RESUMEN
The aim of this study using radio-binding (RB), peroxidase-anti-peroxidase (PAP) and immunoprecipitation (IP) techniques was to investigate the pattern of major histocompatibility complex (MHC) antigen expression in urological malignancies and to compare the results with those seen in established urological human tumour cell lines. The results showed that using PAP technique, the percent bladder cases showing complete loss or cases with greater than 90% of tumour cells negative with W6/32 (detects all class I antigens), HC10 (detects free heavy chain) and BMM.1 (detects beta2-mirogobulin) monoclonal antibodies (Mab) were 16%, 44% and 2% respectively. In a subgroup of 37 cases, the intensity of MHC class II antigen expression for strong, weak and negative cases were 9 (24%), 8 (22%) and 20 (54%) respectively. The expression for class I antigens on testis tumours was mainly negative and when positive, it was present in a small percent of tumour cells. This was also observed for class II antigens where only 8% of cases showed some degree of positivity. Using RB technique, 10 of 12 (85%) of tumour lines expressed class I antigens spontaneously and following interferon gamma (IFNgamma) stimulation, the 2 negative lines one testis (Tera I) and one bladder (Fen) remained negative and 2 lines (both testis lines Tera II and Ep2102) showed a significant class I up-regulation. None of the lines expressed class II antigens spontaneously and following IFNgamma stimulation, 8 of 12 (66%) were induced. The absence of class I and II antigens in the negative lines was confirmed using IP technique. In the case of one class I negative bladder cell line i.e. Fen, the biochemical analysis showed the absence of beta2-m gene product which could not be restored by IFNgamma stimulation. However, transfection of the cells with beta2-m gene resulted in the expression of fully assembled class I antigens, indicating that the loss of antigens was due to the absence of functional beta2-m gene. These results indicated the similarity between the pattern of expression of MHC antigens on tumour biopsies and established tumour cell lines. They also demonstrated that both cytokine stimulation and gene transfection could be used to correct defective class I antigens in tumour cell lines. These approaches might have important implications for pre-selection of bladder cancer patients for cytokine or gene therapies.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Antineoplásicos/farmacología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interferón gamma/farmacología , Neoplasias Testiculares/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Biopsia , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Técnicas para Inmunoenzimas , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Complejo Mayor de Histocompatibilidad/fisiología , Masculino , Pruebas de Precipitina , Ensayo de Unión Radioligante , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/terapia , Transfección , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/terapia , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
The possible role of the immune system in resisting human malignancies has long been debated. Several recent findings from animal and human studies have restimulated interest in the immune surveillance hypothesis for tumor control. These findings have been complied from various disciplines including cytokine therapy, adoptive immunotherapy, and gene therapy. Following the initial euphoria, it is now clear that immunotherapy of selected cancer cases in the early stages of tumor development may make an important contribution to tumor control, particularly in dealing with minimal residual disease after tumor debulking. This review discusses some of these issues and proposes approaches that could pave the way for better selection of the patients best suited for immunotherapy. We would argue that therapies directed at the re-expression of major histocompatibility complex (MHC) class I antigens might improve outcomes in immune-therapy-based treatments.
Asunto(s)
Inmunoterapia , Neoplasias Renales/terapia , Neoplasias de la Vejiga Urinaria/terapia , Línea Celular , Citocinas/fisiología , Receptores ErbB/análisis , Antígenos HLA/inmunología , Humanos , Interleucina-2/uso terapéutico , Neoplasias Renales/inmunología , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/inmunologíaRESUMEN
Tumour cells were separated from 19 renal carcinomas and cultured in vitro. The effect of interferon (IFN) alpha and gamma on cell proliferation was measured and compared to the effect of IFN on the expression of class I and class II major histocompatibility complex (MHC) antigens. When tested within the first 14 days of culture, IFN-alpha inhibited protein synthesis in 12 of 15 and IFN-gamma in four of nine tumours. Reduction in cell counts was in parallel. In six tumours the culture period was extended and in all six the effect of IFN-alpha was lost. Exposure to IFN-alpha induced or enhanced class I antigen expression in eight of 19 tumours and class II expression in two of 19. The analogous figures for IFN-gamma were five and three tumours. In four of five cases where a comparison could be made there was a correlation between the effects of IFN-alpha on cell proliferation and class I antigen expression. The efficacy of IFN in the treatment of renal carcinomas may thus, in part, result from inhibition of cell proliferation and enhancement of antigen expression.
