Ki67 LABELLING INDEX: AN INDEPENDENT PREDICTOR OF PROGRESSION IN PROSTATE CANCER TREATED BY RADICAL PROSTATECTOMY

The clinical course of prostate cancer is highly variable and cannot satisfactorily be predicted by histological criteria alone. Both tumour cell proliferation and neuroendocrine differentiation have been suggested as additional prognostic parameters, neuroendocrine differentiation being considered to enhance tumour cell proliferation. This study investigated the prognostic value of tumour cell proliferation [Ki67 labelling index (LI), MIB 1] and neuroendocrine differentiation and their relationship to each other. One hundred and thirty-seven paraffin-embedded radical prostatectomy specimens were examined. Neuroendocrine differentiation was found in 58 per cent of cases, but was not associated with pTN stage, Gleason score, Ki67 LI, or tumour progression. Ki67 LI was not significantly associated with pTN stage or with Gleason score. High grade ( P =0·0005), advanced local stage ( P =0·0004), positive lymph nodes ( P =0·02), and high Ki67 LI ( P =0·0203) were predictors of tumour progression if univariate analysis was performed, but Cox stepwise regression showed that only advanced local stage ( P =0·0025) and Ki67 LI ( P =0·0105) were independent predictors of tumour progression, the relative risk being 3·6 and 2·5, respectively. It is concluded that Ki67 is an important prognostic marker in prostate cancer with a potential for routine application.     

PROGNOSTIC VALUE OF ANGIOGENESIS IN OPERABLE NON-SMALL CELL LUNG CANCER

Tumour angiogenesis is an important factor for tumour growth and metastasis. Although some recent reports suggest that microvessel counts in non-small cell lung cancer are related to a poor disease outcome, the results were not conclusive and were not compared with other molecular prognostic markers. In the present study, the vascular grade was assessed in 107 (T1,2–N0,1) operable non-small cell lung carcinomas, using the JC70 monoclonal antibody to CD31. Three vascular grades were defined with appraisal by eye and by Chalkley counting: high (Chalkley score 7–12), medium (5–6), and low (2–4). There was a significant correlation between eye appraisal and Chalkley counting ( P <0·0001). Vascular grade was not related to histology, grade, proliferation index (Ki67), or EGFR or p53 expression. Tumours from younger patients had a higher grade of angiogenesis ( P =0·05). Apart from the vascular grade, none of the other factors examined was statistically related to lymph node metastasis ( P <0·0001). A univariate analysis of survival showed that vascular grade was the most significant prognostic factor ( P =0·0004), followed by N-stage ( P =0·001). In a multivariate analysis, N-stage and vascular grade were not found to be independent prognostic factors, since they were strongly related to each other. Excluding N-stage, vascular grade was the only independent prognostic factor ( P =0·007). Kaplan–Meier survival curves showed a statistically significant worse prognosis for patients with high vascular grade, but no difference was observed between low and medium vascular grade. These data suggest that angiogenesis in operable non-small cell lung cancer is a major prognostic factor for survival and, among the parameters tested, is the only factor related to cancer cell migration to lymph nodes. The integration of vascular grading in clinical trials on adjuvant chemotherapy and/or radiotherapy could substantially contribute in defining groups of operable patients who might benefit from cytotoxic treatment.     

Microvessel count predicts metastatic disease and survival in non-small cell lung cancer

