Cells mediating specific in vitro cytotoxicity. II. Probable autonomy of thymus-processed lymphocytes (T cells) for the killing of allogeneic target cells.

In order to investigate whether only T cells are involved in a cell-mediated cytotoxic system in vitro, we tested the cytotoxicity of immune killing cell populations as deprived as possible of B cells. Educated thymus cells, immune spleen cells purified by filtration through a column of beads coated with antimouse Ig antiserum, and finally educated thymus cells further purified by filtration through such a column fully retained their specific cytotoxic activity. This very strongly suggests that only T cells are involved in the killing of target cells by allogeneic immune cells in vitro, in this system. Receptor-bearing cells involved in killing in the present system are thus very probably T cells. This point was further strengthened by the demonstration of specific adsorption, on the relevant monolayers, of each of the three above mentioned killing cell populations.

Stereotactic body radiotherapy of primary and metastatic renal lesions for patients with only one functioning kidney.

BACKGROUND: About 2% of patients with a carcinoma in one kidney develop either metastases or a new primary tumor in the contralateral kidney. Often, renal cancers progress rapidly at peripheral sites and a metastasis to the second kidney may not be the patient's main problem. However, when an initial renal cancer is more indolent yet spreads to the formerly unaffected kidney or a new primary tumor forms there, local treatment may be needed. Stereotactic body radiotherapy (SBRT) has been demonstrated as a valuable treatment option for tumors that cause local symptoms. Presented here is a retrospective analysis of patients in whom SBRT was used to control primary or metastatic renal disease. PATIENTS AND METHODS: Seven patients with a mean age of 64 (44-76) were treated for metastases from a malignant kidney to its contralateral counterpart. Dose/fractionation schedules varied between 10 Gy x 3 and 10 Gy x 4 depending on target location and size, given within one week. Follow-up times for patients who remained alive were 12, 52 and 66 months and for those who subsequently died were 10, 16, 49 and 70 months. RESULTS: Local control, defined as radiologically stable disease or partial/complete response, was obtained in six of these seven patients and regained after retreatment in the one patient whose lesion progressed. Side effects were generally mild, and in five of the seven patients, kidney function remained unaffected after treatment. In two patients, the creatinine levels remained moderately elevated at approximately 160 micromol/L post treatment. At no time was dialysis required. CONCLUSION: These results indicate that SBRT is a valuable alternative to surgery and other options for patients with metastases from a cancer-bearing kidney to the remaining kidney and provides local tumor control with satisfactory kidney function.

Thymidylate synthase expression: an independent prognostic factor for local recurrence, distant metastasis, disease-free and overall survival in rectal cancer.

Several studies have suggested that the intratumoral level of thymidylate synthase (TS) in colorectal tumors correlates with survival. We have studied the correlation between TS expression in primary rectal cancer and locoregional recurrence, distant metastases, and survival. TS enzyme levels were evaluated immunohistochemically using the specific monoclonal antibody TS 106 in paraffin-embedded tumors from 243 patients who had undergone primary surgery for rectal cancer during the years 1980-1993. All patients were included in prospective randomized trials aimed at determining the clinical value of a short preoperative course of local radiation therapy (five doses of 5 Gy each). With a median follow-up of 94 months (range, 43-202 months), it was observed by multivariate analysis that Dukes' stage and TS expression were independent prognostic markers of locoregional recurrence (P < 0.001 and P = 0.038, respectively) distant metastasis (P < 0.001 and P = 0.011, respectively) disease-free survival (P < 0.001 and 0.014, respectively), and overall survival (P < 0.001 and 0.020, respectively). By multivariate analysis, preoperative irradiation therapy showed a borderline improvement in locoregional recurrence (P = 0.051). No other factors, such as age, sex, differentiation of the tumor, or p53 expression, were noted to be independent prognostic factors for clinical outcome in these patients. We concluded that the intratumoral expression of TS in primary rectal cancer is an independent prognostic factor for locoregional recurrence, distant metastases, disease-free survival, and overall survival. Patients with low intratumoral TS expression had a significantly better outcome than those with high TS expression.

Immunohistochemically detected thymidylate synthase in colorectal cancer: an independent prognostic factor of survival.

