response. These data suggest that PAI-1 is not a predictive marker of response to chemotherapy in breast cancer and that its level is not altered by neoadjuvant chemotherapy.     

Flow cytometry in comparison with mitotic index in predicting disease outcome in transitional-cell bladder cancer

The DNA content and S-phase fraction were measured by flow cytometry in 448 tumour biopsy specimens from transitional-cell bladder cancer (TCC). The samples were also analyzed for mitotic index, WHO grade and papillary status, and histological and flow cytometric data were then correlated to clinical behaviour of turnours during a mean follow-up period of 9.9 years. TNM classification, WHO grade, papillary status, mitotic index, DNA ploidy and S phase fraction were significantly interrelated. Twenty-four percent of tumours showed heterogeneous DNA indices when measured from multiple samples (measured in 94 cases). Of the histological parameters, independent predictors of progression in superficial tumours were the S-phase fraction and mitotic index. In superficial tumours, S-phase fraction and the mitotic index included all the available independent prognostic information in survival analysis, whereas in muscle-invasive tumours T category was the most important prognostic factor. The results suggest that DNA ploidy has no independent prognostic value in transitional-cell bladder cancer, whereas proliferation indices (SPF, mitotic index) are important prognostic factors. Accordingly, malignancy classification of papillary bladder tumours can be based on proliferation indices alone. Nodular tumours run an unfavourable course and their malignancy grading by flow cytometry or by mitotic index is not relevant.     

Proliferation kinetics and prognosis in gastric cancer after resection

The influence of proliferation and proliferation kinetics on prognosis in gastric cancer after complete resection are controversial. In a prospective study we investigated the tumour specimens of 111 patients after resection of gastric cancer, who received 200 mg intravenous (i.v.) bromodeoxyuridine (BrdU) pre-operatively. The following biological parameters were analysed in the tumour tissue using flow-cytometry: DNA ploidy, proportion of S-phase cells, BrdU labelling index (LI), DNA synthesis time (T(s)), potential tumour doubling time (T(pot)), proliferating cell nuclear antigen (PCNA) and Ki-67 LI. The median follow-up time was 40 months (range 19-62 months). Besides the established pathohistological prognostic factors, univariate analysis revealed a prognostic influence on survival for BrdU LI, T(pot) and the proportion of S-phase cells. By multivariate Cox analysis of the completely resected cases, only tumour stage and T(pot) had a significant, independent influence on survival. By classification and regression trees (CART) analysis, resection status, tumour stage and T(pot) defined risk groups with significantly different outcomes. A short T(pot) was a predictor of better survival in stage I, II and IIIA tumours. Ploidy and the other investigated proliferation-related parameters failed to demonstrate any influence on prognosis after resection of gastric cancer.     

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 growthrate 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. © 1995 Wiley-Liss, Inc.