The growth of newly formed vessels, or neoangiogenesis, represents an important step in both physiological and pathological situations: in particular, tumour growth and metastasis require angiogenesis. Microvessel count (MC), which represents a measure of tumour angiogenesis, has been associated with metastatic spread in cutaneous, mammary, prostatic, head and neck, and early-stage lung cancer. In this study, the role of tumour angiogenesis as a prognostic indicator was examined in 253 primary non-small cell lung cancer (NSCLC) patients. Microvessels were counted by highlighting endothelial cells with anti-Factor VIII monoclonal antibody (MAb) in methacarn-fixed tumour samples. In univariat analysis, MC (P<0·000001), sex (P=0·0036), histotype (P<0·014), tumour status (P<0·007), and vessel invasion (P<0·019) were significantly related to hilar and/or mediastinal nodal involvement. However, in the stepwise logistic regression analysis, MC (P<0·000003) retained the most important influence on nodal metastasis. The overall survival analysis calculated by the Kaplan-Meier method revealed that tumours with high MC (>25 vessels/field) were significantly associated with increased death risk (log-rank test P=0·00067; Cox's test P=0·00046; Gehan's Wilcoxon test P=0·00108). In 94 patients, the development of metastatic disease during follow-up was significantly related to MC. Indeed, patients who developed metastasis during follow-up showed a higher MC, either as a dichotomous (P=0·01) or as a continuous (P=0·003) variable, than patients who had developed no metastasis at the time of the analysis. Moreover, in the stepwise logistic regression analysis, MC retained the most important influence on distant metastases. Microvessel count, as a method for the quantitation of tumour angiogenesis, has an important prognostic role in non-small cell lung carcinomas.     

Oral cancer-associated tertiary lymphoid structures: gene expression profile and prognostic value

Tertiary lymphoid structure (TLS) provides a local and critical microenvironment for both cellular and humoral immunity and supports effective antigen presentation and lymphocyte activation. However, the gene expression profile and prognostic significance of TLS in oral cancer remain largely unrevealed. In this study, we found the presence of both intratumoral and peritumoral TLSs in a series of 65 patients with oral cancer treated by surgical resection, with positive detection rates of 33.8 and 75.4%, respectively. The presence of intratumoral TLSs, but not peritumoral TLSs, was significantly associated with decreased P53 and Ki67 scores (P = 0·027 and 0·047, respectively). The survival analyses revealed that oral cancer patients with higher grades of TLSs was associated with improved disease-free survival (DFS) and overall survival (OS) (P = 0·037 and 0·031, respectively). Gene expression profiling analysis of the cytokines and chemokines responsible for lymph-node neogenesis identified a three-up-regulated-gene set, i.e. IL7, LTB and CXCL13, which was shown to be correlated with human oral cancer-associated TLSs. This study provides a framework for better understanding of oral cancer-associated TLSs and for delineating future innovative prognostic biomarkers and immune therapeutic strategies for oral cancer.     

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.     

Quantitation and prognostic value of breast cancer angiogenesis: Comparison of microvessel density,Chalkley count,and computer image analysis

In some studies of breast cancer, quantitation of immunohistochemically highlighted microvessel ‘hot spots’ has been shown to be a powerful prognostic tool. However, the antibody used, the number and size of the ‘hot spots’ assessed, and the stratification of patients into high and low vascular groups vary between studies. Furthermore, little is known about the relationship between microvessel density and other vascular parameters. These uncertainties and the laborious nature of the technique make it unsuitable for diagnostic practice. Both manual and computerized image analysis techniques were used in this study to examine the relationship between microvessel density and the vascular parameters in different sized microscopic fields in a pilot series of 30 invasive breast carcinomas. Automated pixel analysis of immunohistochemical staining, Chalkley point counting, and observer subjective vascular grading were also assessed as more rapid methods of measuring tumour vascularity. A Chalkley count was also performed on a further 211 invasive breast carcinomas. Significant correlations were observed between manual microvessel density and luminal perimeter (r=0·6, P=0·0004), luminal area (r=0·56, P=0·002), and microvessel number (r=0·57, P=0·0009) by computerized analysis. There were also significant correlations between the microscopic hot spots of 0·155 mm² and 0.848 mm² for microvessel number (r=0·81, P>0·00005), luminal perimeter (r=0·78, P<0·00005), and luminal area (r=0·65, P=0·0001). In addition, a significant correlation was observed between microvessel density and both subjective vascular grade (P=0·002) and Chalkley count (P=0·0001). A significant reduction in overall survival was observed between patients stratified by Chalkley count in both a univariate (P=0·02) and a multivariate (P=0·05) analysis in the 211 invasive breast carcinomas. These findings show that Chalkley counting is a rapid method of quantifying tumour angiogenesis and gives independent prognostic information which might be useful in diagnostic practice.     

