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.     

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.     

Natural killer cell response to regional lymph node metastases

A determination of natural killer cell activity was performed in 67 individuals with advanced head and neck cancer. The mean activity of 28 patients clinically staged T3 NO or T4 NO was 81 +/- 11 lytic units (LU), significantly higher than 39 patients with palpable lymph node metastases (54 +/- 5 LU). Assessing patients by extent of nodal disease revealed that activity actually increased, though not significantly, with progressive N-staging. A major determinate of increased natural killer cell cytotoxicity in patients with lymph node metastases was extranodal cancer within the neck. The mean activity of nine patients whose tumor was fixed to underlying structures or adherent to skin was 87 +/- 15 LU, significantly higher than the 45 +/- 4 LU mean value of the remaining patients with clinically determined regional nodal disease. The potential clinical implications of these findings are discussed.     

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.     

喉癌引流淋巴结中巨噬细胞对自体肿瘤杀伤活性检测

本实验采作细胞培养技术,从喉癌患者颈廓清淋巴结中提取的巨噬细胞（Mphi）作效应细胞,喉癌患者自体癌细胞及K562细胞作靶细胞,分别混合培养（两组均加PHA作为刺激原）,结果表明：喉癌患者颈部肿大淋巴结Mphi对自体癌细胞（35例）及K562细胞（30例）均有杀伤能力;此外,我们还对比了未转移淋巴结（N0组,18例）与转移淋巴结（N+组,17例）MPhi杀伤能力,结果N0组高于N+组,差异非常显著（P＜0．01）。结果提示,喉癌患者颈部引流淋巴结中尚存在着免疫活性细胞,并具有肮肿瘤免疫潜能,该实验为临床合理实施颈廓清术及将活化的Mphi应用于肿瘤免疫治疗提供理论依据。     

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.     

头颈部肿瘤患者自然杀伤细胞活性及计数分析

本文对25例头颈部肿瘤患者的外周血NK细胞杀伤活性及NK细胞计数进行测定，其中有15例患者手术治疗切除肿瘤，于术后三周复查NK活性及NK计数，并与正常人组对照。实验结果显示：头颈部肿瘤患者NK活性及NK计数均低于正常人，两组比较均有极显著性差异（P＜0．01）；术后患者的NK活性比术前有明显提高，配对t检验P＜0．01，NK计数比术前也有所提高，配对t检验P＜0．05，有显著性差异；NK活性的水平与肿瘤的分期有相关性，晚期（T4期）癌症患者NK活性明显低于早期（T1、T2）患者，可以看出NK活性随着肿瘤的发展而降低，肿瘤对宿主有免疫抑制作用；NK活性与NK数量之间无显著的相关性，二者之间的相关系数r＝0．344。     

Production of lymphokine-activated lymphocytes. Lysis of human head and neck squamous cell carcinoma cell lines

Lymphokine-activated killer cells are thought to be important mediators of host tumor defense. In the present study, the cytotoxic potential of lymphokine-activated lymphocytes against different head and neck squamous cell carcinoma cell lines was investigated. Lymphokine-activated killer cells were derived from peripheral blood lymphocytes. Effector peripheral blood lymphocyte cell suspensions were incubated in the presence or absence of recombinant interleukin-2. Cytotoxicity of incubated cells or fresh peripheral blood lymphocytes was determined in a 3-hour chromium 51 release assay. Target cell lines included K562 (a natural killer-sensitive target) and the following head and neck squamous cell carcinoma cell lines: Cal 27, UMSCC-1, UMSCC-8, UMSCC-16, UMSCC-19, and UMSCC-22a. Fresh peripheral blood lymphocytes and peripheral blood lymphocytes cultured in the absence of added interleukin-2 demonstrated minimal cytotoxic effects against the squamous cell carcinoma targets. In contrast, these fresh and incubated lymphocytes showed significant cytotoxic effects against K562. Cells preincubated in the presence of interleukin-2 demonstrated a statistically significant increase in cytotoxic effects against K562 and all squamous cell carcinoma targets. These investigations support the possible role of lymphokine-activated killer cells in host defense against squamous cell carcinoma. In vitro natural killer cell activity against head and neck squamous cell carcinoma cell lines is low; however, significant lymphokine-activated killer cell cytotoxicity is present.     

