Experimental study of local immunochemotherapy of head and neck cancer]

The mechanism of induction of antitumor activity by local administration of recombinant interleukin-2 (rIL-2) combined with cisplatin (CDDP) was investigated in order to establish a method of immunochemotherapy against head and neck cancer. Local administration of rIL-2 had significantly greater inhibitory effects on tumor growth in both Meth A and C26 tumor bearing mice than did systemic administration. The cytotoxic activity of tumor infiltrating lymphocytes (TILs) obtained from C26 tumor bearing mice was studied. Local injection of rIL-2 around the tumor site for 4 days induced augmentation of the cytotoxicity of TILs not only in NK sensitive tumors but also in NK resistant C26 tumors. This phenomenon was not observed in spleen cells. Both negative selection assay and cold target inhibition assay revealed that the effector cells were tumor nonspecific asialoGM1 positive activated NK cells. Additional experiments were performed to determine the effectiveness of combined immunochemotherapy using CDDP and rIL-2 in C26 tumor bearing mice. The intraperitoneal administration of CDDP following the local administration of rIL-2 was more effective in suppressing tumor growth and in promoting well-survival than the use of CDDP or rIL-2 alone. To investigate the mechanism of antitumor activity, the effects of CDDP on the tumor cells and immunological changes were observed in tumor bearing mice. The susceptibility of tumor cells to effector cells was enhanced after in vitro culture with CDDP. In vivo administration of CDDP augmented the cytotoxic activity of effector cells and responsiveness to IL-2 of TIL. These results suggest that local immunochemotherapy using locally administered rIL-2 combined with CDDP may be available as a therapy for head and neck cancers.     

Perilymphatic injections of recombinant interleukin-2 (rIL-2) partially correct the immunologic defects in patients with advanced head and neck squamous cell carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) are severely immunocompromised. In virtually all such patients who have been studied, reduced numbers of circulating CD3+ T-cell-receptor (TCR)alpha/beta+ T lymphocytes, a reduction of natural killer (NK) activity, and a poor induction of lymphokine-activated killer (LAK) cell activity (following in vitro treatment with recombinant interleukin-2 [rIL-2]) have been detected. Recently, however, it has been demonstrated that perilymphatic injections of low doses of rIL-2 may induce a local reduction of tumor masses in these patients. The present study, a cooperative pilot effort on the clinical effects of this route of administration, showed an activation of the lytic machinery in lymphocytes belonging to the T-cell lineage, as well as a potentiation of NK activity in the peripheral blood. These findings demonstrated that the severe immunodeficiency of HNSCC patients may be at least partially corrected by in vivo administration of rIL-2.     

Immunofluorescent assessment of tumour infiltrating cells in laryngeal carcinoma. Application of monoclonal antibodies

In order to gain some insight into host cell accumulations within primary tumour, frozen sections from surgical specimens of laryngeal carcinoma were subjected to indirect immunofluorescence using a panel of monoclonal antibodies against various human lymphocyte subsets as well as macrophages. In addition, polyclonal antibodies against Ig were used in order to trace B cells. Numerous host cell infiltrates seen at the tumour periphery were composed of T4 (helper) lymphocytes and macrophages. Lymphocytes of OKT8 (suppressor/cytotoxic) and Leu-7 (NK cells) series were intermingled with tumour cells in the case of scanty infiltrates. Infiltrating cells were also linked to the presence of metastases in regional lymph nodes. OKT4-positive abundant infiltrates were usually accompanied by uninvolved nodes, while scanty ones with OKT8 specificity were relatively frequently seen in the patients with evidence of nodal metastases. These differences were not statistically significant, however, B cells as well as plasma cells were infrequently observed and were encountered both in tumour samples with intensive cellular infiltrates as well as in those with scanty ones.     