Asunto(s)
Carcinoma de Células Renales/terapia , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interferón-alfa/farmacología , Interferón beta/farmacología , Neoplasias Renales/terapia , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Recuento de Células , División Celular/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Humanos , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Proteínas de Neoplasias/biosíntesis , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Biopsies from the tumour and the adjacent normal kidney were obtained from 15 patients with renal cell carcinoma (RCC). The proximal convoluted tubules from which the tumour arose expressed major histocompatibility complex (MHC) class I antigen (Ag) in 3 cases and class II in none. By contrast, the carcinoma cells expressed class I Ag in 14 cases and class II Ag in 5 cases. Cells from each carcinoma were established in culture. As the culture period increased, cells from six of eight RCC showed diminished expression of class I Ag and five of six reduced expression of class II Ag. This is similar to the relative loss of class I Ag in synchronous metastases from RCC.
Asunto(s)
Carcinoma de Células Renales/inmunología , Antígenos HLA/análisis , Neoplasias Renales/inmunología , Antígenos HLA-D/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Leucocitos Mononucleares/inmunología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Cells from 22 renal cell carcinomas (RCC) were established in culture. Sensitivity of the tumour cells to doxorubicin alone and in combination with racemic verapamil, which reverses multidrug resistance, was tested using a [75Se]selenomethionine uptake assay to measure protein synthesis. The effect of verapamil was expressed as a potentiation index: LD50doxorubicin/LD50doxorubicin + verapamil. The potentiation index in 15 of these carcinomas was determined for cells within the first 14 days of culture. At 3.3 mumol/l concentration of verapamil, of the tumours sensitive to doxorubicin alone (LD50 < 0.75 microgram/ml) five of seven showed a potentiation index of > 2. For the less sensitive tumours the analogous proportion was seven of eight. Tumour cell expression of glycoprotein P-170, associated with multidrug resistance, was estimated using the monoclonal antibody C-219. Initial expression levels were unrelated to the action of verapamil. In five tumours the proportion of cells expressing P-170 declined as the period of culture increased. This was not associated with any consistent change in the LD50 for doxorubicin or in potentiation of doxorubicin sensitivity by verapamil. Cell cloning associated with prolonged cell growth in vitro could mimic tumour cell cloning which accompanies the formation of metastases. Thus reduced expression of P-170 on prolonged cell growth in vitro may be a pointer to the efficacy of combination therapy in the treatment of patients with metastatic renal cell carcinoma.
Asunto(s)
Carcinoma de Células Renales/tratamiento farmacológico , Doxorrubicina/farmacología , Neoplasias Renales/tratamiento farmacológico , Verapamilo/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Proteínas Portadoras/biosíntesis , Resistencia a Medicamentos , Sinergismo Farmacológico , Humanos , Neoplasias Renales/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Ten renal carcinomas were established in culture and their sensitivity to mitozantrone +/- verapamil, as measured by (75Se) selenomethionine incorporation, was studied. The 5 tumours with spread beyond the kidney were those in which sensitivity to mitozantrone was enhanced by verapamil. With 4 of the 10 tumours and 1 further carcinoma, similar experiments were performed using doxorubicin. This drug was more active, its anti-tumour action being related to concentration. Verapamil was more effective in potentiation of doxorubicin and this action increased in proportion to the concentration of verapamil added.