Immunomodulatory activity in regional lymph nodes

This study is, to our knowledge, the first attempt to evaluate cellular immune mechanisms in regional lymph nodes of patients with head and neck cancer. Twenty lymph nodes from eight patients with stage III-IV squamous cell carcinoma were evaluated using an in vitro culture system. The T-cell mitogenic (concanavalin A) response of patients' peripheral blood mononuclear cells was modulated by the addition of cells from regional lymph nodes removed at neck dissection. Modulatory activity showing augmentation was significantly correlated with the size of the primary tumor and histopathologic grade of the tumor. Modulatory activity did not correlate with the histologic pattern of lymph node reactivity. Although these relationships suggest that regional immunity may be important in tumor-host interactions, further study is necessary to establish their biologic and prognostic importance.     

Combination nonviral cytokine gene therapy for head and neck cancer

OBJECTIVE: To establish the feasibility and efficacy of combination nonviral murine interferon-alpha (mIFN-alpha)and murine interleukin-2 (mIL-2) or murine interleukin-12 (mIL-12) gene therapy for head and neck squamous cell carcinoma in a murine model. STUDY DESIGN: Randomized controlled studies in a murine head and neck cancer model were performed to assess antitumor responses, secondary cytokine expression, and both natural killer (NK) cell and cytolytic T-cell (CTL) activity. METHODS: Tumors were established in the floor of mouth in C3H/HeJ immunocompetent mice. Established tumors were directly injected with polymer-formulated murine interferon-alpha (mIFN-alpha), lipid-formulated mIL-2, and polymer-formulated mIL-12 alone or in combination. Primary and secondary cytokine expression,NK cell activity, and CTL activity were assayed. RESULTS: The use of mIFN-alpha gene therapy in combination with either mIL-2 or mIL-12 resulted in significant antitumor effects as compared with each of the single cytokine and control treatment groups (P = .002).Increased levels of NK cell activity and tumor specific CD8+ cytotoxic T-lymphocyte activity were found in the combination mIFN-alpha and mIL-2 or mIL-12 groups. Augmented immune responses correlated with clinical antitumor effects. CONCLUSIONS: The present study demonstrates that mIL-2 or mIL-12 augments tumor inhibition from mIFN-alpha and increases activation of NK and CD8+ T cells. These data support further investigation of polymer and lipid mediated delivery of cytokine genes for head and neck cancer.     

Multimodality therapy and distant metastases. The impact of natural killer cell activity

One hundred eighty-two previously untreated head and neck cancer patients were stratified by pretreatment-quantitated natural killer (NK) cell activity (less than 60 lytic units [LU] vs greater than or equal to 60 LU) and followed up longitudinally for the development of distant metastases (DMs). Patients with NK activity of less than 60 LU (n = 99) developed DMs at a higher rate than the remaining group. Further stratification of patients on the bases of both regional nodal disease and treatment demonstrated that the risk of DMs predominantly involved one group, ie, patients with histopathologically documented nodal metastases, NK activity of less than 60 LU, and prior treatment with combined surgery and radiation therapy (12[46%] of 26 patients). If one of these three factors was absent, the risk of DMs was not greater than 12%, regardless of the factor. Head and neck cancer patients should be stratified by pretreatment natural immune status to determine the impact of therapy on disease progression.     

The effect of surgery on natural killer cell activity in head and neck cancer patients: in vitro reversal of a postoperatively suppressed immunosurveillance system

Curative surgery diminished natural killer (NK) cell activity in 17 patients with previously untreated squamous cell carcinoma of the head and neck (37% +/- 17%, preoperatively vs. 21% +/- 11%, postoperatively; p less than 0.001). This operatively induced suppression was dependent on the presence of a nylon wool adherent cell population. With the removal of this surgically generated suppressor population from the in vitro assay, postoperative suppression of natural killer activity was significantly diminished (21% +/- 13%, vs. 30% +/- 23%, p less than 0.01). The capability of fully restoring postoperatively suppressed NK cell activity was subsequently demonstrated by the synergistic effect of removing a nylon wool adherent suppressor population and stimulating NK cells with a naturally occurring immunopotentiator, tuftsin (from 21% +/- 13% to 41% +/- 23%; p less than 0.0001). In utilizing biological response modifiers in the perioperative period in the cancer patient, the interaction of these agents and the surgically generated suppressor cell population needs to be considered.     

