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.     

Natural killer cell lysis of head and neck cancer

This study analyzed the capacity of both fresh unseparated peripheral blood lymphocytes and enriched natural killer (NK) cells to lyse head and neck cancer cell lines. In a 6-hour chromium-release assay, only Leu-19+ NK cells mediated significant lysis. Furthermore, cell lines established from poorly differentiated cancers were more sensitive to lysis than were cell lines established from well-differentiated cancers. Cell lines from well-differentiated cancers also less readily inhibited K562 lysis in a cold-target inhibition assay, were not recognized by NK cells in a monolayer absorption assay (unlike poorly differentiated cancers), and failed to form conjugates with NK cells in a single-cell assay. These results indicated that deficient killing of a well-differentiated cancer cell vs a poorly differentiated cancer cell is partly a function of diminished NK cell recognition and tumor binding necessary to initiate lysis. As in previous studies regarding the prognostic implication of quantitated measures of NK cell activity within head and neck cancer patients, the results support the biologic relevance of the NK cell as a defense mechanism against metastatic disease, especially in patients with poorly differentiated, low major histocompatibility complex class I-expressing head and neck cancers.     

Lysis of squamous cell carcinoma via antibody-dependent cellular cytotoxicity

We have developed a system in which antibody-dependent cellular cytotoxicity (ADCC) can be consistently demonstrated against squamous cell carcinoma (SCC) of the head and neck. Serum samples from patients with pemphigus vulgaris (PV) provided antibodies for ADCC. The effector cells were lymphocytes from the peripheral blood of healthy human donors. The SCC cell lines served as targets. Lysis of SCC, as measured by 51Cr release, was significantly enhanced by the presence of serum from patients with PV, but not by healthy human serum. Serum alone produced no target cell killing. Nonepithelial cell lines were not affected by the presence of PV antibodies. The results demonstrate that SCC is susceptible to ADCC. Thus, tumor-specific antibodies may have a role in the treatment of cancer of the head and neck. This system can serve as a positive control for further testing of ADCC against SCC.     

Immunohistochemical identification of squamous carcinoma of the head and neck with monoclonal antibody SQM1

Frozen tissue sections of biopsies from head and neck squamous cancer lesions were examined for immunohistochemical staining with a recently developed monoclonal antibody, designated as SQM1 antibody and directed against the surface membrane of squamous carcinoma cells. SQM1 antibody stained selectively squamous carcinoma, while normal mucosa and cells of the stroma were non-reactive. Positive staining of tumor was found in 33/35 specimens obtained from several major sites of the head and neck area and was observed in primary manifestations and lymph node metastases as well as in recurrences. The most consistent reactivity was seen with carcinomas of the tongue. Well differentiated squamous carcinomas contained a higher proportion of SQM1 positive tumor cells than poorly differentiated carcinomas. We suggest that the SQM1 antibody may aid in the immunohistochemical identification of squamous carcinoma of the head and neck area.     

71例头颈部鳞状细胞癌的多原发癌临床资料分析

目的　了解头颈部鳞癌的多原发癌发病部位和治疗、生存状况。方法　对 71例头颈部鳞状细胞癌 (简称鳞癌 )的多原发癌临床资料做了回顾性分析。结果　发生在头颈部的重复癌 2 7例 ,发生在非头颈部的重复癌 4 2例 ,另有三重癌 2例。本组共有同时性重复癌 4例 ,其中 1例是同时性三重癌。余 6 7例均为异时性重复癌 ,其中 1例为异时性三重癌。 6 7例异时性重复癌中 ,先证癌与重复癌发生的间隔期为 8个月～ 12年不等。 70 %的先证口腔鳞癌发生头颈部重复癌 ;6 2 %的先证下咽癌和 79%的先证喉癌发生非头颈部位的重复癌。非头颈部位以食管和肺部发生的重复癌较多。在所有头颈部鳞癌发生重复癌的部位中 ,以食管重复癌为最多 ,占本组病例的 2 4 %。本组病例总体3年、5年生存率分别为 32 4 %和 2 2 5 % ;重复癌治疗组和未治组的 3年生存率有明显统计学差异 ,治疗组明显高于未治组。结论　头颈鳞癌的重复癌以食管癌最为多见。口腔癌容易发生头颈部重复癌 ,喉癌和下咽癌易发生非头颈部重复癌。细致随访和复查、早期明确诊断和积极有效的治疗 ,可以提高这类患者的生存率。     