Intratumoral thymidylate synthase (TS) expression and M(r) 53,000 phosphoprotein (p53) overexpression were studied immunohistochemically in sections from stored paraffin-embedded primary colorectal cancers in 70 patients who had undergone surgery during the years 1987-1990. These cancers were classified according to Dukes' stage A-D, using monoclonal antibodies TS 106 and DO-7. In patients with Dukes' stage A-C tumors, univariate analyses showed that there was a significant correlation (P = 0.048) between disease-free survival and TS expression and between TS expression and time to death with colorectal cancer (P = 0.038). In patients with Dukes' stage A-D tumors, overall survival was correlated to TS expression (P = 0.015), Dukes' stage (P < 0.001), and level of tumor differentiation (P = 0.044) but not to p53 overexpression. Patients with low intratumoral TS expression survived significantly longer than patients with high expression. Cox multivariate analysis showed that Dukes' stage (P < 0.001) and TS expression (P = 0.043) could independently serve as prognostic factors for time to death with colorectal cancer in patients with Dukes' stage A-D tumors.

Indomethacin modulation of monocyte cytokine release following pelvic irradiation for cancer.

Pelvic irradiation for urogenital cancer reduced monocyte release of tumour necrosis factor alpha (TNF-alpha). Addition of indomethacin to monocyte cultures increased TNF-alpha production after but not before irradiation. E. coli lipopolysaccharide (LPS) increased TNF-alpha release before as well as after radiation therapy and addition of indomethacin to LPS-stimulated monocytes further increased TNF-alpha production following radiotherapy. Spontaneous interleukin-1 (IL-1) release was increased in the cancer patients and was not significantly affected by radiation therapy. LPS increased IL-1 release before as well as after irradiation, but indomethacin did not further change IL-1 secretion. These findings suggest that prostaglandins differentially regulate TNF-alpha and IL-1 release. Administration of cyclo-oxygenase inhibitors during radiation therapy might increase TNF-alpha release in vivo and thereby enhance the host defence against tumours.

Different dose regimens of 5-fluorouracil and interferon-alpha in patients with metastatic colorectal carcinoma.

Three different 5-fluorouracil (5-FU)-interferon-alpha-2b (IFN)-containing regimens were designed for treatment of patients with advanced colorectal cancer. 87 patients with a Karnofsky index > or = 70 were included in three sequential non-randomised phase II trials. Regimen A consisted of 5-FU (750 mg/m2/day) given as a continuous infusion on days 1-5 followed by weekly 1-h intravenous infusions until week 8. IFN (5 MU) was given subcutaneously on days 1, 3 and 5 followed by injections (9 MU) every second day until week 8. The cycle was then repeated. Regimen B consisted of 5-FU (750 mg/m2/day) given as a continuous infusion on days 1-5 followed by 5-min intravenous injections on days 12 and 19. IFN (3 MU) was given subcutaneously on days 1-5 followed by injections (5 MU) on days 11-13 and 18-20. The cycle was repeated every fourth week. Regimen C consisted of 5-FU (750 mg/m2/day) given as a continuous infusion on days 1-5. IFN (3 MU) was given subcutaneously on days 1-5. The cycle was repeated every third week. The objective response rates (complete response (CR) and partial response (PR)) after approximately 4 months of therapy or longer were as follows: regimen A (n = 27) 22% (2 CR, 4 PR), regimen B (n = 33) 42% (4 CR, 10 PR) and regimen C (n = 27) 22% (1 CR, 5 PR). The corresponding response figures for previously untreated patients were regimen A 50%, regimen B 64% and regimen C 38%. Response durations varied from a few weeks up to 142 + weeks. Toxicities were generally mild and reversible, and the treatments were convenient for the patients and cost effective since the 5-day infusions could be given by a portable pump without hospitalisation. Our results are in agreement with those of others showing that 5-FU/IFN combinations can be highly effective in advanced colorectal cancer, and that a number of factors such as doses, dose intensities, infusion rates and timing of the two drugs may be crucial for the anti-tumour activity of this drug combination.

Immunohistochemical determination of thymidylate synthase in colorectal cancer--methodological studies.

