The nude mouse xenograft system: a model for photodetection and photodynamic therapy in head and neck squamous cell carcinoma

Mechanisms for hematoporphyrin derivative (HPD) localization in malignant tissue and in photodynamic therapy (PDT) have not been established. The authors' experience with human cancer xenografts in nude mice as a tumor system for the experimental study of these mechanisms is described. Human mucosal head and neck squamous cell carcinoma was successfully transplanted to nude mice in 29 per cent of trials and serially passed through multiple generations in three tumor lines. Time required for progressive growth averaged 13.9 days, and 300 mm3 tumors were obtained in four weeks. Take rates per passage varied, in part because of infection, and exponential growth rates varied among the three lines. However, within a single line exponential growth rates were similar. Average growth constants for the period of exponential growth were 0.14, 0.13, and 0.09 for the three lines. Fluorescent microscopy of excised xenografts revealed HPD fluorescence principally in connective tissue cells. Minimal fluorescence was seen in malignant epithelial cells. Spectrophotometric measures of HPD uptake in homogenized tissue showed highest values in liver. Tumor HPD levels were higher than those for either skin or muscle. The authors conclude that this tumor system can be used effectively to study HPD and PDT in head and neck cancer.     

Ultrastructure of blebbing phenomenon and phagocytosis of blebs in laryngeal carcinoma

The epithelial-connective tissue junction of invasive squamous cell laryngeal carcinoma was examined electron microscopically. The pleomorphic cytoplasmic protrusions known as blebs were observed on the lateral and basal surfaces of malignant keratinocytes. These blebs were pinched off from the malignant epithelial cells and were then observed in the connective tissue or in the intercellular spaces. After the pinching-off process some blebs were seen to be closely surrounded by pseudopods of phagocytizing histiocytic cells in the lamina propria or by adjacent malignant keratinocytes in the intercellular spaces. Since blebs are believed to be intact parts of viable cells this engulfment can be interpreted as cytophagocytosis. During cytophagocytosis the zeiotic blebs exhibited varying degrees of enzymatic digestion. The process of blebbing was discussed in respect to cellular locomotion of malignant keratinocytes during tumor invasion. This phenomenon seems to occur only in the preliminary stages of malignant tumor growth in the larynx.     

Fluorescent microscopy of hematoporphyrin derivative in the nude mouse tumor model

Mechanisms for localization of hematoporphyrin derivative (HPD) within malignant tissue and for tumor necrosis after photodynamic therapy have not yet been established with certainty. We describe a modified freeze-drying technique to study these mechanisms. Normal tissues and squamous cell carcinoma xenografts were examined in nude mice after administration of HPD. Skin reveals marked fluorescence in connective tissue cells. No fluorescence is visible in surface epithelial cells or keratin. Liver shows diffuse fluorescence only in cells lining sinusoids. In tumor, malignant cells do not fluoresce and connective tissue cells fluoresce brilliantly. This technique provides a clear view of HPD fluorescence, frozen instantaneously in location and time. Fluorescence from connective tissue cells in skin and tumor suggests that localization and photodynamic action may be targeted in part at cells that critically support malignant epithelial cell growth as well as at malignant epithelial cells directly.     

Study of cell adhesion in cancer of the larynx

E48 and Ep-CAM are involved in epithelial cell adhesion in healthy and neoplastic tissues. E48 is associated with desmosomes (desmosome core) and Ep-CAM molecules are present mainly in the basolateral plasma membrane. These molecules are involved in classic cadherin-modulated cell-cell interactions. We used immunohistochemistry (avidin-biotin-peroxidase) to investigate the pattern of expression of both molecules in healthy tissue and squamous-cell carcinoma of the larynx and in metastatic and healthy lymph nodes. Specimens were obtained from 18 patients with squamous-cell carcinoma. In the healthy tissue, every specimen had E48 in the suprabasal layers (15 samples) and expression was intense in half of them. Ep-CAM was expressed in the basal layer of 12 of 15 cases, also with high intensity in a half of them. In squamous-cell carcinoma of the larynx, E48 had a homogeneous distribution while Ep-CAM was prevalent in the outer part of the tumor. Both cell adhesion molecules, E48 and Ep-CAM, are involved in the invasion process of larynx tumor.     