71例头颈部鳞状细胞癌的多原发癌临床资料分析

目的 了解头颈部鳞癌的多原发癌发病部位和治疗、生存状况。方法 对71例头颈部鳞状细胞癌(简称鳞癌)的多原发癌临床资料做了回顾性分析。结果 发生在头颈部的重复癌27例，发生在非头颈部的重复癌42例，另有三重癌2例。本组共有同时性重复癌4例，其中1例是同时性三重癌。余67例均为异时性重复癌，其中1例为异时性三重癌。67例异时性重复癌中，先证癌与重复癌发生的间隔期为8个月～12年不等。70％的先证口腔鳞癌发生头颈部重复癌；62％的先证下咽癌和79％的先证喉癌发生非头颈部位的重复癌。非头颈部位以食管和肺部发生的重复癌较多。在所有头颈部鳞癌发生重复癌的部位中，以食管重复癌为最多，占本组病例的24％。本组病例总体3年、5年生存率分别为32．4％和22．5％；重复癌治疗组和未治组的3年生存率有明显统计学差异，治疗组明显高于未治组。结论 头颈鳞癌的重复癌以食管癌最为多见。口腔癌容易发生头颈部重复癌，喉癌和下咽癌易发生非头颈部重复癌。细致随访和复查、早期明确诊断和积极有效的治疗，可以提高这类患者的生存率。     

头颈部鳞癌颈淋巴结转移诸因素对预后的影响

对382例头颈部原发鳞癌病人颈清扫标本连续切片病理观察结果和临床资料进行回顾性分析，探讨淋巴结转移的各项临床和病理学因素与病人预后的关系。发现总体5年生存率为46.1％，口腔癌、口咽癌、下咽癌和喉癌的5年生存率分别为49．7％，39．7％，35．0％和60．3％。表明颈淋巴结的临床分期、触诊淋巴结大小、病理转移淋巴结情况、颈淋巴解剖分区受累数和最低受累平面与病人5年生存率有密切关系（均P＜0．01），而阳性淋巴的个数对病人预后无明显影响。提示针对影响预后的淋巴结因素应采取相应的治疗措施，以提高病人的5年生存率。     

Squamous cell carcinoma in the immunosuppressed patient: Fanconi's anemia.

The association of immunosuppression and head and neck cancer is supported by numerous reports demonstrating impaired cell-mediated immunity, depressed T-cell function, decreased lymphocyte responsiveness, and elevated circulating immune complexes. Fanconi's anemia (FA) is a rare autosomal recessive syndrome characterized by progressive pancytopenia, skeletal abnormalities, hyperpigmentation, and other congenital anomalies. Increased chromosomal instability and defective DNA repair have been uniform findings. Several reports suggest associated immune deficiencies. There is an increased frequency of leukemia, hepatocellular carcinoma, and squamous cell carcinoma (SCC), including six cases of head and neck SCC. We reported a young girl with FA who developed SCC of the tongue. Initial studies suggest low lymphocyte counts, but normal lymphocyte responsiveness. More precise characterization of the immune system defects in malignancy prone, genetically determined syndromes may provide clues for the diagnosis and treatment of patients with the more usual but more variable risk factors for SCC of the head and neck.     