Asunto(s)
Doxorrubicina/farmacología , Neoplasias Renales/tratamiento farmacológico , Mitoxantrona/farmacología , Verapamilo/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Neoplasias Renales/metabolismo , Proteínas de Neoplasias/biosíntesis , Selenometionina/metabolismo , Células Tumorales CultivadasRESUMEN
High-dose intravenous estrogen therapy was shown to be effective in relieving bone pain due to metastatic disease in 22 of 29 (75.9%) men with advanced hormone-resistant prostate cancer. This clinical response was accompanied by significant falls in serum prostate-specific antigen (PSA) levels in 13 (44.8%) patients. It is suggested that this clinical benefit is due to a direct inhibitory effect of estrogen on prostate cancer cells.
Asunto(s)
Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/sangre , Neoplasias Óseas/secundario , Dietilestilbestrol/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias Óseas/fisiopatología , Dietilestilbestrol/uso terapéutico , Humanos , Inyecciones Intravenosas , Masculino , Dolor/tratamiento farmacológico , Dolor/etiología , Antígeno Prostático Específico , Neoplasias de la Próstata/fisiopatologíaRESUMEN
Tumour cells from 23 renal carcinomas were exposed in vitro to increasing concentrations of medroxyprogesterone acetate (MPA) or one of several cytotoxic agents. Thereafter the uptake of 75Selenomethionine (75SeM) was compared to that of tumour cells not exposed to the drug. Using drug concentrations similar to those seen following in vivo administration in patients, MPA produced greater than or equal to 50% inhibition of 75SeM uptake in 5 of 23 cases. The comparable proportion for doxorubicin was 4 of 5 and for mitozantrone 7 of 12. Methotrexate and Vinblastine were much less effective. In all cases where greater than or equal to 50% inhibition was obtained with doxorubicin and mitozantrone the carcinoma had spread beyond the kidney. Thus drug sensitivity may be associated with a high mitotic rate.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Renales/metabolismo , Medroxiprogesterona/análogos & derivados , Células Tumorales Cultivadas/efectos de los fármacos , Anciano , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Femenino , Humanos , Masculino , Medroxiprogesterona/farmacología , Acetato de Medroxiprogesterona , Metotrexato/farmacología , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Selenometionina/metabolismo , Vinblastina/farmacologíaRESUMEN
Mouse mammary carcinoma cells were exposed in vitro to increasing concentrations of doxorubicin hydrochloride [adriamycin (ADR)] or 5-fluorouracil (5-FU). Uptake of [75Se]selenomethionine (75SeM) in a methionine-deficient medium measured the resulting inhibition of protein synthesis by the tumour cells. This was compared with the ability of the 75SeM labelled tumour cells to localize in mouse lungs and to form pulmonary tumours following intravenous (i.v.) injection into isogenic hosts. These parameters were also related to the ability of the drugs to inhibit pulmonary tumour formation in vivo when injected into mice which had received tumour cells i.v. Results from five different tumours were pooled for analysis. At the highest drug concentration (10 micrograms/ml ADR, 100 micrograms/ml 5-FU) inhibition of protein synthesis was significantly related to the in vivo action of the drugs in limiting formation of pulmonary tumors (P less than 0.02 using the rank difference coefficient). There was also a direct relationship between pulmonary localization of tumour cells following exposure to drugs, their ability to form tumour nodules (P less than 0.025) and the in vivo action of the drugs in inhibiting tumour formation (P less than 0.05). Thus inhibition of protein synthesis in vitro and pulmonary localization following i.v. injection may be of value in predicting the in vivo effect of cytotoxic drugs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Pulmonares/secundario , Neoplasias Experimentales/tratamiento farmacológico , Biosíntesis de Proteínas , Animales , Femenino , Ratones , Ratones Endogámicos CBA , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Selenometionina/farmacocinética , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Controversy still exists as to whether oestrogens exert a direct effect on the prostatic cell. Incorporation of 75Selenomethionine (SeM) was used as a measure of protein synthesis by prostatic carcinoma cells in vitro to investigate the action of hormones on prostatic carcinoma cells in tissue culture. Stilboestrol (DES) and stilboestrol diphosphate (Honvan) inhibited protein synthesis in a proportion of patients, while testosterone was stimulatory. A similar effect was noted in cells from patients with benign hyperplasia (BPH). This work confirms that oestrogens have a direct inhibitory effect on prostatic cells at high concentrations which can be attained in patients given intravenous stilboestrol diphosphate.