Lymphocyte subsets of maxillary mucosa in chronic inflammation

Subsets of infiltrating lymphocytes within maxillary sinus mucosae of patients with chronic sinusitis were investigated by immunoperoxidase staining of frozen sections with the use of monoclonal antibodies. The most commonly observed infiltrating cell type was suppressor/cytotoxic T cells (CD8+) and smaller subpopulations of lymphocytes were helper/inducer T cells (CD4+) and B cells (CD20+). Variable numbers of HLA-DR+ cells were commonly observed in the lamina propria. The fibrous type of chronic sinusitis was found to have more suppressor/cytotoxic T cells (CD8+) and lower CD4/CD8 ratio than the other histopathological types.     

Local administration trials of interleukin-2 for head and neck cancer.

Recombinant interleukin-2 (rIL-2) has been administered locally in 20 patients with head and neck cancer. Two complete responses in lower lip cancer and 1 partial response in lingual cancer have been obtained. Immunohistological study reveals that tumor infiltrating lymphocytes (TILs), including activated T lymphocytes and natural killer cells, are increased after rIL-2 use. Intraarterial chemotherapy, performed subsequently to the local use of rIL-2 results in a dramatic decrease in tumor size within a short time, and a high frequency of CR cases is observed. Local use of rIL-2 is beneficial for treatment of head and neck cancers, and induction immunochemotherapy combining locally used rIL-2 and arterially infused anticancer drugs plays an important role in a multidisciplinary treatment for these cancers.     

Local infiltration of T-lymphocyte subsets as a prognostic indicator in patients with nasopharyngeal carcinoma

We studied tumor-host interactions in 47 patients with NPC. The local infiltration of T-lymphocyte subsets was investigated by an immunoperoxidase technique using monoclonal antibodies. Biopsy specimens of patients without cervical metastasis showed more T-lymphocyte (T11) infiltration. The amount of Leu-3a (helper/inducer) and T8 (cytotoxic/suppressor) cell infiltration did not correlate with the age, sex, clinical stage, and peripheral blood T4 and T8 cells of the patients. A higher incidence of Leu-3a cell infiltration was found in patients with high serum IgA antibody titers to EBV VCA. A trend of better prognosis was revealed in those cases with no or slight stromal T8 cell infiltration. A local immune response was found to exist which may prevent the spread of NPC to the cervical nodes, but this needs further study to evaluate the local infiltration of T-lymphocyte subsets as a prognostic indicator.     

Effect of lymphokine-activated killer cells on head and neck tumours in nude mouse model

The present study was designed to investigate the in vivo effect of local application of lymphokine-activated killer (LAK) cells on the growth of tumours implanted under the renal capsule in nude mice, and especially to test whether large granular lymphocytes (LGL), regarded as natural killer (NK) cells, are the main precursor of LAK cells in vivo. Our results showed that the local application of LAK cells inhibited the growth of tumours in the head and neck region. The growth of tumours implanted under the renal capsule was inhibited by local application of 1 x 10(7) recombinant interleukin-2 (rIL-2) activated non-adherent lymphocytes, but the inhibitory effect was almost the same as produced by 3 x 10(6) rIL-2-activated LGL application. The findings indicate that the rIL-2-activated LGL are the main effectors in inhibiting tumour growth. In addition, rIL-2-activated non-adherent lymphocytes as well as LGL significantly prolonged the number of days of 50% survival and mean survival time of nude mice, in which HLaC78 cells, from a laryngeal tumour cell line, were injected into the subrenal capsule space with effector cells at various effector: target (E:T) ratios. The results indicate that the application of LAK cells may be useful in the treatment of patients with head and neck tumours.     