喉鳞状细胞癌组织体外原代培养的初步研究

目的 应用体外培养技术,对喉鳞状细胞癌组织进行体外培养,探讨喉鳞癌组织原代培养中的各种影响因素,为建立人喉鳞癌组织的细胞系提供实验基础。方法采用体外组织培养技术,对24例人喉鳞状细胞癌组织进行原代培养,观察原代培养中肿瘤细胞的生长与供体的年龄、肿瘤组织的分化程度及不同培养方法的关系,分析在人喉鳞癌细胞的培养中成纤维细胞、微生物污染的影响。结果 24例人喉鳞状细胞癌组织标本,年龄小于60岁组的细胞生长率为31.25％(5/16例),年龄大于60岁组为37.5％(3/8例);高分化组为100％(2/2例),中分化组为30.8％(4/13例),低分化组25％(2/8例);组织块培养法为43.75％(7/16例),酶消化法培养为10％(1/10例);倒置显微镜下观察,在培养的第5～7天,在贴壁组织块周围可见到有上皮样细胞爬出。全部标本中,成纤维细胞的过度生长和微生物的污染是阻碍人喉鳞癌细胞生长的重要因素。结论培养组织的细胞生长率与供体的年龄关系不大;肿瘤组织的分化程度较高者,细胞的生长率较高;与酶消化分离培养法相比,贴壁组织块培养法的细胞生长率较高;成纤维细胞及微生物的污染是阻碍人喉鳞癌细胞系建立的重要因素。     

Zur Ultrastruktur der Tumor-Stromagrenze des invasiven Larynxkarzinoms

Summary The ultrastructural morphology of the hemidesmosomes in malignant epithelial cells of invasive squamous cell carcinoma of the human larynx was studied by electron microscopy. In the more advanced carcinomas the hemidesmosomes were reduced in number or absent over extensive areas of the epithelialstromal junction. Hemidesmosome-containing areas of the basal plasmalemma were seen to invaginate and to appear to pinch off inwardly. The detached hemidesmosomes rounded up with their cytoplasmic attachment plaque lying on the inner circumference of the vesicular structures and were autophagocytized by the malignant keratinocyte. However the detachment of hemidesmosomes and their subsequent downward migration into the deeper layers of the connective tissue space was observed in other areas. These observations indicate that reduction of hemidesmosomes may result in a loss of epithelial-stromal adherence in invasive epithelial cancer of the larynx and may thus facilitate the tumor invasion of the underlying stroma.     

Ultrastructure of blebbing phenomenon and phagocytosis of blebs in laryngeal carcinoma

Summary The epithelial-connective tissue junction of invasive squamous cell laryngeal carcinoma was examined electron microscopically. The pleomorphic cytoplasmic protrusions known as blebs were observed on the lateral and basal surfaces of malignant keratinocytes. These blebs were pinched off from the malignant epithelial cells and were then observed in the connective tissue or in the intercellular spaces. After the pinching-off process some blebs were seen to be closely surrounded by pseudopods of phagocytizing histiocytic cells in the lamina propria or by adjacent malignant keratinocytes in the intercellular spaces. Since blebs are believed to be intact parts of viable cells this engulfment can be interpreted as cytophagocytosis. During cytophagocytosis the zeiotic blebs exhibited varying degrees of enzymatic digestion. The process of blebbing was discussed in respect to cellular locomotion of malignant keratinocytes during tumor invasion. This phenomenon seems to occur only in the preliminary stages of malignant tumor growth in the larynx.The technical assistance of Mrs. S. Csegezi, Mrs. L. Polasek, and Mr. G. Raffesberg is gratefully acknowledged.     

Potential use of cysteine endopeptidase inhibitors in radiodiagnostics of laryngeal and tongue neoplasms

The methods applied up to now do not allow to define the border between the tumor and healthy tissue especially in advanced cases. Microlaryngoscopy facilitates the evaluation of the infiltration superficially, but does not allow for estimation its deeper penetration. CT and MRI often do not answer the question concerning the tumor spread as well. That is why the monoclonal antibodies marked with isotopes directed against specific antibodies of different neoplasms are applied in diagnostic. In the neoplasm development the great role play endopeptidases. They degrade the intercellular matrix and basement membrane and by this way made possible the neoplasm invasion. The high level of the endopeptidase in patients with neoplasm is a bad prognosis. The disposition of endopeptidase in neoplasm tissue is characteristic and gives opportunity to localise the border of healthy tissue thanks their inhibitors application. The inhibitor of cysteine endopeptidase obtained from hen egg and marked by J 125 was used in our study. The reaction between the cysteine endopeptidase inhibitor and malignant cells received directly from patients with tongue and laryngeal carcinoma and cells cultivated in vitro from the carcinoma.     

