Human soluble p66 and p51 tumor-associated antigens promote the suppression of rat mammary tumors in comparison to commercial human albumin

This study examined whether the soluble 66- and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of breast cancer patients, possess specific suppressive effects on chemically-induced rat mammary tumorigenesis in comparison to commercial human albumin. Dimethylbenzanthracene (DMBA, 10 mg/rat, 2 administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of many large tumors, preparations of sTAA (50-60 micro g/rat in 0.5 ml sterile PBS) or commercial human albumin (HA, in the same doses as sTAA) were administered weekly, for 10-14 more weeks. The following groups of mammary tumor-bearing rats were studied: i) control non-treated rats, ii) rats treated with HA, iii) rats treated with sTAA. The experiment was terminated when tumors in 70% of the rats became ulcerous. The treatment with sTAA significantly decreased, compared to controls, the yield and total area of the tumors. In rats treated with sTAA, the appearance of new tumors stopped at week 5 as compared to week 7 in rats treated with HA and week 10 in control rats. In rats treated with sTAA, the time of appearance of ulcerous tumors increased to 8 weeks, as compared to 6 weeks in controls and in rats treated with HA. Duration of the experiment increased from 11 weeks in controls to 12 weeks in rats treated with HA and to 14 weeks in rats treated with sTAA. We conclude that sTAA have tumor-suppressive properties, which are well-defined if the treatment is begun on small tumors.     

Response of T- and B-lymphocytes in the spleen of mammary tumor-bearing rats to treatment with tamoxifen and soluble tumor-associated antigens

We analyzed the role of T- and B-lymphocytes in the antitumor effects of the anticancer drug tamoxifen and soluble tumor-associated antigens (sTAA) on rat mammary carcinogenesis. Studies were performed on the spleen from the following groups of mammary tumor-bearing rats. i) Rats in group 1 were not exposed to DMBA and served as age-related controls. Rats in other groups were exposed to DMBA and received different types of treatment; ii) rats in group 2, received no additional treatment, and served as carcinogen-related controls; iii) rats in group 3 were treated with the commercial hormone-dependent anticancer drug tamoxifen by weekly subcutaneous (s.c.) injections of 10 mg dissolved in 0.5 ml distilled water per rat; iv) rats in group 4 were vaccinated s.c. weekly with a preparation of sTAA (50 micro l/rat) dissolved in 0.5 ml of phosphate-buffered saline; v) rats in group 5 were treated with tamoxifen and were also vaccinated with a preparation of sTAA. Different zones of the spleen were measured and their T- and B-cell contents were analyzed immunohistochemically. The treatment with tamoxifen significantly increased the total number of lymphocytes in the follicles, PALS (periarterial lymph sheath) and red pulp relative to all other groups. The combined treatment with tamoxifen and sTAA increased the areas of white pulp, the PALS, and marginal zone. The number of B-cells was higher in the marginal zone of spleens from age-related controls, as well as from rats treated with sTAA and those treated with tamoxifen and sTAA. The number of CD4+ lymphocytes in the PALS was higher in rats treated with sTAA and tamoxifen, and notably so in those treated with sTAA alone. The number of CD8+ lymphocytes was significantly lower in the PALS of spleens from all tumor-bearing rat groups compared to the unexposed age-related control rats. We suggest that the tumor-suppressive effect of sTAA and tamoxifen is accompanied by the activation of B- and T-lymphocyte production.     

Rat soluble tumor-associated antigens inhibit chemically-induced mammary tumorigenesis in syngeneic rats

This study examined whether soluble 66 and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of rats with mammary cancer, possess specific suppressive effects on chemically-induced mammary tumorigenesis in syngeneic counterparts. Dimethylbenzanthracene (DMBA, 10 mg/rat, two administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of numerous tumors, preparations of sTAA (50 to 60 microg/rat in 0.5 ml sterile PBS) obtained from breast cancer patients (heterologous sTAA) or from syngeneic mammary tumor-bearing rats (syngeneic sTAA) were administered weekly for 12 weeks. The following groups of mammary tumor-bearing rats were studied: groups 1 and 3, control rats treated with saline; group 2, rats treated with heterologous sTAA; and group 4, rats treated with syngeneic sTAA. The experiment was terminated when tumors in 50% of the rats became ulcerous. The treatment with both types of sTAA significantly decreased, compared to controls and initial values, the yield and total area of the tumors. We conclude that syngeneic sTAA have tumor-suppressive properties, which are very similar to those in heterologous sTAA.     