Asunto(s)
Dietilestilbestrol/farmacología , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Próstata/metabolismo , Testosterona/farmacología , Ciproterona/análogos & derivados , Ciproterona/farmacología , Acetato de Ciproterona , Dietilestilbestrol/análogos & derivados , Estradiol/metabolismo , Humanos , Masculino , Selenometionina/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Neoplastic tissue was obtained at operation from 10 renal cell carcinomas, from the adjacent 'normal' kidney in 6 cases and from 1 other normal kidney. The biopsies were snap frozen in liquid nitrogen and sections were subsequently stained with monoclonal antibodies against major histocompatibility complex (MHC) antigens, class I and II, and several types of mononuclear cell, by the indirect immunoperoxidase method. The degree of staining or the number of cells stained was estimated as heavy 4, through moderate 3, few 2, occasional 1, or nil 0. MHC Ag were consistently expressed, grade 2-4, by the glomeruli and proximal convoluted tubules of normal kidney, but were absent in 8 of 10 carcinomas. There was a grade 3-4 mononuclear cell infiltration in the stroma of normal kidney and between the carcinoma cells which was composed principally of macrophages. However in the two carcinomas expressing MHC Ag there was also a grade 2-3 infiltration with T lymphocytes. The absence of MHC Ag on carcinoma cells mitigates against attempts to potentiate the patient's immune response to his tumour, e.g. by renal artery embolisation.
Asunto(s)
Carcinoma de Células Renales/inmunología , Antígenos HLA/inmunología , Neoplasias Renales/inmunología , Leucocitos Mononucleares/inmunología , Anciano , Carcinoma de Células Renales/patología , Femenino , Antígenos HLA-D/inmunología , Humanos , Neoplasias Renales/patología , Macrófagos/patología , Masculino , Persona de Mediana Edad , Linfocitos T/patologíaRESUMEN
Tissue from four mouse mammary carcinomas was enzymatically disaggregated and cells from the resulting cell suspension were fractionated on a discontinuous density gradient column (5-20%) of Nycodenz (Nycomed A.S. Oslo). The cell fractions separating at the 10-15% and 15-20% interfaces (density 1.082 and 1.110 g/ml respectively) contained a mean of 83.2 +/- 10.8 (s.d.) and 79.9 +/- 17.4 tumour cells. Compared with the original cell suspension these cell bands contained less cell aggregates and cell debris. The cells in the bands also showed an equivalent ability to grow in tissue culture and to form pulmonary tumours on i.v. injection into isogenic mice, when compared with the tumour cells in the original suspension. The relatively pure preparations of carcinoma cells thus separated may be of value in limiting the unwanted effect of normal cell contamination when testing neoplastic cells in vitro for sensitivity to drugs or hormones.
Asunto(s)
Neoplasias Mamarias Experimentales/patología , Animales , Recuento de Células , Separación Celular , Centrifugación por Gradiente de Densidad , Femenino , Neoplasias Pulmonares/etiología , Ratones , Ratones Endogámicos , Trasplante de NeoplasiasRESUMEN
Exfoliated colorectal carcinoma cells were obtained by lavage of 27 freshly resected tumor-bearing segments of human bowel with the use of either Hartmann's solution or medium 199. The tumor cells were isolated from the lavage fluid on Nycodenz (Nyegaard, Oslo, Norway) columns. Their proliferative and metastatic potentials were investigated by their ability to incorporate tritiated thymidine ([3H]dThd) and by their ability to form experimental pulmonary tumors following iv injection into immune-deprived stain A mice. Tumor cells from 7 of 12 patients incorporated [3H]dThd as detected by autoradiography of the cells. Pulmonary nodules of colorectal carcinoma were seen in a single mouse after iv injection of tumor cells from 6 of 17 patients and were histologically distinct from spontaneous lung cancers seen in 3 other animals. No tumors were seen in 12 immune-deprived mice receiving tissue culture medium 199 only. Thus exfoliated colorectal carcinoma cells can undergo further division and might give rise to implantation metastases in humans.