Diagnostic sensitivity of 18fluorodeoxyglucose positron emission tomography for detecting synchronous multiple primary cancers in head and neck cancer patients

We assessed the sensitivity of positron emission tomography (PET) for detecting synchronous multiple primary cancers, particularly synchronous esophageal cancers in head and neck cancer patients. We retrospectively reviewed 230 head and neck cancer patients. All the patients routinely underwent the following examinations: urinalysis, occult blood, tumor marker detection [squamous cell carcinoma (SCC), cytokeratin fragment (CYFRA), and carcinoembryonic antigen (CEA)], esophagogastroduodenoscopy, colonoscopy (when CEA was high or occult blood was positive), abdominal ultrasonography, plain chest computed tomography (CT), and PET. Bronchoscopy was performed when CT revealed lung shadow of central region. Synchronous multiple primary cancers were detected in 42 (18.2%) patients. The diagnostic sensitivity of PET for synchronous primary cancers was as follows: esophagus, 7.6% (1/13); stomach, 25.0% (2/8); lung, 66.7% (4/6); head and neck, 75.0% (3/4); colon, 0% (0/1); kidney, 0% (0/1); and subcutaneous, 100% (1/1). The sensitivity of PET for detecting synchronous esophageal cancers is low because these are early-stage cancers (almost stage 0–I). Therefore, it is necessary to perform esophagogastroduodenoscopy for detecting synchronous esophageal cancers. PET is an important additional tool for detecting synchronous multiple primary cancers because the diagnostic sensitivity of PET in synchronous head and neck cancer and lung cancer is high. But PET has the limitation of sensitivity for synchronous multiple primary cancers because the diagnostic sensitivity of PET in synchronous esophageal cancer is very low.     

The in vivo biologic effect of interleukin 2 and interferon alfa on natural immunity in patients with head and neck cancer

Given the association of deficient natural immunity with the risk of metastatic disease, the ability to activate natural killer cell function may have a therapeutic significance. The effect of continuous infusion of interleukin 2 plus intramuscular interferon alfa on natural immune status was, therefore, analyzed in eight patients with head and neck cancer. Also evaluated was the effect of interleukin 2-interferon alfa therapy on lymphokine-activated killer cell activity as well as total lymphocyte count, percent of lymphocyte subsets, and levels of both circulating immune complexes and antibody classes. Both the percent and absolute number of natural killer cells (ie, CD56+ CD3- lymphocytes) within peripheral blood as well as natural killer cell activity against K562 targets increased significantly with treatment. The remaining immune parameters were not significantly altered. The demonstrated capacity to modulate natural immune function supports the potential use of interleukin 2-containing regimens as a preventive measure against metastatic disease in patients with head and neck cancer.     

Responses of killer cells in head and neck cancer patients

Summary The immunological functions of 60 patients with head and neck cancers were evaluated using in vitro natural killer cell (NK) activity, lymphokine activated killer cells (LAK) and OK-432 activated killer cells, as well as by in vivo purified protein derivative (PPD) and Su-PS skin tests. There were signifcant differences of the above three activities between stage I cancer patients and stage III and/or IV. The PPD skin test corresponded significantly to LAK activity, while Su-PS corresponded significantly to OK-432 activated killer cell activity. Responses to interleukin-2 and OK-432 of stage IV patients with head and neck cancers were different from those of stage IV with gastrointestinal cancer. These in vitro assays were very useful for the evaluation of immunological function in head and neck cancer patients, especially before any biological response modifying treatment.     

Report of a clinical trial in 12 patients with head and neck cancer treated intratumorally and peritumorally with multikine

BACKGROUND: There is cumulative evidence suggesting that cells of the immune system recognize and may participate in eradicating neoplastic cells. As a result, immune modulation, first with interleukin 2 and later with other cytokines, has been tried in the clinical setting as part of antitumor therapy. OBJECTIVE: To examine the effectiveness and toxicity of a combination of natural interleukins in patients with squamous cell head and neck cancer. METHODS: Twelve previously untreated patients with various head and neck cancers were treated by peritumoral injection of a combination of cytokines (Multikine), in addition to zinc sulfate, indomethacin, and a single dose of cyclophosphamide, which were administered systemically. Response was evaluated clinically and histopathologically. T-lymphocyte determinants were studied by fluorescence-activated cell sorter analysis (against controls). RESULTS: Two patients showed complete regression and another 2 showed partial regression. There were no serious adverse effects of treatment. Pathological study results showed tumor fragmentation and the appearance of multinucleated macrophages. Fluorescence-activated cell sorter analysis showed lymphocyte activation, reflected by an unusually high number of cytotoxic T-lymphocyte activation 4 cells and natural killer cells. CONCLUSION: Multikine warrants further investigation for inclusion in the pharmacotherapeutic armamentarium of head and neck cancer.     

