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Natural killer cell activity in long term survivors of testicular cancer: Influence of cytostatic therapy and initial stage of disease
Abstract
Patients with testicular cancer can be cured by cisplatin-based chemotherapy in many cases. Thus, concern about possible late toxicities of treatment is warranted.
In this investigation, the absolute number of natural killer (NK) cells according to their CD56+ phenotype, NK cell activity, and antibody dependent cellular cytotoxicity (ADCC) were investigated in 29 patients with seminomas or nonseminomatous germ cell tumors (NSGCT) a median of 31 months (range, 5-73 months) after termination of chemotherapeutic treatment.
No difference in the absolute number of NK cells, NK cell activity, and ADCC was found between patients with testicular cancer after either standard polychemotherapy consisting of bleomycin, etoposide, and cisplatin (BEP) or monotherapy with carboplatin and healthy control subjects. When patients were analyzed further using multivariate analysis, a significant (P < 0.05) detrimental influence of BEP polychemotherapy versus carboplatin monotherapy on NK cell activity was found. Moreover, NK cell activity also depended significantly (P < 0.05) on the time elapsed after chemotherapy was administered, but normalized with time. Because the absolute number of NK cells was not affected, is was assumed that polychemotherapy induced a functional defect. In a subanalysis of patients with NSGCTs, metastases at diagnosis resulted in a significant (p < 0.05) and persistent stimulation of NK cell activity but not of ADCC.
Cytostatic chemotherapy in patients with testicular cancer did not lead to compromised lytic effector cell function as assessed by NK cell activity and ADCC. However, a short, time-dependent reduction was found that also depended on the intensity of chemotherapeutic treatment. This finding related to the observation of a long-lasting stimulus of NK cell activity by initial metastases in patients with NSGCTs.
... Other reports have stated that tumours propagated as a result of NK activity depression, and that metastasis could be inhibited by elevating NK activity [41±43]. There have also been a number of cases of metastatic-free survival extension in tumour patients with high NK activity [44,45]. Since the effects of immune surveillance and the antitumour activities of NK cells can affect tumour growth, it is possible that the NK cells activated by DCs possess potent antitumour activity, and may be applicable to the clinical treatment of cancer patients. ...
Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1.8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-gamma secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-gamma was markedly reduced. IFN-gamma production was completely blocked by an anti-IL-12 antibody, indicating that IFN-gamma secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40-CD40L or CD28-B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response.
The observation that cancer patients who experienced a feverish period after surgery survived significantly longer than patients
without fever and the fact that spontaneous tumor remission was observed mostly after a fever period was the rationale for
the artificial induction of fever (“fever therapy”). The history and rationale for fever therapy are presented and the immunological
basis for endo- and exotoxin-induced tumor regression are discussed on the basis of nearly 800 citations of research literature.
The effects and clinical research of different biological inductors of hyperthermia like Coley’s Toxin (MBV), Propioni Bacteria,
Corynebacterium parvum, Bacillus Calmette Guerin (BCG), OK-432, Staphylococcus protein A and Streptokinase are described.
Though the biological effects of fever on tumors are well characterized and interesting biological and immune ological results
are obtained, and some clinical observational studies and small randomized trials show very promising results, larger controlled
GCP-conform trials are still lacking. In combination of moderate and extreme whole body hyperthermia with chemotherapy, radiotherapy
or immunotherapy with monoclonal antibodies significant improvement in outcome of the treatment of cancer patients is to be
expected. The toxicities of active “fever therapy” or passive “fever-range whole body hyperthermia” are tolerable.
Assessment of immunocompetence in non-seminomatous testicular cancer (NSTC) patients.
In the peripheral blood of 43 patients with NSTC, lymphocyte subsets, lymphocyte reactivity to mitogens, NK cell activity as well as neutrophil and monocyte phagocytic functions were studied.
The proportion and number of T- and B- lymphocytes, lymphocyte reactivity to mitogens as well as neutrophil and monocyte phagocytic functions were equal in patients with localized disease and in those with metastatic disease and they not differ from those in controls. The NK cell number and NK cell activity, however, were significantly impaired in both patient groups. The patients with metastatic disease had a lower NK cell activity than those with localized disease. One year after the completion of anticancer treatment, the NK cell number and cytotoxic activity recovered in both groups of patients.
These results suggest that patients with NSTC are primarily deficient in NK cell number and their cytotoxic function influence the host's ability to control the proliferation and spread of tumor cells.
To investigate the impact of polychemotherapy on cellular immunity in patients with testicular cancer.