Rapid and prognostically valid quantification of immunohistochemical reactions by immunohistometry of the most positive tumour focus. A prospective follow-up study on breast cancer using antibodies against MIB-1, PCNA,ER, and PR

The prognostic significance of immunohistochemical markers for cell proliferation [MIB-1, proliferating cell nuclear antigen (PCNA)] and hormone receptor analysis [oestrogen receptor (ER), progesterone receptor (PR)] was tested by means of immunohistometry in a series of 103 breast cancer patients in comparison with the lymph node status N, the tumour size T, histomorphological grading and the biochemical ER and PR status. Immunohistochemical reactions were performed on 2 μm sections from paraffin-embedded tissue, using an indirect peroxidase method. The proportion of immunostained tumour cell nuclei was determined using a TV-image analysis system. Measurements were performed using a 20×objective on 40 viewing fields (1·94 mm², MIB-1 and PCNA) or 20 viewing fields (0·97 mm², ER and PR). The mean immunopositivity of all viewing fields and the value of the most immunopositive viewing field (MIB-1_max, PCNA_max, PR_max, ER_max) were calculated. The mean values and the maximal values were highly correlated (r=0·903, P<0·001). After 1:2:1 quantilization, 84·2 per cent of the 412 single measurements revealed mean and maximal values in the same category (P<0·0001). For each of the four immunohistochemical markers, the prognostic significance of the maximal values was higher than that of the mean values. The highest prognostic significance was found for MIC-1_max (P=0·0002), followed by PR_max (P=0·0046), ER_max (P=0·0154), and PCNA_max (P=0·0161). From the results of a Cox model, a ‘prognostic index (PI)’ was developed, ranging from −1 to 8: PI=2×N+T+MIB-1_max–PR_max. The four groups of patients with PI values of <2, 2–3, 4–5, and >5 revealed significantly different 7·5-year survival probabilities (P<0·0001). The simplicity of the PI makes it a clinically useful, routinely applicable, and understandable parameter in the surgical pathology of breast carcinoma. © 1998 John Wiley & Sons, Ltd.     

Tumour growth fraction and apoptosis in salivary gland acinic cell carcinomas. Prognostic implications of Ki-67 and bcl-2 expression and of in situ end labelling (TUNEL)

bcl-2 protein and Ki-67 (MIB-1) were studied in 32 acinic cell carcinomas (ACCs), all with a minimum of 5 years' clinical follow-up. Tumour apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and by morphological criteria. Five patients died of their disease. Patients with stage I tumours had significantly better survival compared with other stages (P<0·05). Patients with MIB-1-negative tumours had significantly better survival than patients with MIB-1-positive tumours (P=0·05). This study confirms a previous report that MIB-1 is an independent prognostic factor for survival in patients with ACC. Stage I tumours had high expression of bcl-2 protein, but there was no difference when compared with other stages. TUNEL positivity was most prevalent in stage I tumours, compared with stages II, III, and IV (P<0·05), probably indicating more apoptosis. This could imply a capacity of stage I tumours (‘early tumours’) for early selection of tumour cells for elimination by apoptosis. There was no significant difference between expression of bcl-2 and TUNEL, between these parameters and clinical outcome, or between any parameter and morphological subclassification. We conclude that MIB-1 has prognostic value in ACC. Clinical staging, bcl-2, and TUNEL are also potentially useful as prognostic markers. © 1997 John Wiley & Sons, Ltd.     

Standardized AgNOR analysis of the invasive tumour front in oral squamous cell carcinomas

In the last decade, silver staining of nucleolar organizer region-associated proteins (AgNORs) has been widely used in tumour pathology both for diagnostic and for prognostic purposes. However, a reliable and reproducible assessment of these proteins on routinely processed archival tissues has only become possible since the recent introduction of standardized staining method and computer-aided morphometric analysis. In the present study, the AgNOR content at the invasive front of 80 squamous cell carcinomas of the floor of the mouth/tongue was investigated using this novel approach, with regard to prognosis and a variety of clinico-pathological parameters. All standardized AgNOR parameters [mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV) of both AgNOR number and area] were statistically significantly associated with the clinical course. The strongest correlation was found for the AgNOR-area univariate analysis (P=0·006). In multivariate analysis, the mean of AgNOR number could independently predict both overall (P=0·01) and disease-free survival (P=0·001). It is concluded that standardized staining and computer-aided analysis of AgNORs are prerequisites for an objective and reproducible AgNOR assessment, which has potential as a supplementary diagnostic and prognostic tool in oral cancer. © 1997 John Wiley & Sons, Ltd.     