Cytopathologic assessment of gloves and instruments after major head and neck surgery

PurposeTo investigate the potential for cancer cells to be transferred between anatomic sites via instruments and other materials.Materials and methodsPilot prospective study from April 2018–January 2019 at Rush University Medical Center. Glove and instrument washings were collected from 18 high-risk head and neck cancer resection cases (36 samples total). Each case maintained at least one of the following features in addition to a diagnosis of squamous cell carcinoma or sarcoma: palliative/salvage surgery, positive margins, extensive tumor burden, and/or extra capsular extension (ECE). Surgical gloves and four main instruments were placed through washings for blind cytological assessment (2 samples/case).Results18 patients undergoing surgical tumor resection for biopsy-proven squamous cell carcinoma with at least one of the aforementioned characteristics were included. 26.7% of cases had ECE, 40.0% had positive final margins and 46.7% had close final margins. Tumor locations included: oral cavity (10), neck (4), parotid gland (2), and skin (2). Malignant cells were isolated on glove washings in 1 case (5.5%). No malignant cells were isolated from instrument washings. The single case of malignant cells on glove washings occurred in a recurrent, invasive squamous cell carcinoma of the scalp with intracranial extension. Anucleated squamous cells likely from surgeon skin were isolated from 94.4% of washings. Squamous cells were differentiated from mature cells by the absence of nuclei.ConclusionsMalignant squamous cells can be isolated from surgical glove washings, supporting the practice of changing of gloves after gross tumor resection during major head and neck cancer resections.     

Confocal microscopic evaluation of epithelia of the larynx

BACKGROUND: To evaluate mucosal changes of the true vocal cord, e. g. leukoplakia, microlaryngoscopy is needed to obtain tissue for histopathologic investigation. But there is a discrepancy between the amount of representative tissue necessary for pathology and the postoperative voice function. Thus, a diagnostic procedure that enables the surgeon to differentiate inflammation from dysplasia and carcinoma in situ intra-operatively is urgently needed. We are now reporting on our results using laser scanning microscopy to visualize larynx epithelia. METHODS: The laser scanning microscope used in this ex vivo study is a combination of the Heidelberg Retina Tomograph HRTII and the Rostock Cornea Module. A 63 x water immersion objective lens was used for imaging. PATIENTS: Seven larynx preparations of patients (age 54 to 62 years) with a T4 larynx carcinoma who underwent laryngectomy between 2003 and 2006 were examined. RESULTS: Areas of epithelia of the true vocal cord of macroscopically normal appearance supplied images of healthy, but also inflamed laryngeal epithelia. The corresponding histopathologic sections prepared horizontally confirmed the regularity and homogeneity of the mucosa. The different layers of the lamina propria can be separated. In contrast to healthy and inflamed laryngeal mucosa, dysplasia, precancerous lesions and carcinoma-atypical cells were visible. In the latter entity a loss of cellular junctions characterized by lack of visualization of the cell membrane was apparent. CONCLUSIONS: LSM supplies information of healthy, inflamed, but also pre-malignant and malignant mucosa changes of the larynx. The pilot study shows that both hyperkeratosis and extensive hyperplasia confine the visualization of the basement cell layer. Research projects have been initiated with the objective to develop a rigid endoscope with integrated LSM technology.     

Cytokeratin-17 as a potential marker for squamous cell carcinoma of the larynx

To assess cytokeratin-17 (CK17) as an immunohistochemical marker for squamous cell carcinoma of the larynx, we stained 63 tissue samples from 63 consecutive patients who were believed or suspected to have squamous cell carcinoma of the larynx for CK17 and analyzed them by computerized histomorphometry. The mean staining intensity for CK17 was significantly stronger (p < .01) in cancerous cells, dysplasia, and normal epithelium proximal to the tumor than in distal normal epithelium and polyps. The percentage of stained area, within samples taken from a single patient, was significantly higher in malignancy and dysplasia as compared to distal normal epithelium and in malignancy as compared to dysplasia and proximal normal epithelium (p < .001). The integrated optical density was significantly higher in the malignant epithelium, dysplasia, polyps, and proximal normal epithelium than in distal normal epithelium (p < .0001). We conclude that CK17 is a highly sensitive and specific immunohistochemical marker for premalignant and malignant transformation in the larynx. Further investigation is warranted in order to assess the role of CK17 in determining safe resection borders.     