Lymphocyte subpopulations, lymphoproliferative responses to mitogenic stimulation, and mitogen-induced release of soluble interleukin-2 receptor from peripheral blood mononuclear cells were investigated in 15 patients with testicular germ cell tumors a median of 61 months (range 7 to 73) after polychemotherapy with bleomycin, etoposide, and cisplatin (BEP).
The numbers of peripheral blood T cells (CD3+), CD4+ and CD8+ subsets, and lymphoproliferative responses to pokeweed mitogen, phytohemagglutinin, and concanavalin A in patients were comparable to those of healthy control subjects. When two groups of patients were formed according to elapsed time from BEP polychemotherapy and study onset (group A, 12 months and group B, 69 months after termination of BEP), a significant increase in lymphoproliferative response to concanavalin A (P <0.05) was found in group A 1 year after chemotherapy.
BEP chemotherapy administered to patients with testicular cancer does not result in impairment of cellular immunity but rather leads to a significant increase in the capacity of patients' lymphocytes to respond to mitogenic stimulation up to 1 year after polychemotherapy. Moreover, the increased T-cell activity found after BEP therapy may contribute to the high rate of long-term complete remission.
Immunotherapy using a monoclonal antibody against the human epidermal growth factor receptor 2 protein, HER-2/neu, has proven to be clinically efficacious in about one-half of breast cancer patients who exhibit strong (3+) plasmalemmal immunoreactivity for this protein. The tumoricidal effect of this antibody relies in part upon antibody-dependent cell-mediated cytotoxicity. This report provides observations on certain factors or circumstances which could have an impact on this aspect of the therapeutic approach. These include: (1) concurrent medications; (2) the composition (immunophenotype) of peritumoral lymphocytes and the generally limited numbers of intratumoral T-lymphocytes/natural killer (NK) cells, monocytes, and neutrophilic granulocytes; (3) the presence of circulating HER-2/neu antigens which might bind the exogenous antibody and lead to immune complex formation; (4) the variable co-expression in the tumor of cytokines known to downregulate NK cell function (ie, transforming growth factor-beta1 [TGF-beta1] and platelet-derived growth factor [PDGF]-AB); and (5) the tumoral and/or stromal immunoreactivity for angiotensin-converting enzyme, which forms a part of one of the pathways for the activation of latent TGF-beta1 and for the biosynthesis of PDGF-AB. These observations provide an immunologic perspective for the use of monoclonal antibody therapy in HER-2/neu protein-receptor-positive breast carcinoma and suggest a role for the clinical laboratory in identifying potential avenues for additional manipulations of the immune system in individual cases in order to enhance the therapeutic response.
Accumulating evidence exists for (1) an inverse correlation between the incidence of infectious diseases and cancer risk and (2) an inverse correlation between febrile infections and remissions of malignancies. This review is part of an effort of the Office of Alternative Medicine at the National Institutes of Health to examine this evidence.
A review of the literature to a key word search was undertaken, using the following key words: fever, infectious diseases, neoplasm, cancer incidence and spontaneous remission.
The data reviewed in this article support earlier observations on the topic, i.e. that the occurrence of fever in childhood or adulthood may protect against the later onset of malignant disease and that spontaneous remissions are often preceded by feverish infections.
Pyrogenic substances and the more recent use of whole-body hyperthermia to mimic the physiologic response to fever have successfully been administered in palliative and curative treatment protocols for metastatic cancer. Further research in this area is warranted.
Sulfur mustard (2,2-dichloroethyl sulfide, SM) has been documented as an alkylating agent. It has been widely used as a chemical weapon during the last two decades. Despite extensive worldwide research, no effective therapy has yet been devised for the treatment of patients exposed to SM. A severe suppression of the immune system still remains as the major cause of opportunistic infections, septicemia and death in such patients. The aim of this study was to determine the possible effect of SM on natural killer (NK) cells in patients suffering from SM injuries. Patients were classified into three groups: mild, moderate and severe. Blood sample obtained from each patient was examined using flowcytometric technique. Results showed that the percentage of NK cells (CD45+/CD56+) is significantly lower in severe patients than that of the control group (P<0.05). It was also observed that the activity of NK cells (CD56+/CD25+) in severe alkylating group is noticeably higher compared with the control group (P<0.1).