Prognostication of astrocytoma patient survival by Ki-67 (MIB-1), PCNA,and S-phase fraction using archival paraffin-embedded samples

The prognostic power of three proliferation estimation methods, Ki-67 (MIB-1) and PCNA immunohistochemistry, and flow cytometry (S-phase and S+G2/M fractions, respectively), were evaluated in 50 cases of astrocytoma. Each proliferation index showed a strong association with the grade of malignancy (grades I-IV). The MIB-1 labelling index (LI) provided additional information, as it could be used for the discrimination of grade II and grade III astrocytomas (P=0·0357). All three proliferation estimation methods also had strong prognostic potential (MIB-1 LI: P<0·0001; PCNA Li: P<0·0001; S-phase: P=0·0004; S+G2/M: P=0·0124). According to the receiver operating characteristics (ROC) curve, the MIB-1 LI showed generally the best sensitivity and specificity in placing the patients correctly into groups of survivors and non-survivors, which was further confirmed in the multivariate analysis. Only 4 per cent of the patients having high MIB-1 scores (>15·3 per cent) were alive after 2-years' follow-up. In contrast, 72 per cent of patients with tumours of low proliferation activity survived. It appears that Ki-67 (MIB-1) immunolabelling using archival paraffin-embedded samples is of value in predicting prognosis in astrocytic tumours.     

High-affinity monomeric 67-kd laminin receptors and prognosis in pancreatic endocrine tumours

Cell-surface high-affinity monomeric 67-kD laminin receptors have been proposed to promote the invasion and metastasis of a variety of tumours, but there are, as yet, no data regarding the expression of these molecules in pancreatic endocrine tumours (PETs). The prognosis of these very rare tumours is problematic and the only irrefutable evidence of their malignancy still continues to be the occurrence of local invasion and metastases. In this retrospective investigation, 34 functioning and 48 non-functioning sporadic PETs were evaluated for the expression of the MLuC5 monoclonal antibody, which specifically recognizes the 67-kD laminin receptors. Laminin receptors were found in 42/82 cases (51 per cent) and their expression was associated with metastatic disease (P<0·001), high proliferative activity expressed by a Ki-67 index above 5·0 per cent (P<0·001), absence of progesterone receptors (P=0·013), immunoreactivity for hormones other than insulin (P<0·001), a tumour diameter more than 3·0 cm (P=0·001), and a fatal clinical outcome (P<0·001). Laminin receptors were also expressed by most metastatic foci and all intravascular emboli of tumour cells. Positivity for laminin receptors was associated with shorter survival in functioning (P=0·026) and non-functioning (P=0·042) tumours, as well as in the whole series of pancreatic endocrine tumours (P<0·001). On multivariate analysis, laminin receptor expression was not an independent prognostic factor, while a Ki-67 index above 5·0 per cent was the most powerful predictor of survival. However, the association of laminin receptor expression and Ki-67 index could identify a group of malignant PETs with low proliferative activity characterized by an intermediate prognosis. In conclusion, these data suggest that monomeric laminin receptors may play a role in the invasion and metastasis of PETs and that their expression may be an additional prognostic factor, along with proliferative activity. © 1997 by John Wiley & Sons, Ltd.     