喉鳞状细胞癌中hMLH1和hMSH2的表达及与细胞凋亡的相关性

目的检测人类错配修复基因hMLH1（human mutl homolog 1）和hMSH2（human muts homolog 2）蛋白在喉鳞状细胞癌（简称喉癌）组织中的表达及与细胞凋亡的相关性,从而探讨其与喉癌发生发展的关系。方法应用免疫组织化学方法检测40例喉癌组织、19例癌旁组织和12例非癌喉黏膜组织（声带息肉、乳头状瘤患者喉黏膜）中hMLH1和hMSH2的表达情况。应用流式细胞术的方法定量检测40例喉癌组织、40例癌旁组织和15例非癌喉黏膜组织中hMLH1和hMSH2的含量并检测其凋亡率。结果 hMLH1和hMSH2蛋白在喉癌组织中的定量及定性表达均明显低于癌旁组织及非癌喉黏膜组织,两者在喉癌组织中的表达与淋巴结转移、临床分期、病理分级有相关性,喉癌组织细胞凋亡率与hMLH1和hMSH2蛋白表达均呈直线正相关。结论 hMLH1和hMSH2蛋白可能与喉癌的发生、发展及转移有关,可能通过促进细胞凋亡而与抑制肿瘤发生发展有关。     

喉癌人类组织相容性抗原Ⅰ缺失与癌组织中淋巴细胞浸润的研究

OBJECTIVE: To detect the deletion of human leucotyte antigen (HLA) class I antigen expression in laryngeal carcinoma tissue and discuss the relationship between its expression and tumor infiltrating lymphocyte (TIL) cell infiltration or/and malignancy of tumor cell. METHODS: From Feb 2001 to May 2001 specific mouse antihuman HLA class I monoclonal antibody was combined to examined tissue, 64 samples of laryngeal carcinoma were detected by streptavidin-peroxidase (SP) method immunohistochemistry. RESULTS: All of 64 cases, 57 were positive HLA class I antigen and 7 were negative, the negative expression rate was 10.9% (7/64). CD3+ and CD8+ T cell infiltrated in HLA class I positive tumor mass were significantely more than those in negative tissue. the malignant degree elevated along with the declination of HLA class I antigen expression. By 3 years follow up, the mortalitywas not statistically significant in both HLA- group and HLA+ group (P > 0.05). CONCLUSION: There is deletion of HLA class I antigen in laryngeal tissue, which favours penetration of cytotoxic lymphoid cells into tumor mass; alteration in HLA class I expression may be used by cancer cells to avoid immune surveillance.     

上颌窦恶性肿瘤侵及骨壁的CT影像与组织病理对比观察分析

目的　通过术前CT影像与术后组织病理检查对比分析,探索和评价CT诊断上颌窦恶性肿瘤的窦内外侵犯的应用价值和受累骨壁的准确性。方法　无淋巴结及远处转移的上颌窦鳞状细胞癌患者11例,术前给予组织病理学检查、增强CT扫描,根据CT影像特征和范围实施上颌骨部分切除或全切除,标记切下的骨组织标本的部位和方位,给予常规固定、脱钙、包埋、切片和HE染色,光镜下观察各壁上颌骨的骨组织病理学改变。结果　11例患者鼻腔鼻窦增强CT扫描显示上颌窦内侧壁均有破坏吸收（4例内壁缺失）,其中侵犯前壁6例,上壁7例,底壁3例,后外壁9例;组织病理学检查见除外内壁缺失患者外,上颌窦内壁均有肿瘤细胞侵犯,且前壁和底壁均有肿瘤细胞侵及,其中上壁4例,后外壁4例;术前CT表现为骨质内壁虫蚀样改变但骨壁连续无中断且伴有增厚硬化的骨壁,术后病理验证无肿瘤侵及。结论　术前CT骨壁的破坏并不意味着骨质被肿瘤侵犯,而且上颌骨各壁侵犯的机率是不同的;综合分析发现术前CT表现为骨质内壁呈虫蚀样改变但骨壁连续无中断且伴有增厚硬化的“改建性骨破坏”者,术后病理验证无肿瘤侵及骨壁;术后常规选取上颌骨骨组织病理分析,可以补充术前CT诊断上颌窦恶性肿瘤侵犯范围的不足,从而精确判断肿瘤的T分级,可能为选择微创的手术方式及术者术后评估手术效果、更科学的制定术后综合治疗提供更有力的依据。     

The expression and prognostic relevance of indoleamine 2,3-dioxygenase in tongue squamous cell carcinoma

Conclusion IDO might be useful for predicting progression of primary tumor stage T2 and T3 in tongue squamous cell carcinoma (TSCC), but does not seem like a specific biomarker for diagnosing TSCC and predicting patient survival. Objectives Indoleamine 2,3-dioxygenase (IDO) is expressed in many cells and it catabolises the essential amino acid tryptophan to kynurenine. IDO acts as an immune modulator through suppression of T-cell immunity and other pathways. In cancer cells, IDO has been proposed to promote tumor progression by enabling malignant cells to escape from the immune system. The aim of this study was to evaluate the association and prognostic relevance of IDO expression in TSCC. Method One hundred and eight retrospective tongue and lymph node specimens were stained immunohistochemically with monoclonal antibody anti-indoleamine 2,3-dioxygenase. The relative abundance of IDO positive epithelial cells, IDO staining intensity, and inflammation were assessed semi-quantitatively with light microscopy. Results IDO was expressed stronger in tongue hyperplasia than in TSCC. However, IDO expression associated with poor survival in the sub-groups with primary tumor stage T2–T4 and in the sub-group with strong inflammation in tumors’ invasive front.     