The senior author of this comprehensive review observed and reported in 1969 that his lymphocytes killed allogeneic tumor cells in vitro. Some of his research associates and technicians and other healthy individuals also yielded such killer lymphocytes. The team considered pre-immunization to cancer occurring in individuals after in-family or professional exposure to patients with cancer (in an era when the concept of viral etiology of cancer was receiving major support); or that lymphocytes can acquire through blastic transformation immune reactivity to allogeneic cells anew in vitro. The phenomenon was eventually referred to as 'lymphocytes practicing Burnet's immunosurveillance.' Project site visitors of the USA NCI first viewed these observations as a matter of 'in vitro artifacts' being in opposition to strong tumor- specific cytotoxicity of tumor-bearing patients' lymphocytes recognized in the vast majority of other assays. After NCI funds were released for intramural studies on the phenomenon of non-specific cytotoxicity by lymphocytes, recipients (other than the senior author) of these NCI funds later characterized (1973-1975) the 'indiscriminately' cytotoxic lymphocyte populations as those of 'natural killer (NK) cells.' In this article, the original observations made in 1969-1971 are reviewed based on genuine material preserved by the senior author and are explained in view of recent discoveries that were not available at the time of the original observations. NK cells display a fascinating history arising first in urochordates during the cambrian explosion. At that level, NK cells protected their hosts from incompatible cell colony fusions and against intracellular, especially viral, pathogens. Since then, viruses evolved evasive maneuvers to escape NK cell attack on the infected cells. NK cells persisted after the evolution of adaptive immunity in cartilaginous fish fitting seamlessly into the new system. In mammals, NK cells assumed the role of chief arbitrators between the fetal trophoblast and maternal immune reactions to the semi-allograft fetus. Tumors induce in NK cells the same (inactivating; mediating Th2-type immunity) reactions the fetal trophoblast engenders in utero, but NK cells may overcome the host's tolerance to its tumor and kill tumor cells, especially when converted into lymphokine-activated killer (LAK) cells by molecular mediators of Th1-type immunity. The authors prepare and utilize LAK cells and IL-2 for adoptive immunotherapy of patients with metastatic melanoma and kidney carcinoma. A patient with malignant ascites due to ovarian carcinoma entered remission on LAK cell therapy. Just as dendritic cells, the major antigen presentors, may undergo malignant transformation, NK cells are also subject to transformation into FasL-producer virulent lymphoma-leukemia cells. The senior author reported in 1970 a patient with 'lymphosarcoma cell leukemia' whose circulating lymphoma cells killed indiscriminately human sarcoma and carcinoma cells. The exemplary case history of another patient with NK cell lymphoma-leukemia treated by the authors is presented.
Immune competence was evaluated in cured patients who had been treated by irradiation for carcinoma of the laryngopharynx, and compared with that of similar patients treated by surgery alone, and normal controls. Cellular immunity was determined by quantitation of in vitro phytohemagglutinin (PHA)-induced lymphocyte reactivity and peripheral blood thymus-dependent lymphocyte (T cell) levels. Humoral immunity was assessed by measurement of serum immunoglobulin levels and by the effect of serum on in vitro normal lymphocyte reactivity to PHA. In 21 patients who were studied for 4–23 years (mean 8.4) after surgical treatment alone for laryngopharyngeal carcinoma, neither cellular nor humoral immunity differed from that of 44 controls. In contrast, 14 patients who had been irradiated and subsequently cured for 4–15 years (mean 9.0) prior to evaluation displayed significantly impaired cellular immune competence when compared to normals and patients treated by surgery alone. Since previous determinations of the effects of radiation therapy have been in patients who received irradiation to the thymic region or large area of bone marrow, this study indicates that radiation therapy for cancer administered via portals that encompass a minimal area of the immune system may be associated with prolonged impairment of cellular immunity.
Natural killing (NK) in humans, as well as in other species, has been shown to be specific for antigenic determinants present on the surfaces of a variety of tumor cells. Physical separation of NK cells from K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC), has not been successful; however, there is indirect evidence suggesting that these activities are distinct. To further study the relationship between NK and K cells, competitive inhibition techniques were employed.
NK cells can be blocked via two mechanisms: 1) by direct inhibition with NK-sensitive tumor cells binding to NK receptor sites present on the effector cells and 2) by steric inhibition resulting from the binding of antibody-coated cells to the FcR on the effector cells. K cells, however, lack the NK receptor site(s) but are FcR+, and can therefore be blocked only by antibody-coated cells. We therefore postulate that NK and K cells are two separate lymphoid populations. NK cells bear receptor site(s) for NK determinants and FcR, whereas K cells bear only FcR.