DNA amplification for the in vitro detection of Candida albicans in head and neck squamous cell carcinomas

DNA was extracted from whole cells of Candida albicans and digested with HindIII restriction enzyme. After electrophoresis in a segment of the lane containing between 800 and 1200 base pairs (bp) of DNA fragments, a 1.1-kilobase (kb) fragment was found that hybridizes to biopsied tumor cells from head and neck squamous cell carcinomas (SCC). From the nucleotide sequence of the putative gene locus, primers were synthesized for use in a polymerase chain reaction (PCR) with DNA extracted from 18 SCC of the upper aerodigestive tract. After 30 cycles of amplification all tumors were found to contain sufficient amplified DNA to be detected in polyacrylamide or agarose gels. In contrast, template DNA from lymph nodes and malignant lymphomas failed to generate positive signals under these conditions. However, samples of DNA obtained from head and neck SCC cells in vitro, Candida glabrata, and Candida parapsilosis after PCR were found to contain homologous sequences. Application of this technique to head and neck SCC biopsies may help to identify quickly the presence of concurrent candidal species.     

Abrasive esophageal cytology for the oncological follow-up of patients with head and neck cancer.

OBJECTIVES: The occurrence of a second primary cancer in the esophagus in patients with head and neck squamous cell carcinoma is frequent and is associated with a poor prognosis. The aim of this study was to evaluate the yield of abrasive esophageal cytology as a means of screening for metachronous cancer of the upper aerodigestive tract. STUDY DESIGN: We retrospectively reviewed the results of abrasive esophageal cytology performed twice yearly for the screening of patients with prior head and neck cancer. METHODS: From 1987 to 1996, 320 patients treated for head and neck cancer underwent 1,673 abrasive cytology examinations of the esophagus during a mean follow-up period of 4 years. Cytological results were classified as negative, suspect, or positive for malignancy. RESULTS: Twenty-five patients without symptoms had one or more suspect or positive cytologic findings, leading to 29 endoscopic examinations. These revealed 20 premalignant or early malignant lesions of the esophagus (2 dysplasias, 18 squamous cell carcinomas), 2 glandular carcinomas, and 10 clinically unsuspected oral or pharyngeal carcinomas. In seven patients, positive cytological results were associated with clinically visible head and neck cancer. Of the 34 patients with suspect cytological results for malignancy, 10 had no evidence of tumor at endoscopy and 24 had no endoscopic examination because of refusal or because suspected cells were not found in additional examinations. Negative results on cytological examination were found for 254 patients throughout their follow-up, and none of them developed esophageal cancer during a mean follow-up period of 3 years. CONCLUSIONS: For patients with head and neck cancer, abrasive sponge cytology is useful for detecting esophageal cancer at an early stage. In addition, it may reveal unsuspected second primaries or recurrences in the head and neck region.     

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.     

Oncofetal antigen expression in head and neck carcinoma.

Oncofetal antigens (OFA) are proteins expressed during periods of embryonal/fetal development and on malignant cells. Previous investigations detected the presence of phase-specific 44- and 200-kd OFA, using a monoclonal antibody (MoAb 115). This study will determine if primary head and neck squamous cell carcinomas express the 44-kd OFA, and will assess the possible role of OFA in oncogenesis. Fifteen primary human head and neck squamous cell carcinomas (HNSCC) and six HNSCC cell lines were tested for OFA expression. Thirteen primary cancers and three cell lines demonstrated various degrees of OFA positivity. Oncofetal antigens are proposed to represent a proto-oncogene active during fetal development and malignant growth in head and neck squamous cell carcinomas.     