Asunto(s)
Neoplasias del Colon/patología , Siembra Neoplásica/patología , Células Neoplásicas Circulantes/patología , Neoplasias del Recto/patología , Animales , Autorradiografía , División Celular , Neoplasias del Colon/cirugía , Femenino , Humanos , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos , Ratones Desnudos , Neoplasias del Recto/cirugía , Timidina/metabolismoRESUMEN
Serial frozen sections were prepared from 22 colorectal carcinomas. Additional samples were obtained from the adjacent normal bowel in 10 patients, from 6 concomitant adenomas in 5 patients, and from another 4 isolated adenomas. Mononuclear cell infiltrates were stained by the indirect immunoperoxidase technique with the use of a panel of 6 mouse monoclonal antibodies to human leukocyte antigens. The degree of infiltration was graded from 4 (heavy) to 0 (nil). The colorectal carcinomas and adjacent normal bowel showed an equal degree of leukocyte infiltration (HLe-1), graded 3-4 in 8 cases and 2-3 in the other 2 cases. In 7 carcinomas cytotoxic-suppressor T-lymphocytes (UCHT-4) graded 2-3 predominated over helper T-cells (OKT-4) graded 0-1. By contrast, in the adjacent normal bowel cytotoxic and helper cells were present in equal numbers. Among the adenomas leukocyte infiltration was grade 4 in 9 and grade 3 in 1. In 9 of the 10 adenomas cytotoxic cells graded 2 predominated over helper cells graded 0-1. The number of helper cells was equivalent among 6 concomitant adenomas and carcinomas from 5 patients. Adenomatous epithelial cells expressed class II major histocompatibility complex antigens (OKIa-1). However, carcinomatous or normal epithelium showed only faint staining with OKIa-1. The similarity in cell infiltration is consistent with an adenoma-carcinoma sequence. The predominance of cytotoxic cells in carcinomas that expressed class I major histocompatibility complex supports the association between lymphocyte infiltration and a favorable prognosis.
Asunto(s)
Adenoma/inmunología , Carcinoma/inmunología , Neoplasias del Colon/inmunología , Antígenos HLA/inmunología , Monocitos/metabolismo , Neoplasias del Recto/inmunología , Adenoma/patología , Anticuerpos Monoclonales , Carcinoma/patología , Neoplasias del Colon/patología , Citotoxicidad Inmunológica , Epitelio/patología , Antígenos HLA/clasificación , Humanos , Neoplasias del Recto/patología , Valores de Referencia , Linfocitos T Reguladores/patologíaRESUMEN
A total of 77 patients with invasive TCC of the urinary bladder each received a single arterial injection of tumor-immune pig lymph node cells (LNC), into the tumor blood supply. The patients were divided into 4 groups: those receiving pig LNC as the only treatment (16 patients); those who received pig LNC on relapse (judged by EUA cystoscopy and biopsy) following radical radiotherapy, 5,500 cGy (34 patients); those who received pig LNC followed after an interval of six weeks by radiotherapy, 4,000 cGy (10 patients); as in Group 3 but with the dose of radiotherapy increased to 5,500 cGy (17 patients). Complete remission was characterized by complete disappearance of the tumor (for a varying time) following treatment. Partial remission was defined as: a reduction in the level of symptoms and a decrease in tumor size on EUA and/or cystoscopy. There were 1 complete and 3 partial remissions among the patients in Group 1, 5 complete and 7 partial in Group 2, 6 complete and 2 partial in Group 3, and 8 complete and 1 partial in Group 4. Of the 20 patients showing complete remission, 7 lived for more than five years after treatment.