With the aid of specific monoclonal antibodies, an immunohistochemical technique has recently been developed for the detection of intratumoral thymidylate synthase (TS). This technique can be applied to paraffin-embedded material suitable for retrospective studies. In order to examine this technique further, the TS enzyme activity of lysates from frozen-stored colorectal cancer (CRC) specimens were compared with their immunohistochemical TS staining intensity (arbitrarily graded from 0 to 3). A statistically significant correlation between these two methods on a total of 25 tumour specimens (P < 0.001) was observed. The staining intensity in different areas of 48 paraffin-embedded CRCs was examined. Sixty-seven per cent of the tumours were homogeneously stained (either grades 0-1 or 2-3), 33% showed a heterogeneity in TS staining. Increased TS expression correlated with more advanced Dukes' stage (P < 0.001). It is concluded that TS immunostaining intensity reflects TS enzyme activity in colorectal tumours and is well suited for paraffin-embedded material. The TS immunostaining pattern is heterogeneous in up to one-third of the tumours.

Changes of the blood lymphocyte population following 131I treatment for nodular goiter.

The blood lymphocyte population was examined in 34 patients who were treated with 131I for toxic or atoxic nodular goiter. The patients received one to three doses of 300-550 MBq of 131I administered at 1 week intervals. Lymphocyte counts were significantly reduced both 1 and 6 weeks after treatment. This reduction was accompanied by a changed composition of the lymphocyte population. The frequency of lymphocytes expressing membrane receptors for C'3 (EAC-rosette forming) was significantly reduced 1 and 6 weeks after 131I-administration. At 6 weeks there was a slight but statistically significant increase of the frequency of T-cells as identified by Leu 1 monoclonal antibodies. This was largely caused by an increased proportion of helper/induced T-cells as identified by Leu 3a monoclonals. 131I-treatment also reduced the capacity of lymphocytes to secrete immunoglobulins (Ig) upon PWM-stimulation. The most pronounced effect was observed for IgM. Secretion of IgG and IgA were less reduced. Mitogenic stimulations of lymphocytes with PHA and ConA were not significantly changed. We conclude that these changes observed, with the exception of mitogen reactivity, are essentially similar to those occurring after external radiation therapy for cancer. We speculate that blood lymphocytes passing through the continuously irradiated gland are damaged mainly by emitted beta-particles.

Long term effects on the immune system following local radiation therapy for breast cancer. I. Cellular composition of the peripheral blood lymphocyte population.

Local radiation therapy for breast cancer depletes the blood of various subsets of lymphocytes. Previous studies showed that the recovery is still incomplete at 30 months. To further elucidate the recovery we examined blood lymphocyte counts of 138 disease-free women and various lymphocyte subsets in 102 of these patients. These patients, 5-6 and 10-11 years earlier, had entered a clinical trial in which preoperative irradiation (45 Gy) was evaluated against postoperative irradiation (45 Gy) or surgery only. Patients who had undergone surgery only served as controls. Total lymphocyte counts of the irradiated patients were still significantly reduced 10-11 years after treatment. This reduction was mainly attributable to a subnormal level of T-cells as determined by the monoclonal antibody Leu-1 and the ability to form rosettes with sheep erythrocytes, whereas the number of non-T cells, expressing C'3 receptors, did not differ significantly from the controls. Within the T-cell population a subset with helper/inducer phenotypes, detected by Leu-3a antibodies, was significantly reduced even 10-11 years after irradiation. T-cells with suppressor/cytotoxic phenotypes, stainable with Leu-2a antibodies, however, had already recovered 5-6 years after irradiation. The duration of the radiation induced reductions of different lymphocyte subsets may be related to the physiological turn-over of the cells or a changed distribution of cells in the body.

Bestatin treatment of human lymphocytes increases the frequency of sheep red blood cell-binding cells.

Treatment of cancer patients with Bestatin, a new immunomodulator increases the frequency of peripheral lymphocytes forming rosettes with sheep red blood cells (SRBC). This study has shown that treatment of lymphocytes from healthy donors with Bestatin in vitro increases the frequency of such cells and there is a slight decrease of C'3-receptor bearing cells. The proportions of TM and TG cells were not significantly changed. The results indicate that the increased frequency of SRBC-binding cells during Bestatin medication is not due to a mobilisation of T-cells but rather to an improved binding capacity of certain T-cells for SRBC.