Effects of tamoxifen and soluble tumor-associated antigens on ovarian structure in mammary tumor-bearing rats

Previously, we showed that the 66 and 51 kDa soluble tumor-associated antigens (sTAAs) have distinct suppressive effects on chemically induced mammary cancer in rats, both alone and in combination with the hormone-related anticancer drug tamoxifen. Here, we describe the effects of both sTAA and tamoxifen on the histological structure of ovaries in mammary tumor-bearing 30- to 34-week-old rats. Central ovary sections were pooled, the number of the healthy and degenerated follicles were counted, and the size of the corpora lutea was estimated. In follicular development primordial, primary, preantral and antral stages were recognized. Only healthy follicles with visible nuclei were counted. Follicular degeneration was estimated as the number of atretic follicles with follicular remnants. Treatment with tamoxifen alone or in combination with sTAA significantly increased the number of primordial follicles and atretic follicles in the ovaries, and promoted the formation of small follicular cysts. Total area of the corpora lutea decreased. sTAA participated in this process by increasing apoptosis in degenerated follicles.     

Occurrence of monocytoid B lymphocytes in lymph nodes of patients treated by chemotherapy

Occurrence of monocytoi B lymphocytes (MBL) in the lymph nodes of patients receiving preoperative chemotherapy for cancer was examined and compared to lymph nodes in controls who had not received chemotherapy. Number of patients receiving and not receiving preoperative chemotherapy were 3 and 10 cases in ovarian cancer, 7 and 11 in testicular cancer, and 22 and 8 in lung cancer, respectively. Chemotherapeutic agents for ovarian, testicular, and lung cancer consisted of cisplatin, Adriamycin, and cyclophosphamide; cisplatin, vinblastine, and bleomycin; and cisplatin, vindesin, and mitomycin, respectively. MBL were defined morphologically as having abundant pale cytoplasm with distinct cell borders and small nucleus. Immunohistochemistry revealed a B-cell nature of these cells, i.e., CD20⁻ and/or MB-1⁺ together with negative reactivity for antibodies for T lymphocytes (CD43, CD45RO, OPD4) and macrophages (KP-1, PGM-1). Monocytoid cells in two cases showed a positive reactivity for CD43 together with CD20. The occurrence rate of MBL in patients with ovarian, lung, and testicular cancer receiving and not receiving chemotherapy was 67% (2/3) and 10% (1/10), 59% (13/22) and 75% (6/8), and 43% (3/7) and 9% (1/11), respectively. The occurrence rate in the total patients receiving chemotherapy (56%) was significantly higher than for those not receiving chemotherapy (28%) (P < 0.05). These findings suggest that chemotherapy-induced depressed immune function is causative for the occurrence of MBL in the lymph nodes. MBL might be found more frequently in nodes from patients who have received chemotherapy in certain settings. © 1996 Wiley-Liss, Inc.     

Immunohistochemical evidence of immune responses to tumor-associated antigens in lymph nodes of colon carcinoma patients.