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.     

Histopathological features of occult metastasis detected by sentinel lymph node biopsy in oral and oropharyngeal squamous cell carcinoma.

OBJECTIVES: Sentinel lymph node biopsy has been introduced for head and neck cancer with promising results. Research in breast cancer has revealed different histopathological features of occult lymph node metastasis with possibly different clinical and prognostic implications. The aim of the study was to evaluate the histopathological features of occult metastasis detected by sentinel lymph node in oral and oropharyngeal squamous cell carcinoma. STUDY DESIGN: Prospective. METHODS: According to Hermanek (5), occult metastasis was differentiated into isolated tumor cells and infiltration of lymph node parenchyma smaller than 2 mm in diameter (micrometastasis) and larger than 2 mm in diameter (metastasis). RESULTS: Occult metastases were found in 6 of 19 (32%) sentinel lymph nodes. Three patients showed micrometastasis with a mean size of 1.4 mm (range, 1.2-1.5 mm), the first with three separate micrometastases within the same sentinel lymph node, the second with an additional cluster of isolated tumor cells within the same sentinel lymph node, and the third with an additional micrometastasis in one lymph node of the elective neck dissection. Two patients had macrometastasis (3.4 and 8 mm), both with multiple metastases in the elective neck dissection. One patient had two clusters of isolated tumor cells in the sentinel lymph node and an additional cluster of isolated tumor cells in one lymph node of the elective neck dissection. CONCLUSIONS: Occult metastasis can be subdivided histopathologically in isolated tumor cells, micrometastasis, and macrometastasis. We present the first study describing a great variety of these subtypes in sentinel lymph nodes from head and neck squamous cell carcinoma. Because the independent prognostic factor and clinical relevance of these subtypes is still unclear, we emphasize the importance of reporting these findings uniformly and according to well-established criteria.     

Cytotoxic T lymphocytes in peripheral blood and regional lymph nodes in laryngeal cancer patients

Cytotoxic T cells is an unique lymphocyte subpopulation able to recognize in specific manner and kill tumor cells. Therefore they constitute an important cells engaged in anti-tumor defense. The aim of the study was to determinate cytotoxic T cells frequency in peripheral blood and among lymphocytes isolated from regional lymph nodes. The study group consisted of twenty patients diagnosed with laryngeal cancer subjected surgical treatement. Cytotoxic T cells were estimated using three color flow cytometry based on CD3(+)CD8(+)CD28(-) GranzymeB(+) phenotype. Additionally TCR zeta chain expression and spontaneous apoptosis considered as a potential markers of immunosupressive effect exert by tumor were determined. In patients with laryngeal cancer significant increase of CD3(+)CD8(+)CD28(-) lymphocytes in peripheral blood in comparison to healthy control was observed. In lymph nodes the content of those cells was much lower, less than 10%, however in a group bearing metastases to regional lymph nodes higher than in a group without metastases. Cytotoxic T cells were also the main population subjected spontaneous apoptosis. The role and specificity of cytotoxic T cells in laryngeal cancer patients still remain to be elucidated, especially in respect to specificity of recognition tumor cell. Understanding details of this process may rise significant progress in an approach to diagnosis and therapy in laryngeal cancer patients.     

Induction of cytotoxic activity from regional lymph node lymphocytes in head and neck malignant tumor]

Cytotoxic activities of rIL-2-stimulated effector cells from peripheral blood mononuclear cells (PBMC-LAK), and those from the regional lymph node cells (LN-LAK) from patients with head and neck malignant tumor were examined by 4-hour 51Cr release assay. Cytotoxicity of LAK cells from involved lymph node (LN (+)-LAK) were significantly lower than those of PBMC-LAK. LAK cells from non-involved lymph node (LN (-)-LAK) had a significantly higher cytotoxicity against Daudi cell than PBMC-LAK. Cytotoxic activities against K562 cell and autologous tumor cells mediated by LN (-)-LAK were not significantly different from those by PBMC-LAK. However, autologous tumor cell lysis by LN (-)-LAK from lymph nodes showing follicular lymphoid hyperplasia (FLH) pattern was higher than that by PBMC-LAK. The effector cells against autologous tumor cells were characterized CD56+ cells and CD8+ cells (CD8+CD11b- cells) by phenotypic analysis and negative selection assay using immunomagnetic isolation technique.     