An immunohistologic study of the distribution and status of activation of head and neck tumor infiltrating leukocytes

We examined tumor infiltrating leukocytes (TIL) in frozen sections of 28 biopsies from squamous cell carcinomas of the head and neck (SCCHN). In so doing, we used monoclonal antibodies (MoAb) directed against various leukocyte antigens. As defined by HLe-1+ cells, leukocyte infiltration was present in all biopsies. The amount of HLe-1+ cells was more often greater in stage III than in stage IV lesions. Most of the TIL were identified as CD5+ T-lymphocytes. In contrast, CD19+ B-cells were sparse in most biopsies. CD14+ monocytes/macrophages were found in only a few specimens. The relative proportion of CD4+ T-helper cells was higher than or at least equal to CD8+ suppressor/cytotoxic cells in all samples tested. Interleukin-2 (IL-2) receptor+ lymphocytes were evident in 13 of 22 biopsies stained for CD25 reactivity, and were more often observed in stage III than in stage IV tumors. All biopsies from recurrent tumors had no detectable IL-2 receptor+ cells. Our findings provide evidence for a positive correlation between a greater amount of TIL in earlier stages of SCCHN. The presence of IL-2+ lymphocytes suggests that SCCHN may be capable of activating resting lymphocytes for further IL-2-induced proliferation.     

An immunohistologic study of the distribution and status of activation of head and neck tumor infiltrating leukocytes

Summary We examined tumor infiltrating leukocytes (TIL) in frozen sections of 28 biopsies from squamous cell carcinomas of the head and neck (SCCHN). In so doing, we used monoclonal antibodies (MoAb) directed against various leukocyte antigens. As defined by HLe-1+ cells, leukocyte infiltration was present in all biopsies. The amount of HLe-1+ cells was more often greater in stage III than in stage IV lesions. Most of the TIL were identified as CD5+ T-lymphocytes. In contrast, CD19+ B-cells were sparse in most biopsies. CD14+ monocytes/ macrophages were found in only a few specimens. The relative proportion of CD4+ T-helper cells was higher than or at least equal to CD8+ suppressor/ cytotoxic cells in all samples tested. Interleukin-2 (IL-2) receptor+ lymphocytes were evident in 13 of 22 biopsies stained for CD25 reactivity, and were more often observed in stage III than in stage IV tumors. All biopsies from recurrent tumors had no detectable IL-2 receptor+ cells.Our findings provide evidence for a positive correlation between a greater amount of TIL in earlier stages of SCCHN. The presence of IL-2+ lymphocytes suggests that SCCHN may be capable of activating resting lymphocytes for further IL-2¡nduced proliferation.     

Differences of sensitivity to autologous cytotoxic lymphocytes between primary tumor and its cervical lymph node metastases

Antigenic differences between primary tumors and their cervical lymph node metastases of 12 patients with head and neck cancers were examined by measuring their sensitivity to cytotoxic lymphocytes (CL). Cytotoxicity was induced by autologous mixed lymphocyte (CL). Cytotoxicity was induced by autologous mixed lymphocyte tumor cell culture (MLTC), and further cultivation with recombinant interleukin-2 (rIL-2). The effector cells which were used in this study consisted of OKT3+8+ and OKT3+4+ subpopulations. Their cytotoxic nature was different from lymphokine activated killer cell (LAK cell) activity. Cytotoxicity of CLs stimulated by autologous primary tumor cells (CLP) was observed in 7 out of 12 patients (58.3%). In contrast, cytotoxicity of CLs stimulated by metastatic tumor cells (CLM) was observed in 4 out of 12 patients (33.3%). In the cases in which both CLP and CLM were successfully induced, cross-reactivity tests and cold target inhibition tests were performed. These results suggested that a reduction in immunogenicity had occurred at the metastatic site, and sensitivity against autologous CL was different between primary and metastatic tumor cells.     

Functional and phenotypic analysis of lymphocytes in head and neck cancer.