Apoptosis in colorectal carcinoma occurring in patients aged 45 years and under: relationship to prognosis,mitosis, and immunohistochemical demonstration of p53, c-myc and bcl-2 protein products

The aim of this study was to ascertain whether apoptotic counts have prognostic significance in colorectal cancer and if such counts are related to the expression of proteins implicated in cell cycle regulation. Material from a cohort of patients aged 45 years or less with colorectal carcinoma was re-examined to determine apoptotic and mitotic counts by light microscopy, in addition to assessing p53, c-myc, and bcl-2 protein status by immunohistochemistry. The apoptotic index in the 74 patients who were alive or who had died of colorectal carcinoma ranged from 1·2 per cent to 12·3 per cent and exhibited independent prognostic significance, with high counts predicting better survival (P=0·02). Mitotic counts were not related to survival, despite a close correlation with apoptosis (r=0·85). Tumours regarded as not staining with the CM1 antibody for p53 protein demonstrated higher apoptotic counts, compared with those that stained (medians 5·2 and 4·0 per cent, respectively; P=0·03), but p53 expression was found not to be related to survival. The 68 tumours which stained for c-myc appeared to exhibit higher mitotic counts than those that did not. bcl-2 was detected in only four tumours. The latter two proteins exhibited no apparent relationship to the apoptotic index or survival. Although these results indicate a potential role for apoptotic counting in prognostic prediction in colorectal tumours, this is an uncommon group of patients who exhibited some atypical features. The likelihood of a proportion of cases arising within hereditary non-polyposis colorectal cancer syndrome may limit the application of the findings to a more general population with cancer of the colon and rectum. Further work is required, including critical measurement of reproducibility and assessment of the relative impact of this parameter compared with ‘traditional’ prognostic markers. © 1997 John Wiley & Sons, Ltd.     

Expression of p53 protein has no independent prognostic value in breast cancer

A series of 392 female breast carcinomas was analysed immunohistochemically for expression of p53 protein with special emphasis on the role of p53 as an independent prognostic factor. Altogether, 54·8 per cent of the carcinomas expressed p53 protein, with the mean [standard error (SE)] fraction of positive nuclei being 17·1 per cent (1·2 per cent). Expression of p53 protein was independent of tumour metastasis at diagnosis, axillary lymph node status, tumour diameter, histological type, tubule formation, proportion of intraductal growth, margin formation, necrosis, DNA ploidy, and S-phase fraction. A high fraction of p53-positive nuclei was significantly related to patient age under 70 years, high grade, severe nuclear pleomorphism, dense infiltration of tumour by lymphocytes, high mitotic index, and high apoptotic index (for all, P<0·05). Impaired survival probability in the entire cohort (P=0·05) and in the axillary lymph node-positive (ANP) tumours (P=0·015) was associated with a fraction of p53-positive nuclei less than 25 per cent, while in the axillary lymph node-negative (ANN) tumours, expression of p53 had no prognostic value. In multivariate analysis, independent prognostic predictors included axillary lymph node status, tumour diameter, and mitotic index. In the ANN tumours, tumour diameter, fraction of p53-positive nuclei, and tumour grade were independent prognostic factors, whereas in the ANP tumours, diameter and mitotic index were the two independent prognostic factors. The results suggest that abnormal expression of p53 protein is only a weak independent prognostic factor in female breast cancer.     

Angiogenic cytokines in mesothelioma: a study of VEGF,FGF-1 and -2, and TGF β expression

Vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factors (FGF-1 and -2), and transforming growth factor β (TGFβ) are potent angiogenic cytokines. Malignant mesothelioma of the pleura presents with a high intra-tumoural microvascular density (IMD) which also has prognostic relevance. This study was designed to verify the immunohistochemical expression of the angiogenic cytokines in mesothelioma as well as in non-neoplastic human mesothelial cells and to study the individual as well as the combined expression of these cytokines in mesothelioma in relation to both IMD and prognosis. In addition, four mesothelioma cell lines were studied by ELISA for the secretion of VEGF and FGF-2 in their supernatants and were shown to contain high levels of both of these cytokines. Immunohistochemically, VEGF, FGF-1 and -2, and TGFβ immunoreactivity was present in 81, 67, 92 and 96 per cent of mesotheliomas, and in 20, 50, 40, and 10 per cent of samples of the non-neoplastic mesothelium, respectively. Co-ordinate expression of the cytokines was observed whereby mesotheliomas expressed more than one cytokine. The combined immunohistochemical expression levels for all four cytokines correlated significantly with both IMD ( p=0·01) and prognosis ( p=0·0013). When studied individually, high FGF-2 expression correlated best with more tumour aggressiveness and worse prognosis for mesothelioma ( p=0·0011). There was no significant correlation between prognosis and immunoexpression of VEGF ( p=0·07), FGF-1 ( p=0·3), or TGFβ ( p=0·1), or between IMD and any of the cytokines studied individually. These data support the assertion that selective angiogenic cytokines might contribute to the progressive changes of mesothelioma by tumour angiogenesis. Copyright © 1999 John Wiley & Sons, Ltd.     