The authors investigated by immunohistochemical study the drainage of three tumor-associated antigens in unaffected regional lymph nodes of colon cancer patients. The study was conducted using monoclonal antibodies (MoAb) directed against different epitopes of the tumor-associated glycoprotein, TAG-72 (CC-49, CC-83, B72.3), of the carcinoembryonic antigen (CEA) (COL-4, COL-12), and of the colon-associated antigen, CAA (anti-CAA). The authors detected immunohistochemical reactions of MoAb CC-49 and anti-CAA with antigen-presenting cells (APC), such as peritumoral and sinus macrophages and lymphatic endothelial cells and with specific areas of germinal centers in lymph nodes draining 11 of 24 colorectal carcinomas studied. The corresponding primary tumors expressed the TAG-72 and CAA antigens. No immunostaining was detectable in lymph nodes using the anti-CEA MoAb, even when the primary tumors strongly expressed the specific epitopes. In germinal centers of regional lymph nodes, the immunostaining was often distributed at the periphery with a characteristic crescentic or circular pattern, which strongly suggested the exposure of the specific epitopes defined by MoAb CC-49 and anti-CAA on follicular dendritic cells. This would indicate that these epitopes are selectively recognized and presented to germinal center B-cells. This phenomenon may have clinical and diagnostic implications.     

Activated T lymphocytes in infiltrates and draining lymph nodes of nasopharyngeal carcinoma

Lymphocytes isolated from the tumors and draining lymph nodes of nasopharyngeal carcinoma (NPC) patients exhibit the following characteristics of immune activation: (1) stable E rosette formation, (2) natural attachment to various human cells, (3) sensitivity in vitro to the lytic effect of glucocorticoids. Although the NPC T cells attach in vitro to various cells they kill only EBV-genome-carrying targets. These findings suggest the occurrence of a local cellular immune response in NPC, possibly directed to EBV-determined antigens.     

Proliferative patterns of lymphocytes in lymph nodes during tumour development: involvement of T and B cell areas

DNA-synthetizing lymphocytes were identified in the lymph nodes regional and more distal to the site of developing P-815 tumours by incorporation of [3H]-thymidine followed by autoradiography of lymph node sections. It appeared that not only T but also B cell areas of draining and to a lesser extent of distal lymph nodes were stimulated by the growing tumour. This result was unexpected since neither humoral nor tumour cell-bound antibody could be identified so far as a functional correlate of B cell stimulation. In general the proliferative response of lymphocytes followed a biphasic pattern with an early peak of reactivity on days 2-3 and a second peak around day 12-15 after tumour cell inoculation. In the draining (axillary) lymph node the second peak of reactivity was suppressed, possibly as a consequence of metastatic tumour cells in this node when tumour cells were inoculated in the flank. The pattern of lymphocyte stimulation revealed larger individual variations after tumour cell inoculation in the flank than the foot pad. These results were associated with a slower and less regular drainage of carbon particles from the flank to the axillary and exceptionally the brachial lymph node than from the foot pad to the popliteal node after injection of India ink.     

Antibodies and soluble tumour-specific antigens in blood and lymph of rats with chemically induced sarcomata

In confirmation of other studies it has been shown that antibody directed against the tumour specific transplantation-type antigens (TSTAs) cannot be detected in rats with a tumour growing intramuscularly but appears within a few days after excision of the tumour. Circulating antibody is found in the serum of rats with lymph node metastasis following excision of the primary. Absorption by the intramuscular tumour of circulating antibody does not account for the absence of antibody since the antibody levels in thoracic duct lymph in rats with tumours in the leg are also very low and rise rapidly after tumour excision. Antibody is released by the draining nodes directly into the lymph and must pass through the thoracic duct before entering the blood. Under these conditions low levels of antibody activity in lymph cannot be ascribed to absorption by the tumour.     

Activation and in vitro expansion of tumor-reactive T lymphocytes from lymph nodes draining human primary breast cancers.

The feasibility of in vitro activation of lymphocytes from the draining lymph nodes (DLN) of breast cancer patients was examined. Lymphocytes isolated from 48 DLN from 12 patients were examined for their proliferative responses to rIL-2, autologous tumor cells, or rIL-2 plus tumor cells. Three general patterns of cellular responses were observed. Cells from some DLN (17%) were unresponsive to any stimuli. Lymphocytes from 52% of the DLN responded moderately to rIL-2 alone. The combination of rIL-2 and tumor antigen had a synergistic effect on the proliferation of cells from 31% of the DLN assayed. Phorbol dibutyrate and ionomycin plus rIL-2 stimulated expansion of DLN lymphocytes by up to 850-fold after 35 days. These expanded cell populations, as well as those stimulated with antigen plus rIL-2, were predominantly CD3+ and CD16- cells, varying in proportions of CD4+ and CD8+ subsets. Both populations were cytotoxic against autologous tumor, MCF-7, and K562 target cells.     