We compared the phenotype and antitumor effector function of lymphocytes obtained from tumor tissues, lymph nodes, and the peripheral blood of patients with head and neck cancer. Freshly isolated tumor-infiltrating lymphocytes were deficient in CD4+ T cells in comparison with lymph node lymphocytes (LNL) and peripheral blood lymphocytes. A significantly higher CD4/CD8 ratio observed in LNL vs tumor-infiltrating lymphocytes and peripheral blood lymphocytes was attributable to both a significant enrichment in CD4+ T cells as well as a decrease in CD8+ T cells. The percentage of natural killer cells (CD3-CD56+) was uniformly low in both tumor-infiltrating lymphocytes and LNL. In patients with cervical metastases, LNL contained an increased proportion of CD16+ cells. Tumor-involved lymph nodes were not enriched in the CD8+C11b+ subset of T "suppressor" lymphocytes compared with uninvolved lymph nodes. Also, tumor-involved lymph nodes had significantly fewer CD4+ T cells than did uninvolved lymph nodes. In comparison with peripheral blood lymphocytes, freshly isolated tumor-infiltrating lymphocytes and LNL were depleted of cytotoxic effector cells, as indicated by low or absent cytotoxic activity against tumor cell targets. The ability to generate lymphokine-activated killer cells was significantly reduced in LNL in comparison with peripheral blood lymphocytes. In patients with head and neck cancer, depressed local and regional antitumor responses are associated with a deficiency of functional cytotoxic effector cells rather than an increase in suppressor T lymphocytes.     

Phenotyping of mononuclear cells from tonsils and corresponding biopsies using a cytofluorimeter. A comparative study

A series of B, T, natural killer cell (NK) and monocyte-specific monoclonal antibodies was used to determine the distribution of lymphocyte subpopulations in cell suspensions obtained from whole tonsils as well as their corresponding biopsies. The majority of the mononuclear cells stained with OKB-7 and anti-Dr. A consistent lower percentage of the B lymphocytes stained with OKB-2. The T lymphocytes were composed of a majority of OKT4+ lymphocytes. Only a minority of the lymphocytes stained with Leu-7, whereas Leu-llb+ lymphocytes were virtually absent. A significant proportion of the mononuclear cells stained with OKM-1. There was a highly significant correlation between the distribution of the cells stained with the different monoclonal antibodies obtained from the whole tonsil and the corresponding biopsy (r2 = 0.95). This study shows that biopsies offer a reliable source to study the lymphocyte subsets in tonsils and that cytofluorimetry can be applied to study the lymphocyte distribution in this organ.     

Identification of lymphocyte subsets and natural killer cells in head and neck cancers. An immunohistological study using monoclonal antibodies

We investigated host-immune defenses against head and neck cancer cells by using various monoclonal antibodies with an immunoperoxidase technique to define lymphocyte subsets and natural killer (NK) cells. By so doing, we were able to identify lymphocyte subsets and NK cells in various head and neck cancers. We found that the majority of these cells infiltrate in or around nests of cancer cells and are stained with Leu-1 antibody. They include both Leu-2a and Leu-3a positive cells, which show equally intense levels of infiltration. Leu-7 positive cells were only scattered in the peripheral portion of the cancer nests in some cases. We also found a tendency for T-cells to infiltrate more intensely in poorly differentiated squamous cell carcinomas (SCC) than in moderately or well-differentiated SCC. Similarly, T-cells were more prevalent in maxillary carcinomas than in laryngeal carcinomas. These findings suggest the presence of a host-immune defense mechanism against cancer cells in patients with head and neck cancers.     

Identification of lymphocyte subsets and natural killer cells in head and neck cancers

Summary We investigated host-immune defenses against head and neck cancer cells by using various monoclonal antibodies with an immunoperoxidase technique to define lymphocyte subsets and natural killer (NK) cells. By so doing, we were able to identify lymphocyte subsets and NK cells in various head and neck cancers. We found that the majority of these cells infiltrate in or around nests of cancer cells and are stained with Leu-1 antibody. They include both Leu-2a and Leu-3a positive cells, which show equally intense levels of infiltration. Leu-7 positive cells were only scattered in the peripheral portion of the cancer nests in some cases. We also found a tendency for T-cells to infiltrate more intensely in poorly differentiated squamous cell carcinomas (SCC) than in moderately or well-differentiated SCC. Similarly, T-cells were more prevalent in maxillary carcinomas than in laryngeal carcinomas. These findings suggest the presence of a host-immune defense mechanism against cancer cells in patients with head and neck cancers.     