Standard chemotherapy for disseminated germ-cell tumors includes a combination of cisplatin, vinblastine, and bleomycin, but this regimen produces substantial neuromuscular toxicity. In a randomized clinical trial in 261 men with disseminated germ-cell tumors, we substituted etoposide for the vinblastine in this regimen in half the patients to compare the efficacy and toxicity of the two treatments. Among 244 patients who could be evaluated for a response, 74 percent of those receiving the regimen including vinblastine and 83 percent of those receiving the regimen including etoposide became disease-free with or without subsequent surgery (P not significant). Among the 157 patients with high tumor volume, 61 percent became disease-free on the regimen that included vinblastine, as compared with 77 percent on the regimen that included etoposide (P less than 0.05). Survival among the patients who received etoposide was higher (P = 0.048). The regimens were similar in terms of myelosuppressive effects and pulmonary toxicity. However, the etoposide regimen caused substantially fewer paresthesias (P = 0.02), abdominal cramps (P = 0.0008), and myalgias (P = 0.00002). We conclude that etoposide with cisplatin and bleomycin is superior to vinblastine with cisplatin and bleomycin in the treatment of disseminated germ-cell tumors because of diminished neuromuscular toxicity and, among patients with advanced disease, better efficacy.
Between 1979 and 1984, 195 evaluable patients were entered in an international multicenter study comparing two regimens for patients with completely resected pathological Stage II testicular cancer (that is, with positive retroperitoneal lymph nodes). All patients had undergone orchiectomy and dissection of the retroperitoneal lymph nodes. They were randomly assigned to be treated with two cycles of immediate adjuvant cisplatin-based chemotherapy or to be observed monthly with treatment at relapse. The median follow-up period was four years. Of the 97 patients assigned to adjuvant chemotherapy, 6 (6 percent) had a recurrence; however, only 1 had received adjuvant chemotherapy before the recurrence. Three died (one of testicular cancer), and 94 of the 97 survived. Of the 98 patients who were observed, 48 (49 percent) had a relapse. However, almost all patients with relapses were effectively treated, and 93 of the 98 are alive and disease-free; 3 have died of testicular cancer. No identifiable factors were strongly associated with the risk of relapse. We conclude that two courses of cisplatin-based adjuvant chemotherapy will almost always prevent relapse in pathological Stage II testicular cancer treated with orchiectomy and retroperitoneal-lymph-node dissection. However, when surgery, follow-up, and chemotherapy are optimal, observation with chemotherapy only for relapse will lead to equivalent cure rates.
The role of clinical status and therapeutic intervention on natural cell-mediated cytotoxicity in breast cancer was ascertained by monitoring natural killer (NK) cell activity in peripheral blood samples. Patients with localized disease on chemotherapy showed significant reductions in NK activity concomitant with reduced lymphocyte numbers, when compared to untreated patients (18.1% versus 32.7%, P less than 0.005). Lymphocyte counts were included in a calculation of the absolute proportion of NK activity that incorporates a correction factor for the leukopenia that occurs as a result of cytotoxic therapy and disease progression. This calculation more accurately reflects the significant reduction of NK activity that occurs in patients with localized and systemic disease on chemotherapy when compared to untreated patients with no current evidence of disease (10.3% and 14.9% versus 30.7%, respectively; P less than 0.001). Different chemotherapeutic regimens were found to selectively affect NK cell function. The levels of both actual and absolute NK activity were significantly reduced in patients receiving 5-fluorouracil and L-phenylalanine mustard; cyclophosphamide, methotrexate, and 5-fluorouracil; and vincristine, Adriamycin (doxorubicin), and 5-fluorouracil, whereas only the levels of absolute NK activity were significantly reduced in patients receiving mitomycin, Megace (megestrol acetate), and Adriamycin when compared to untreated cancer patients. In contrast, tamoxifen-treated patients demonstrated levels of actual and absolute NK activity observed with untreated cancer patients. Patients receiving tamoxifen showed significantly elevated NK activity when compared to patients on all other chemotherapies. These results indicate that monitoring NK cell function may be useful in assessing the immunosuppressive effects of chemotherapeutic intervention.