Depressed responder and stimulator capacities of peripheral lymphocytes from patients with advanced breast cancer in the mixed lymphocyte culture.

Peripheral lymphoid cells of patients with carcinoma of the breast were examined for reactivity (responders) against allogeneic lymphoid cells in the one-way mixed lymphocyte culture. The latter cells were also tested for response against the patients' lymphocytes (stimulators). The responder and stimulator capacities of lymphoid cells of patients with advanced cancer were found to be significantly reduced compared to cells from healthy subjects. Such defects were not observed in disease-free women previously treated for primary carcinoma of the breast. The results support the view that there is an abnormality of the lymphocyte-monocyte pool of cells in patients with advanced cancer.

Interferon and spontaneous cytotoxicity in man. III. Effect of interferon on lymphocytes and target cells in vitro.

Human leukocyte interferon (IF) was tested for its capacity to modify the spontaneous cytotoxicity of human peripheral lymphocytes for allogeneic target cells in vitro. Pretreatment of lymphocytes with IF augmented their cytotoxicity whereas pretreatment of the target cells did not increase but possibly decreased their sensitivity to the spontaneous cytotoxicity of lymphocytes.

Changes of blood T cell subsets following radiation therapy for breast cancer.

The changes of the size of various T cell subsets in the blood, as defined by monoclonal antibodies and Fc-receptors for IgG and IgM, were examined in 11 women after postoperative radiation therapy (45 Gy) for breast cancer. All subsets of T cells were significantly reduced at completion of irradiation. The most extensive depletion was noted in a subset with receptors for IgG, which may exert suppression. Approximately 1 year later this subset had recovered significantly in parallel with another subset, also rich in suppressor T cells, which was detected by monoclonal antibodies. On the contrary, T cell subsets rich in cells with helper activity did not exhibit any significant recovery.

Prostaglandin sensitivity of the PHA-response of blood lymphocytes following radiation therapy for breast cancer.

The reduction of mitogen responses of blood lymphocytes which occur after radiation therapy for breast cancer, known to be largely due to inhibitory monocytes, can partly be reverted by indomethacin which is an inhibitor of prostaglandin (PG) synthesis. This may indicate that PG-synthesis by blood monocytes is increased after irradiation or that PG-sensitivity of the lymphocyte population is increased. To test the latter possibility the sensitivity of the PHA-response of blood lymphocytes to varying concentrations of PGE2 and PGD2 was examined in 15 patients with breast cancer before and up to 6 months after local radiation therapy (46 Gy). The results showed that the sensitivity of the PHA-response was not significantly changed after treatment suggesting that the immunosuppression observed after irradiation is partly due to an increased production of PGs rather than an increased PG-sensitivity of lymphocytes.

Long-term effects on the immune system following local radiation therapy for breast cancer. 4. Proliferative responses and induction of suppressor activity of the blood lymphocyte population.

The long-term effect of local irradiation for breast cancer on the blood lymphocyte population was examined in 149 women who had been disease-free for 5-6 and 10-11 years. The patients were included in a clinical trial aiming at determining the value of pre- and post-operative irradiation (45 Gy) compared to surgery only. It was observed that the relative mitogen responses of lymphocytes to phytohaemagglutinin (PHA) and Concanavalin (Con A) and in a mixed lymphocyte culture (MLC) were significantly lower in irradiated compared to unirradiated patients at least a decade after treatment. The prolonged reductions of mitogen responses after irradiation could partly be due to an increased proportion of lymphocytes which may express suppressor function since the Con A-inducible suppressor activity of lymphocytes was significantly higher in irradiated compared to unirradiated patients.

In vitro capacity of various cyclooxygenase inhibitors to revert immune suppression caused by radiation therapy for breast cancer.