Alterations of the basement membrane and connective tissue antigens in human metastatic lymph nodes

Antigens of the basement membrane (type-IV collagen and laminin) and the connective tissue (type-Ill collagen and fibronectin) were studied by immunofluorescence in 16 lymph nodes draining colorectal carcinomas and 6 lymph nodes draining breast carcinomas. A comparison was also made between 7 primary colorectal carcinomas and 9 lymph nodes draining these tumors. Anti-type-IV collagen and anti-laminin rarely stained the basement membrane of metastatic tumors. In contrast, we detected type-IV collagen in the peritumoral stroma, although similar images were rarely seen in primary tumors. When tumoral cells were in the vicinity of lymphoid cells, they were occasionaly separated by a barrier stained by the four antisera, or only by antifibronectin and anti-type-lll collagen. In other cases no barrier was observed between both types of cells which were in close contact. On the whole the above alterations were more marked in the lymph nodes draining breast carcinomas, in comparison to those draining colorectal carcinomas. Tumor cells were never stained by antitype-IV collagen or antilaminin serum. Some cells found either in the lymphoid or in the tumor area of metastatic lymph nodes were stained not only by these antisera, but also by a monoclonal antibody against Willebrand Factor, which is a marker of endothelial cells. Thus the labelled cells were characterized as being derived from the capillary wall.     

A case of recurrent colon cancer with urinary bladder, para-aortic lymph nodes, and spleen metastases successfully treated with CPT-11

The patient was a 71-year-old woman with sigmoid colon cancer with urinary bladder invasion, for which sigmoidectomy with D 3 lymphadenectomy and partial cystectomy was performed. After surgery, the patient was started on 4 courses of 6-week systemic chemotherapy (500 mg/m(2) 5-FU and 200 mg/m(2) l-LV weekly). However, 4 months later, CT revealed local recurrence in the urinary bladder and recurrence in the para-aortic lymph nodes and spleen. Therefore, low-dose CPT-11 therapy (40 mg/m(2) once per week) was instituted, which achieved a complete response as revealed by CT for response evaluation 5 months after the start of therapy. Up to the present, after 8 months no recrudescence or recurrent lesions in other organs have been observed. The patient developed mild side effects such as grade 1 nausea, anorexia, and leukopenia, but has a well-maintained QOL.     

Secretion of immunoglobulin by neoplastic B lymphocytes from lymph nodes of patients with lymphoma.

An investigation has been made into the ability of neoplastic B lymphocytes obtained from lymphoid tissue of patients with non-Hodgkin''s lymphoma (NHL) to secrete immunoglobulin (Ig) in vitro. The majority of the cell populations secreted IgM (17/24 patients), identified as pentameric in three cases examined, and free monotypic light chains (23/24 patients) of the same type as the surface Ig. Secretion of IgD (6/21 patients) and IgG (3/21 patients) was found less frequently. The amounts of Ig secreted were variable and there was no significant difference in the patterns of secretion of cells from NHL patients when compared to previous studies of chronic lymphocytic leukaemia (CLL), nor was there any clear correlation with the histological type. For four of the patients, anti-idiotypic antibody was produced and was used to demonstrate the idiotypic nature of the secreted Ig, and also to show its presence in the serum. The level of idiotypic IgM was measured in one patient during chemotherapy and appeared to correlate well with disease. Such idiotypic Ig must be taken into account when planning treatment of B cell neoplasms with antiidiotypic antibody since it could act as a block to antibody attack. Assessment of the ability of tumour cells to secrete Ig in vitro provides a useful preliminary screen when choosing such patients since a high secretion rate together with extensive disease could lead to unacceptable levels of serum idiotypic Ig.     