Electron microscopic observation of killer cells induced by mixed culture of lymphocytes with autologous cancer cells and further culture with recombinant interleukin-2.

Peripheral blood lymphocytes obtained from 2 patients with hypopharyngeal cancer were cultured with mitomycin C treated autologous tumor cells (autologous MLTC) for 10 days and further cultured with recombinant interleukin 2 (rIL-2). In one case 10-day MLTC induced increase of CD25-positive lymphocyte count, indicating that IL-2 receptors were expressed dominantly by the autologous tumor stimulation, and further culture with rIL-2 differentiated killing activity against autologous tumor cells. In the other case, however, MLTC alone induced killing activity against autologous tumor cells, indicating that the tumor cells from this patient might possess stimulatory activity sufficient to induce mature killer cells. Electron microscopic observation of the morphological features of lymphocytes cultured for 10 days revealed mostly small lymphocytes with low incidence of cytoplasmic granules. Further culture with rIL-2, however, induced slightly larger lymphocytes with well-developed microvilli, and cytoplasmic granules were found in many of the cells. Lymphokine activated killer (LAK) cells induced by culture of lymphocytes with rIL-2 alone were much larger and had long microvilli and abundant cytoplasmic granules, and were apparently morphologically different from the killer cells initiated by MLTC. The small lymphocytes induced by autologous MLTC alone might be autologous tumor specific cytotoxic T lymphocytes (CTL) and/or CTL precursors. Further culture with rIL-2 induced maturation of the CTL. However, the nature of the cytoplasmic granules remains obscure.     

Expression of intercellular adhesion molecule (ICAM)-1 in adenoid cystic carcinoma of the head and neck

OBJECTIVES/HYPOTHESIS: Adenoid cystic carcinoma of the head and neck (ACCHN) is characterized by late recurrence and frequent distant metastasis. Tumor attack by cytotoxic T lymphocytes and macrophages is mediated by the interaction of leukocyte function-associated antigen (LFA)-1 on lymphocytes with intercellular adhesion molecule (ICAM)-1 on the tumor surface. Thus, the reduced expression of ICAM-1 on tumor cells could contribute to their escape from host immune surveillance. To investigate the relationship between the clinical features of ACCHN and host immune surveillance, the expression of ICAM-1 and infiltration of T/natural killer (NK) cells and macrophages were immunohistochemically examined. STUDY DESIGN: Retrospective analysis of immunohistochemical tumor characteristics and clinical outcome. METHODS: Immunohistochemical study of ICAM-1, T/NK cells, and macrophages was performed on paraffin sections of 42 patients with ACCHN. The expression of T/NK cells and macrophages was represented by T-cell-restricted antigen (TIA)-1 and CD68 expression, respectively. The expression of these molecules and clinical features were analyzed. RESULTS: Of 42 ACCHN cases, 15, 9, and 15 patients were classified as ICAM-1 high, TIA-1 high, and CD68 high, respectively. The TIA-1 expression scores in ICAM-1-low patients were significantly lower than those in ICAM-1-high patients (1.3 +/- 3.7 vs. 8.3 +/- 12.7, P =.0031). The CD68 expression scores in ICAM-1-low patients were also significantly lower than those in ICAM-1-high patients (9.6 +/- 9.6 vs. 21.1 +/- 17.6, P =.0047). Moreover, ICAM-1-high patients had a significantly better disease-free survival rate (P =.043). CONCLUSIONS: Reduced expression of ICAM-1 may promote immune evasion and metastasis, resulting in poor prognosis in ACCHN.     