Interactions and biological mechanisms of action of molecular signal peptides. II. Interleukin 2 (IL-2)

Interleukin 2 (IL-2) is predominantly produced by T-helper cells (TH1) having the phenotype CD4+, and by subpopulations of thymocytes after antigenic or mitogenic stimulation. IL-2 causes an indefinite growth of T-cells, and its function depends on binding to IL-2 receptors (IL-2R alpha and IL-2R beta). Thus the immune response of T cells is controlled through the expression of the IL-2 receptors and the IL-2 binding. IL-2 receptors are expressed not only by T-cells but also by B-cells, NK cells, monocytes, thymocytes, thymic stroma cells, oligodendrocytes and endothelial cells. This explains the various functions of IL-2, such as increased immunoglobulin production, growth of certain B-cell subpopulations, macrophage-dependent cytotoxicity, growth and differentiation of oligodendrocytes and proliferation of lymphokine activated killer (LAK) cells. Abnormal production of IL-2 may lead to autoimmune diseases, immunodeficiencies and, under certain circumstances, to T-cell leukemia. With antibodies against the IL-2 receptors the binding of IL-2 may be blocked to avoid auto-aggressive destruction in autoimmune diseases. LAK cells increase the growth of NK cells and T-cell cytotoxicity against transformed cells. LAK cells, especially those from tumor infiltrating lymphocytes, in conjunction with IL-2 have already been used with promising initial results in the treatment of distant metastases. In the future LAK cell therapy with IL-2 may be adopted to prevent metastases and second primary tumors in high-risk patients with head and neck cancer.     

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.     

Influence of genetic variation in the major histocompatibility complex on head and neck cancer susceptibility

BACKGROUND: While cigarette smoking and alcohol consumption are risk factors for squamous cell carcinomas (SCC) of the head and neck, genetic factors are also significant. The gene of tumor necrosis factor (TNF) is located in the major histocompatibility complex class III and the cytokine has pleiotropic actions some of which are anticarcinogenic. As this gene complex is polymorphic with microsatellite markers identified it is a further candidate for head and neck cancer susceptibility. METHODS: We used a case-control approach to study the influence of polymorphism at the A-D markers on susceptibility in 113 controls, 265 laryngeal and 123 oral cavity/pharyngeal SCC cases. Genotypes were identified on polyacrylamide gels in an automated DNA sequencer after amplification with fluorescently-labeled primers. RESULTS: We found no differences in allele frequencies between controls and oral cavity/pharyngeal SCC cases but the frequency of B3 was greater in the laryngeal SCC cases than controls (p = 0.004, odds ratio 2.8). Homozygosity for B3 conferred an increased risk of laryngeal cancer compared with controls (p = 0.021, odds ratio 10.8). CONCLUSIONS: The data provide the first evidence that allelism at MHC class III microsatellite markers is associated with risk to laryngeal SCC.     

Diagnosis of recurrences of head and neck carcinoma with the tumor marker SSC-antigen

BACKGROUND: The follow-up of squamous cell carcinomas of the head and neck is often challenging. Due to tissue alteration and anatomic changes after primary treatment or submucosal tumor growth, recurrences are sometimes detected very late. The tumor marker SCC-Antigen (SCC-Ag) may provide additional information for early detection of such tumor recurrences. PATIENTS: Serum levels of SCC-Ag in 578 patients with primary squamous cell carcinomas of the head and neck were assayed by SCC-RIA and IMx-SCC before treatment and every 2-3 months during follow-up. During the observation period of 30-84 (mean 50) months, 179 recurrences were verified by histologic examination. RESULTS: Seventy-seven patients (43%) with tumor recurrence showed elevated serum levels of SCC-Ag (< 2.0 ng/ml). Fifty-eight (32%) of them exhibited elevated levels of SCC-Ag up to 11 months (mean 6.1 months) prior to histopathologic diagnosis. This mainly became evident in 48 (83%) patients whose SCC-Ag serum levels were elevated before treatment. CONCLUSION: Use of SCC-Antigen in head and neck tumors follow-up can provide early evidence of almost one third of all recurrences of squamous cell carcinomas of the head and neck. For clinical purposes, we recommend an initial analysis of the SCC-Ag serum level in every patient with primary squamous cell carcinoma of the head and neck. The SCC levels of all SCC positive patients should be closely monitored. Elevated SCC should be regarded as a potential early sign for recurrence and therefore indicates the need for intensified follow-up. Depending on the individual situation this should include ultrasonography, CT, MRI and especially frequent endoscopy in general anesthesia with multiple biopsies of suspicious regions.