Neck management in patients undergoing postradiotherapy salvage laryngeal surgery for recurrent/persistent laryngeal cancer

OBJECTIVE: To determine a plan for the management of cervical lymph nodes in patients undergoing salvage laryngeal surgery (SLS) for recurrent/persistent laryngeal cancer after primary radiotherapy (RT). STUDY DESIGN:: Retrospective chart review. METHODS: Charts of 51 consecutive patients who had salvage total or supracricoid laryngectomy with or without neck dissection for recurrent/persistent laryngeal squamous cell carcinoma after primary RT from 1988 to 2005 in our institution were reviewed. No patients received concomitant or neo-adjuvant chemotherapy. Thirty-four patients underwent SLS along with unilateral or bilateral neck dissection, whereas 17 patients underwent the SLS without neck dissection. Reports of preRT and preSLS staging of the primary tumor and the neck, recorded using the TNM system, were reviewed. Reports of the final histopathologic examination for the excised laryngeal cancer and cervical lymph nodes were reviewed. RESULTS: Thirty-four patients underwent SLS with unilateral or bilateral neck dissection. The preRT staging of the primary tumor for those 34 patients showed that 32 (94%) were staged T-1 (14) and T-2 (18), whereas the preSLS staging of the primary tumor for those 34 patients showed that 29 (85%) were staged T-3 and T-4. The postSLS final histopathologic examination of the excised lymph nodes in those 34 patients demonstrated that 30 (88%) did not have any evidence of nodal metastasis. On comparing patients with and without nodal metastasis (on their postSLS final histopathology), we found that the preSLS neck staging, based on computed tomographic (CT) scanning of the neck, was significantly associated with the negative/positive postSLS status of nodal metastasis (P = .006). Of 29 patients staged preSLS as N-0, 28 (97%) patients did not have nodal metastasis on their postSLS final pathology (negative predictive value = 97%, confidence interval, 82.2-99.9). PreRT neck staging, preRT and preSLS staging of the primary tumor, along with laryngeal subsite involvement (supraglottis, glottis, subglottis) did not significantly correlate with the status of neck metastasis on final postSLS histopathology (P = .68, 0.78, 0.49, and 0.42, respectively). None of the 34 patients had any neck tumor recurrence in the postSLS follow-up period (median, 3 yr). In addition, all 17 patients who underwent SLS without neck dissection were staged N-0 both before RT as well as preSLS, and none developed neck disease in the postSLS follow-up period (median, 2.5 yr). CONCLUSION: Management of the neck in patients undergoing salvage total or supracricoid laryngectomy for laryngeal cancer recurrence/persistence after primary RT should be based on the preSLS CT staging of the neck. Patients staged N-0 preSLS are not likely to harbor occult nodal metastasis and therefore may not require elective neck dissection.     

喉癌和下咽癌颈淋巴结转移临床对比分析

目的：探讨喉癌、下咽癌患者颈淋巴结转移的特点和分布规律。方法：对全喉切除术同期及复发后第1次行颈淋巴结清扫的129例喉癌、下咽癌患者的临床资料进行回顾性对比分析，研究不同类型的喉癌、下咽癌患者颈淋巴结的转移情况。结果：声门上型喉癌、下咽癌患者易发生早期淋巴结转移；下咽癌患者的转移淋巴结融合率高，颈静脉下区出现阳性淋巴结的比率高；声门上型喉癌、下咽癌患者原发病灶分化差的比率相对偏高；同期与复发后行颈淋巴结清扫的患者原发病灶分期差异无显著性意义。结论：对T2期及以上的声门上型喉癌及下咽癌患者，尤其当细胞分化比较差时，即使颈淋巴结阳性体征不明显亦应积极考虑颈淋巴结清扫问题，对下咽癌患者行颈淋巴结清扫时应考虑彻底清扫颈静脉下区的淋巴结。