Sensitivity test to chemotherapeutic agents and cytotoxicic test against immuno-effecter cells of established malignant fibrous histiocytoma cell line]

A cell line was established from a patient with malignant fibrous histiocytoma, which had originated in the maxillary sinus. Using this cell line, sensitivity to chemotherapeutic agents and cytotoxicity against various immunoeffecter cells were tested. Results: This MFH cell line was sensitive in some degree to adriamycin, 5-fluorouracil, cisplatin, peplomycin, and methotrexate at high doses, but insensitive to mitomycin-C, vincristine and cyclophosphamide. Furthermore, this cell line showed no sensitivity to cytolytic cytokines, such as tumor necrosis factor-alpha and interferon-gamma. Lymphokine activated killer (LAK) cells and lymphokine activated tumor infiltrating lymphocytes (LA-TIL), induced by culture of peripheral blood lymphocytes and TIL respectively with rIL-2 showed high NK and LK activities and remarkable anti-autologous tumor ability.     

Lymphocyte subsets in normal airway mucosa of the human nose

The distribution and number of lymphocytes, monocytes/macrophages, and cells expressing HLA-DR antigen were studied in frozen biopsy sections of nasal mucosa from 40 healthy adults, using monoclonal antibody avidin-biotin immunoperoxidase techniques. The lymphocyte to monocyte/macrophage ratio was estimated to be 10:1; the T cell to B cell ratio was 3:1; and the T helper/inducer cell to T suppressor/cytotoxic cell ratio averaged 2.5:1. Regional differences were observed with a relatively increased number of T suppressor/cytotoxic cells around submucosal glands, and a relatively large number of B cells in lymphocyte aggregates in the lamina propria. The HLA-DR antigen was expressed in epithelial cells, suggesting involvement of surface epithelium of human airway in local immune responses.     

Administration of a prostaglandin synthetase inhibitor associated with an increased immune cell infiltrate in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck produces a prostaglandin, PGE2, a potent inhibitor of cellular immune responses. We tested the effects of prostaglandin synthetase inhibition on the infiltration of squamous cell carcinoma of the head and neck with host lymphocytes. Tumor tissue samples were obtained from six patients (age range, 51 to 72 years) who presented with squamous cell carcinoma of the head and neck before and after 14 days of treatment with indomethacin (50 mg administered orally three times a day). Tumor-infiltrating immune cells were assayed in frozen tissue samples by indirect immunofluorescence. An eightfold increase in CD2+ lymphocytes compared with pretreatment tissue was observed. The number of CD4 and CD8 lymphocytes increased similarly. CD57 lymphocytes increased 15-fold and CD11b cells increased 11-fold. No infiltrating B-cell populations were evident. Double-labeling studies revealed that the mononuclear cells were located outside blood vessel walls, indicating that they had infiltrated the tumor parenchyma. Our findings demonstrate that the administration of indomethacin is associated with the increased immune cell infiltration of squamous cell carcinoma of the head and neck. This suggests that inhibition of PGE2 synthesis as it occurs in the tumor and or systemically may contribute to the homing of mononuclear cells to the tumor. These data suggest a mechanism to account for the clinical response to indomethacin previously reported in squamous cell carcinoma.     

An immunohistochemical study of the endolymphatic sac in patients with acoustic tumors

Intraosseous endolymphatic sacs obtained from patients with acoustic neuromas who had undergone total labyrinthectomy during tumor removal were examined for the presence of T helper/inducer and T suppressor/cytotoxic lymphocytes, B lymphocytes, plasma cells, and macrophages. Immunoperoxidase staining of cryostat sections revealed the presence of T helper/inducer lymphocytes, T suppressor/cytotoxic lymphocytes, and macrophages. The number of B lymphocytes and plasma cells was much smaller than the number of T lymphocytes. The number of T suppressor/cytotoxic lymphocytes was higher than the number of T helper/inducer lymphocytes. This study supports the notion of local immune responsiveness in the human inner ear. This is the first immunohistochemical study to analyze lymphocyte subpopulations; specifically, to provide insight into T-cell function in the endolymphatic sac.