Asunto(s)
Carcinoma/terapia , Inmunización Pasiva , Ganglios Linfáticos/citología , Neoplasias de la Vejiga Urinaria/terapia , Animales , Carcinoma/inmunología , Carcinoma/radioterapia , Terapia Combinada , Humanos , Ganglios Linfáticos/inmunología , Invasividad Neoplásica , Porcinos , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/radioterapiaAsunto(s)
Adenoma/patología , Carcinoma/patología , Neoplasias del Colon/patología , Neoplasias del Recto/patología , Adenoma/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T , Antígenos de Neoplasias/análisis , Antígenos de Superficie/análisis , Carcinoma/inmunología , Neoplasias del Colon/inmunología , Humanos , Leucocitos/inmunología , Neoplasias del Recto/inmunologíaRESUMEN
A-strain mice received 1 X 10(5) A-strain mammary carcinoma, B72, cells iv on Day 0. At the same time fragments of the same tumor were implanted into the ileal mesentery of a pig together with fragments of cellulose sponge. The middle third of the pig mesenteric lymph node chain was resected. Sponge fragments of equivalent size, shape, and total weight were implanted alone into the jejunal mesentery. Seven days later, separate cell suspensions were made from the (disconnected) proximal (nonimmune) and distal (tumor-immune) segments of lymph node chain. Cells were also expressed by digital pressure from the sponges removed from the jejunal (nonimmune) and ileal (immune) segments of mesentery. A comparison was made between the antitumor action of immune lymph node cells and the two categories of sponge cell. Cells (4 X 10(6] were injected iv into the mice on Day 7 and the number of pulmonary tumors counted after killing the mice on Day 14. Cells obtained from the "immune" sponges were significantly more effective in reducing the number of pulmonary tumors than cells from the "nonimmune" sponge, or from the immune lymph nodes. The antitumor action of all cell suspension was abolished when 4 X 10(6) cells from the "interface" layer, following centrifugation on a Ficoll-triosil column, were injected into the mice. Antitumor activity was found to be correlated with the presence of "blast" cells in the pig mononuclear cell suspensions.
Asunto(s)
Neoplasias Pulmonares/terapia , Monocitos/fisiología , Animales , Separación Celular , Recuento de Leucocitos , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Neoplasias Mamarias Experimentales/terapia , Mesenterio/citología , Ratones , Ratones Endogámicos A , Neoplasias/inmunología , PorcinosRESUMEN
The viability of tumour cells shed into the intestinal lumen was determined in 49 patients with carcinoma of the large bowel. Preoperative colorectal lavage was performed in 19 patients and irrigation of the cut ends of the operative specimen in 30 patients. The resulting cell suspensions were centrifuged on a Nycodenz linear density gradient column so that tumour cells, being larger, were concentrated in a band at the top. In 14 of 19 colorectal lavage cases viable tumour cells were recovered, as assessed by their characteristic morphology and ability to exclude trypan blue. A median of 0.78 X 10(6) viable tumour cells was recovered. The median percentage cell viability in the suspension was 92, i.e. 8 per cent of the tumour cells were dead (stained with trypan blue). In eight specimens viability was confirmed by the ability of tumour cells to hydrolyse fluorescein diacetate. In 17 of 30 proximal resection margin irrigations a median of 0.55 X 10(5) viable tumour cells was recovered, with a median percentage viability of 92.5. In 15 specimens the neoplastic cells showed fluorescence. In 21 of 25 distal resection margin irrigations a median of 1.92 X 10(5) viable tumour cells was recovered with a median percentage cell viability of 79.3, and fluorescence was observed in all specimens. The number of viable tumour cells did not correlate with the stage, differentiation, diameter or fixity of the tumour. However, the number of tumour cells recovered from the distal resection margin was inversely related to the distance of the tumour from that margin (Rank Difference Coefficient R = -0.6). Thus viable exfoliated tumour cells were demonstrated in 52 of 74 specimens (70 per cent). Their presence in large numbers at the site of intestinal anastomoses supports a potential role in the aetiology of suture-line recurrence.