53 patients with clinical stage I non-seminomatous germ-cell testicular tumours were entered into a prospective study to receive no treatment other than orchidectomy until unequivocal clinical evidence of metastases was established. Of this group, 9 men (17%) have relapsed, 8 within six months of orchidectomy. All 9 are alive and disease-free after chemotherapy. The relapse rate was higher in patients with malignant teratoma undifferentiated (embryonal carcinoma) primary tumours than in those with malignant teratoma intermediate (teratocarcinoma); 42.8 and 3.4%, respectively. The results were compared with those from 157 men treated by orchidectomy and radiotherapy for stage I disease. In this group, 49 patients (25.8%) relapsed and 85% of relapses occurred within one year of orchidectomy. The tempo relapse was identical for embryonal carcinoma and teratocarcinoma. Of 32 patients in whom serum markers were measured before orchidectomy, 24 (75%) had raised levels of alphafetoprotein and/or beta human chorionic gonadotropin. These preliminary results imply that routine lymphadenectomy or lymph node irradiation in clinical state I testicular non-seminoma may be unjustifiable.
The results of treatment of 126 patients with non-seminoma germ cell tumours (malignant teratoma) of the testis are presented. Of this group 81% are alive and 70% disease-free. Of 98 patients who had had no prior treatment 83% are alive and 76.5% disease-free. Of 43 early-stage patients 41 (95%) are alive and tumour-free despite a 21% relapse rate after radiotherapy. The disease-free rate in patients with advanced disease treated with chemotherapy, and in some cases radiotherapy and/or surgery, ranged from 100 to 9% and was strongly dependent upon the volume of metastatic tumour and the histology. Patients with malignant teratoma undifferentiated (embryonal carcinoma) showed a significantly higher disease-free survival rate than those with malignant teratoma intermediate (teratocarcinoma). Twenty-six men with advanced disease received radiotherapy after chemotherapy and of these 23 (88%) are alive and 21 (81%) are disease-free. No difference in disease-free survival rate was seen between advanced-stage patients receiving vinblastine and bleomycin and those receiving, in addition, cis-platinum. The overall drug-related mortality was 4.5%. Serum alpha-fetoprotein (AFP) and beta-human chorionic gonadotrophin (beta-HCG) levels were valuable monitors in advanced disease but not prognostically significant if allowance was made for tumour volume. Fifteen patients had surgical resection of residual masses and one died post-operatively.
The combination of platinum, vinblastine, and bleomycin was first used at Indiana University in 1974. Thirty of 47 patients (64%) survived for 5 years, and 27 (57%) are currently disease free (NED) and cured of their neoplasm. From 1976 to 1978, 78 consecutive patients were entered on a random prospective study that indicated that equal therapeutic results could be achieved with a lower dosage (0.3 mg/kg) of vinblastine. Fifty-two (67%) patients are continuously NED, and 57 (73%) are currently NED for 2 or more years. Our third-generation study, done in conjunction with the Southeastern Cancer Study Group, tested the hypothesis of whether maintenance vinblastine was necessary to ensure optimal cure rates in disseminated testicular cancer. One hundred thirteen patients entered this maintenance study, and the results demonstrated that cure in a far-advanced cancer could be achieved with only 12 weeks of therapy (remission induction) because the relapse rate in such patients was only 7%. The cure rate for patients presenting with locoregional disease (Stages A and B) should approach 100%. Platinum, vinblastine, and bleomycin will regularly produce a 70% complete remission rate, and a further 10% of patients will be rendered NED with surgical resection of residual disease. The relapse rate with four courses of remission induction therapy in a large cooperative group study (Southeastern Cancer Study Group) was only 7%. The high success rate in disseminated disease has allowed the option of high cure rate in Stage B disease (positive retroperitoneal nodes) with or without adjuvant chemotherapy. At Indiana University, 137 patients have been followed with Stage A and B nonseminomatous testicular cancer from 1973 to 1979 with a minimum follow-up of 2 years, and currently 135 are alive and well. Successful treatment strategies in testicular cancer have yielded a cure rate unparalleled in cancer treatment.
A group of 17 patients, having undergone modified radical mastectomy for breast cancer, received 12 cycles of chemotherapy with methotrexate, 5-fluorouracil, and chlorambucil during 17 months. The number of circulating T and non T lymphocytes, as defined by E, EAC, and ME rosette formation, were reduced during treatment. The Non-T lymphocytes, however, were reduced to the highest relative extent. Relative phytohemagglutinin and mixed lymphocyte culture responses of the cells decreased, whereas purified protein derivative responses were unchanged. Serum concentrations of IgM were reduced, but IgA and IgG concentrations were unchanged or slightly increased. Antibody titres to morbilli and herpes simplex were not changed, whereas the antibody activity against cytomegalovirus (CMV) increased in several seropositive patients. None of these patients, however, developed signs of a CMV infection.