Radiation therapy triggers blood monocytes to an increased secretion of immunosuppressive prostaglandins (PGs), which in part can explain the post-irradiation impairment of lymphocyte blastogenesis. Since low mitogen responses of lymphocytes in irradiated breast cancer patients is linked to a poor prognosis a clinical trial is planned to examine if treatment with inhibitors of PG-synthesis during irradiation can counteract immunosuppression and increase survival. In the present investigation we have compared nine different inhibitors of PG-synthesis for capacity to enhance phytohemagglutinin responses of blood lymphocytes before and after irradiation for breast cancer. Five of the drugs (aspisol, indomethacin, meclofenamic acid, ketoprofen and diclofenac) enhanced the reactivity to more than 150%. In general, the strongest enhancements were observed in lymphocyte preparations obtained at completion of irradiation when reactivity was most depressed followed by those obtained at one month and before irradiation.

Possible role of prostaglandin producing monocytes in the depression of mitogenic responses of blood lymphocytes following radiation therapy.

Previous studies have shown that the depression of mitogenic responses of cultured blood lymphocytes following radiation therapy can largely be explained by immunosuppressive cells. In this investigation we have shown that the addition of silica, a monocyte toxic agent, to the cultures enhance the PHA- and PPD-responses of the cells at completion of irradiation. Such an effect, however, was not noted before radiation treatment was started. Similar results were obtained by adding indomethacin, an inhibitor of prostaglandin synthesis, to the cultures. The results thus indicate that immunosuppressive monocytes which mediate their activity by prostaglandins are involved in the reductions of mitogen responses of blood lymphocytes following irradiation.

High dose rDNA human alpha 2 interferon therapy in patients with advanced colorectal adenocarcinoma: a phase II study.

Eighteen patients with advanced and inoperable colorectal adenocarcinomas were treated with high doses of alpha 2 Interferon (Schering-Plough Corporation). The patients were randomized to receive either subcutaneous injections of 20 X 10(6) I.U./m2 three times weekly for 3 months, or pulsed treatments of 50 X 10(6) I.U./m2 daily, given intravenously, for 5 consecutive days every 4 weeks. No objective tumour regression was seen in any patient. The side effects were considerable.

Blood lymphocyte subsets in operable breast-cancer - relation to extent of tumor disease and prognosis.

Previously we have reported that a high frequency of E-rosette forming cells (T-cells) in the blood of newly diagnosed breast cancer patients was associated with the development of distant metastases and a short survival. In the present investigation, comprising 204 untreated breast cancer patients, we showed that the proportion of the total T-cell population (CD2 and CD3 positive cells) and the proportion of helper/inducer T-cells (CD4 positive) was positively linked to spread of cancer cells to axillary nodes which in turn Was strongly correlated to prognosis. The latter subset also correlated significantly to time to development of distant metastases. Cox multivariate regression analysis showed that the frequency of these lymphocytes, independently of other variables, predicted prognosis. Our present as well as our previous results do not support the view that a high proportion of T-cells in the blood forecast a good prognosis.

Does early administration of thyroxine reduce the development of Graves' ophthalmopathy after radioiodine treatment?

The roles of thyroid hormones and thyrotropin (TSH) in the development of Graves' ophthalmopathy are not clear. Some studies suggest a protective effect of thyroid hormones on experimental exophthalmos and an adverse effect of increased TSH levels. In September 1988 we introduced early thyroxine (T4) administration after 131I therapy for hyperthyroidism caused by Graves' disease. We carried out a retrospective study of records from all patients with this disease treated with 131I for 4 years. During the first 2 years 248 patients were treated (group A). They received T4 when the serum concentration of TSH and/or T4 indicated hypothyroidism. During the next 2 years 244 patients were treated (group B). They were all given 0.05 mg of T4 daily, starting 2 weeks after therapy, and 0.1 mg after a further 2 weeks. With a follow-up of 18 months, 45 patients (18%) in group A and 27 patients (11%) in group B developed or deteriorated in an already present ophthalmopathy (p = 0.03, relative risk = 1.64, 95% confidence interval = 1.05-2.55). Twenty-six patients in group A required specific therapy for the ophthalmopathy (e.g. antithyroid drugs, steroids, etc.) compared to 11 patients in group B (p = 0.02, relative risk = 2.33; 95% confidence interval = 1.18-4.60). Our results suggest that early administration of T4 after 131I therapy reduces the occurrence of Graves' ophthalmopathy.