EXPRESSION OF CATHEPSIN D IN TRANSITIONAL CELL BLADDER TUMOURS

Biopsy specimens from 177 bladder tumours were analysed by immunohistochemical methods for the expression of cathepsin D. Strong expression of cathepsin D was detected in transitional carcinoma cells in 40 per cent of cases. Umbrella cells were positive in 29 per cent of cases and a cathepsin D-positive cell zone composed of tissue macrophages was detected at the invasion front in 34 per cent of tumours. Strong expression of cathepsin D was related to muscle invasive growth phase (T⩾2) ( P =0·019), grade 2–3 histology ( P =0·008), S-phase fraction over 10 per cent ( P =0·032), and overexpression of epidermal growth factor receptor (EGFR) ( P <0·001). Umbrella cells were positive in low-grade ( P =0·03) papillary tumours ( P =0·02) with an S-phase fraction ⩽10 per cent ( P =0·02). Cathepsin D was expressed in macrophage-like cells in the invasion front in tumours which were densely infiltrated by inflammatory cells ( P =0·017) and in tumours overexpressing EGFR ( P =0·017) or p53 protein ( P =0·007). Progression in N- ( P =0·04) and M-categories ( P =0·01) was related to strong expression of cathepsin D in cancer cells and in univariate survival analysis; this was weakly related to poor outcome ( P =0·09). In multivariate analysis, papillary status ( P =0·055) and S-phase fraction ( P =0·079) predicted prognosis in Ta-1 tumours. In T2–4 tumours, T-category ( P <0·001), papillary status ( P <0·001), S-phase fraction ( P =0·028), and the presence of cathepsin D-positive tissue macrophages ( P =0·017) were independent prognostic factors.     

Primary cutaneous T-cell lymphoma: Clinicopathological features and prognostic parameters of 35 cases other than mycosis fungoides and cd30-positive large cell lymphoma

Within the group of primary cutaneous T-cell lymphomas (CTCLs), mycosis fungoides (MF), Sézary's syndrome (SS), and CD30-positive lymphomas have been delineated as clinicopathological entities. Primary CTCLs that do not belong to one of these entities represent a heterogeneous and ill-defined group of neoplasms. This paper describes the clinical and histological features of 35 of such cases. The object of t his study was to define prognostic parameters for this group of primary CTCLs. Using a slightly modified version of the updated Kiel classification, a subdivision was made into CTCL, pleomorphic, small cell type (n = 3); plemorphic, medium-sized cell type (n = 6); pleomorphic, large cell type (n = 18); and immunoblastic lymphomas (n = 8). Altogether, these lymphomas had a poor prognosis with estimated 2- and 4-year survival rates of 53 and 22 per cent, respectively. Patients with pleomorphic, small and medium-sized cell lymphomas (n = 9) proved to have a significantly better survival that those with pleomkorphic, large cell lymphomas (P = 0·032) and immunoblastic lymphomas (P = 0·008). Primary cutaneous immunoblastic lymphomas had the worst prognosis with an estimated 2-year survival rate of 14 per cent. Other parameters including age (P = 0·345), sex (P = 0·345), sex (P = 0·662), extent of skin lesions at presentation (P = 0·0854), and mode of initial treatment (P = 0·609) had no significant effect on the survival time. The results of this study suggest that primary CTCLs other than classical MF, SS, and CD30-positive lymphomas have a poor prognosis in most cases, and that the current classification may be a useful means of predicting the clinical behaviour in these lymphomas.     