Soluble low-molecular-mass tumor-associated antigens promote the suppression of rat mammary tumors by tamoxifen and prevent its toxic effect.

This study examined whether the soluble tumor-associated antigens (sTAA) of 66 kDa and 51 kDa could promote suppression of chemically-induced rat mammary tumorigenesis by the hormone-related anticancer drug tamoxifen and prevent the drug's toxic side-effects. Dimethylbenzanthracene (DMBA, 10 mg/rat, 3 administrations) was used to induce mammary tumors in 8-week-old Wistar rats. Then, for 13-17 more weeks, preparations of sTAA (50 microg/rat) and tamoxifen (10 mg/rat) were administered, separately or in combination, on a weekly basis. The experiment was continued for 18 weeks and was terminated when the number of dead rats reached 50% in each group. Treatment with tamoxifen inhibited tumor growth and their malignance: the number of rats without malignant tumors significantly increased compared to controls, 27.3% and 5.6%, respectively. Treatment with sTAA resulted in a significant increase in the number of regressed tumors to 10.1% compared to 0% and 1.4% in control and tamoxifen-treated rats, respectively. Moreover, the period of 50% survival increased from 13 weeks in tamoxifen-treated rats to 17 weeks, and as a result, rats treated with sTAA were involved in the experiment for an average 14.3 weeks compared to 10 and 10.4 weeks in control and tamoxifen-treated groups. In rats treated simultaneously with tamoxifen and sTAA, the time of appearance of each new tumor increased from 4.5 weeks to 6.6 weeks with a significant increase to 14.3% in the number of regressed tumors. The period to 50% survival increased to 18 weeks, and these rats were involved in the experiment for up to 16.4 weeks. The number of rats without malignant tumors increased to 22.2% and the time of appearance of malignancy increased to 9.6 weeks, as compared to 7.3 weeks in controls. The results demonstrated that sTAA have tumor-suppressive properties, and also enhance the anticancer effects of tamoxifen and prevent its toxic side-effects.     

Canine transmissible venereal sarcoma: distribution of T and B lymphocytes in blood, draining lymph nodes and tumours at different stages of growth.

The levels of T, B and null lymphocytes in the peripheral blood, draining lymph nodes, and tumour masses at different growth stages in dogs transplanted with canine transmissible venereal sarcoma (CTVS) were determined by immunofluorescence techniques. The tumours were classified at excision into "progressor", "steady state", and "regressor" stages of growth. The percentage of B cells in the lymphocytes infiltrating into the progressively growing tumours (n = 10, 37.3 +/- 7.4%) was significantly higher (P less than 0.025) than that in regressing tumours (n = 21, 26.1 +/- 1.9%). In contrast, the percentage of T cells in the lymphocytes infiltrating into the regressing tumours (n = 21, 61.2 +/- 2.6%) was significantly higher (P less than 0.005) than that in the progressively growing tumours (n = 10, 34.0 +/- 5.1%). The tumours at the steady-state growth stage (n = 9) had 50.8 +/- 5.7% infiltrating T-cells, which was significantly higher (P less than 0.005) than the progressors and lower (P less than 0.005) than the regressors. The percentage of null cells of progressors (n = 10, 26.0 +/- 6.9%) was significantly (P less than 0.025) higher than in regressors (n = 21, 13.5 +/- 2.9%). The draining lymph nodes of progressor dogs (n = 5) had significantly fewer (P less than 0.025) B cells (8.2 +/- 2.3%) than in normal (n = 5, 16.1 +/- 3.1%), regressors (n = 12, 19.1 +/- 1.7%) and steady-state dogs (n = 5, 15.8 +/- 2.6%). Although there was slight lymphopenia and fluctuation of null cells, no significant differences in T- and B-lymphocyte levels were noted in the peripheral blood of the tumour dogs (n = 44) studied.