Thymidine labeling index in laryngeal squamous cell carcinoma in correlation with pTNM,age, histological grade and recurrence

In this study, our aim was to investigate the prognostic features of the thymidine labeling index (TLI) in laryngeal squamous cell carcinoma (SCC). The TLI values in tumor tissues and adjacent healthy tissues were assessed in 40 patients who had a pathological diagnosis of laryngeal SCC. The data were correlated with age, pTNM, histological grade and recurrence. The tissues (tumor and adjacent tumor-free tissue) obtained during surgery were labeled with (3)H-thymidine. A statistically significant difference was observed between tumoral tissue and adjacent tumor-free tissue (p < 0.05), but we could not confirm any statistically significant correlation between TLI and age, pTNM, histological grade and recurrence. In conclusion, TLI in laryngeal SCC had no significant relation with the clinicopathological features, probably due to the variation in tissue sampling and for tumor-dependent reasons. TLI may assist in differentiating malignant from benign lesions.     

Establishment of carcinoma cell lines from larynx and submandibular gland

Summary For many techniques in the oncology of head and neck tumors large amounts of pure tumor cells are required.Although larynx and salivary gland tumors are common in man, no report exists on isolation and purification of tumor cells of which malignancy was proved.The present paper describes in vitro cultivation of living human malignant tumor cells from a larynx and a submandibular gland carcinoma. Carcinoma cells were freed from all non-tumor cells and cloned thus indicating that cultures contained only a single cell type. Transplantation of grown cells s.c. into athymic (nu/nu) mice induced rapidly growing tumors of which malignancy was demonstrated by histology.Dedicated to Prof. Dr. L. Seiferth on the occasion of his 80th birthday     

Natural killer cell activity in patients with carcinoma of the larynx and hypopharynx

Natural killer (NK) cells have recently gained much attention as potential effector cells in antitumor and antiviral immune defense mechanisms. The role of NK cells in patients with squamous cell carcinoma has not yet been described. In our study of larynx and hypopharynx cancer patients, no correlation was found with the extent of the disease expressed by the TNM classification or the TNM staging. Furthermore, no change of the spontaneous natural killer cell mediated K562 lysis was shown in advanced malignant disease or in the postoperative period up to 24 months following surgery. When the NK cell activity was compared with two control groups of healthy volunteers, aged 20 to 30 or 55 to 65 years, both with high alcohol and tobacco consumption, no alterations of the killing capacity for K562 targets were observed during aging, with or without a tumor. NK cell cytotoxicity was significantly lower in tumor draining lymph nodes of the neck than the values found in peripheral blood. In cancer patients as well as in healthy donors, the spontaneous killer cell activity was significantly enhanced by incubation with beta-interferon. The differences, however, between natural killing in peripheral blood and lymph nodes were increased in the head and neck cancer population mainly by an overall reduced NK cell cytolysis in the tumor draining lymph nodes. Although none of the lymph nodes used was infiltrated by malignant cells as seen under microscopic examinations, the diminished NK cell activity in laryngeal and hypopharyngeal cancer patients seems to be caused by factors or the presence of locoregional suppressor cells capable of reducing natural cell mediated immunity.     

头颈肿瘤组织标本库构建的初步研究

目的 探讨头颈肿瘤组织标本的采集、保存,组织库构建方法,以及信息化管理技术。方法 收集手术切除和活检的头颈肿瘤组织、癌旁组织、远端正常组织及患者和正常人外周血标本(分离出血清、血浆、淋巴细胞、干细胞等),置于液氮或-80℃低温冰箱冷冻保存;同时建立一套系统管理组织标本库,使标本库规模化、信息化、智能化。结果 共收集新鲜组织标本220份,血液标本476份。其中肿瘤组织120份(包括肿瘤组织、癌旁组织);非肿瘤组织100份。肿瘤患者血液标本316份,非肿瘤患者血液标本160份。结论 标本库的建立使少见的头颈肿瘤组织标本集约化、规模化,极大提高了标本利用的效率,达到了资源共享的目的。