The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma

Micrometastases have been detected by immunocytochemical means in the lymph nodes of patients with otherwise node-negative cancer of the colon and rectum. This study examines the incidence and prognostic significance of nodal micrometastases in Dukes' B carcinoma. Five hundred and fifty-nine lymph nodes from 77 cases of Dukes' B carcinoma were examined for lymph node micrometastases by immunocytochemical staining for cytokeratin AE1:AE3. Micrometastases were detected in 19 cases (25 per cent). Cell clusters were present in ten cases, the remaining nine cases displaying only single cells. The presence of micrometastases was unrelated to age (P = 0·06), sex (P = 0·32), tumour site (P = 0·37), tumour size (P = 0·67), or tumour differentiation (P = 0·66). Ten-year survival estimates by the Kaplan–Meier lifetable method was 47 per cent in patients with and without micrometastases (χ² = 0·35 and 1 df, P = ns). The presence of nodal micrometastases detectable only by immunocytochemistry in patients with Dukes' B colorectal cancer does not justify reassignment to a more advanced disease stage.     

Expression and prognostic value of the CD44 splicing variants v5 and v6 in gastric cancer

In the present study, the expression and prognostic role of the CD44 splicing variants v5 and v6 were immunohistochemically investigated in 418 curatively resected gastric carcinomas. CD44v5 was expressed in 65·3 per cent (n=273) and CD44v6 in 77·0 per cent (n=322) of the tumours. Whereas the expression of CD44v5 was correlated with advanced pT categories, with lymph node involvement, and with the presence of blood and lymphatic vessel invasion, such a correlation could not be found for the variant v6. As shown by univariate analysis, patients with CD44v5-positive tumours had a significantly shorter overall survival than patients with CD44v5-negative tumours (P=0·049). In contrast, expression of CD44v6 had no impact on prognosis (P=0·574). In a multivariate analysis including the prognostic parameters pT category and pN category, as well as blood and lymphatic vessel invasion, the prognostic impact of CD44v5 expression could not, however, be maintained. Although in the present study the expression of CD44v5 was correlated with a more aggressive tumour type, these data suggest that neither CD44v5 nor CD44v6 can predict survival in patients with gastric cancer, nor is their expression a suitable tool for identifying subgroups of patients who may be at higher risk. © 1997 John Wiley & Sons, Ltd.     

Apoptotic death of pancreatic cancer cells induced by polyunsaturated fatty acids varies with double bond number and involves an oxidative mechanism

Polyunsaturated fatty acids (PUFAs), reported to be cytotoxic at micromolar concentrations for cancer cells in vitroand in vivo, are currently being tested in clinical trials as anti-cancer agents. This study has shown that seven PUFAs all inhibited the growth in vitroof three pancreatic cancer cell lines and the HL-60 leukaemic cell line. Five PUFAs induced cell death within 20–30 h, but two less potent PUFAs induced death between 50 and 75 h. Apoptosis was demonstrated to be the mode of cell death by light, UV fluorescence, and electron microscopy, together with studies of DNA fragmentation. In a time–course study of PUFA-treated Mia-Pa-Ca-2 cells, apoptosis accounted for an average of 80 per cent of the loss of viability, with ‘secondary necrosis’, a feature of late apoptosis, apparently accounting for the remainder. Correlations were found between the number of fatty acid double bonds and the proportion of cells undergoing apoptosis induced in both Mia-Pa-Ca-2 cells (R=0·88, P=0·0001) and HL-60 cells (R=0·85, P=0·0001) and inversely with the micromolar concentrations of PUFAs required for 50 per cent inhibition of growth (IC₅₀) of Mia-Pa-Ca-2 cells (R=−0·73, P=0·05). Cell death was preceded by progressively increasing lipid peroxidation. The extent of PUFA-induced lipid peroxidation, measured as malondialdehyde (MDA), also correlated with the proportion of apoptosis induced in Mia-Pa-Ca-2 cells (R=0·69, P=0·025) or HL-60 cells (R=0·64, P=0·043), as well as with the number of fatty acid double bonds (R=0·82, P=0·0015). PUFA-induced apoptosis was oxidative, being blocked by both vitamin E acetate and sodium selenite, the latter in a critically time-dependent manner. The cytotoxic effects of exposure to a PUFA and to γ-irradiation simultaneously with, or prior to, the addition of PUFA. © 1998 John Wiley & Sons, Ltd.