Flow cytometric analysis of DNA content in laryngeal cancer

DNA content was measured by flow-cytometric analysis in 30 paraffin embedded sections from patients with laryngeal squamous cell carcinoma. The morphological characteristics and N staging of the tumours as registered in their clinical charts were correlated with their DNA content. Eighty per cent of the tumours were found to have a predominantly aneuploid distribution of DNA values. There was no correlation between the N stage of the tumour or degree of cell differentiation and DNA content. A multiploid pattern correlates with non-metastatic laryngeal tumours and we suggest that this pattern may be a good indicator for biological activity.     

喉癌及喉正常黏膜组织的差异蛋白质组学分析

目的 建立喉癌与喉正常黏膜组织的双向凝胶电泳图谱,筛选喉癌异常表达的蛋白质.方法 应用双向凝胶电泳、基质辅助激光解析电离飞行时间质谱分析和生物信息学等技术对9例喉癌和喉正常黏膜组织进行差异蛋白质组学分析.结果 ①获得了重复性较好的喉癌及喉正常黏膜组织的双向凝胶电泳(2-DE)图谱.②PDquest 2-DE图像分析软件对同一病例的癌组织和喉正常黏膜上皮组织各3块凝胶分析,蛋白质斑点数分别为1504±122和1235±164,平均匹配点数分别为1398±143和1136±82,匹配率分别为93%和92%.③从差异蛋白质斑点中取34个进行MS分析,获30个蛋白的肽质量指纹图.④查询数据库鉴定了30个差异蛋白质点,其中一些蛋白质与细胞代谢、信息转导格细胞增生等有关.结论 鉴定了30个喉癌异常表达的蛋白质,为筛选喉癌标志物以及揭示喉癌发病机制提供理论依据.     

Human papillomavirus in laryngeal papillomas and in adjacent normal epithelium

Eleven adults with laryngeal papillomas were studied for the presence of human papillomavirus (HPV) DNA by in situ hybridization. As well as from the papillomas, three additional biopsies were taken from the normal-appearing mucosa as follows: the involved vocal cord, the opposite vocal cord (when the papilloma was unilateral), and from the ventricular fold on the side of the lesion. These normal tissues were analysed by polymerase chain reaction (PCR) to detect HPV DNA. All except one of the 11 papillomas contained HPV DNA; nine were HPV 6/11 DNA positive and one positive for HPV 16 DNA. The normal-appearing laryngeal mucosa harboured HPV DNA in eight out of 11 patients. The present results strongly support the concept that the adult-type laryngeal papilloma is an HPV-induced lesion, mostly due to HPV types 6 and 11. The persistence of HPV DNA in the adjacent normal epithelium is consistent with the frequent recurrence of these lesions.     

Relation between the DNA content in laryngeal cancer cell and its clinical behaviour

The DNA contents of 45 cases of laryngeal cancer have been analyzed by flow cytometry (FCM). The DNA index (DI) ranged from 0.9 to 3.39; proliferative index (PI) was 27.3%. Among them, 13 cases (28.8%) were diploid and 31 (68.8%) distinctly aneuploid. Correlation has been found between DI and the histopathologic grades of the tumors. The larger the DI, the higher the histopathologic grade (P less than 0.05). The mean value of the PI in supraglottic types was higher than that of glottic types. DNA content analysis also showed that diploid tumor had a better prognosis and longer survival than those with higher DI values. In addition, the proliferative activity of cancer cell was found to be markedly reduced after irradiation which demonstrates that preoperative radiotherapy is favourable for laryngeal cancer.     

晚期喉癌边缘DNA含量和增殖细胞核抗原表达

目的探讨晚期喉癌边缘ＤＮＡ含量和增殖细胞核抗原（ＰＣＮＡ）表达与喉癌手术预后的关系。方法通过ＰＣＮＡ表达和ＤＮＡ含量检测，对３４例晚期喉癌边缘进行观察。结果肿瘤边缘１．０、０．５、０ｃｍ处粘膜，ＰＣＮＡ指数分别为８．６２％、１７．７６％和５０．３２％。ＤＮＡ指数分别为０．９８、１．０８２和１．４３６。统计学分析，１．０、０．５ｃｍ处粘膜与０ｃｍ处粘膜差异有显著性（Ｐ＜０．０１）。结论中、重度非典型增生的上皮在分裂，增殖等生物学行为方面已与轻度非典型增生和单纯性增生的上皮细胞不同，手术切缘应在中度非典型性增生的粘膜上皮以外，即０．５ｃｍ以外。     

晚期喉癌边缘DNA含量和增殖细胞核抗原表达

目的 探讨晚期喉癌边缘ＤＮＡ含量和增殖细胞核抗原（ＰＣＮＡ）表达与喉癌手术预后的关系。方法 通过ＰＣＮＡ表达和ＤＮＡ含量检测，对３４例晚期喉癌边缘进行观察。结果 肿瘤边缘１．０、０．５、０ｃｍ处粘膜，ＰＣＮＡ指数分别为８．６２％，１７．７６％和５０．３２％。ＤＮＡ指数分别为０．９８、１．０８２和１．４３６。统计学分析１．０、０．５ｃｍ处粘膜０ｃｍ处粘膜差异有显著性（Ｐ〈０．０１）。结论 中、重度非     

Changes in nuclear DNA content in nasopharyngeal epithelium]

The nuclear DNA content was measured by flow cytometry (FCM) in 167 specimens with normal nasopharyngeal epithelium, paracarcinoma tissue and nasopharyngeal carcinoma (NPC). Thirty eight (38%) of 101 NPC, seven (58%) of 12 recurrent and six (30%) of 20 paracarcinoma tissues were nondiploid. There was no nondiploid at all in 20 specimens of normal nasopharyngeal epithelium. Two of six patients with VCA-IGA(+) were nondiploid. Cellular proliferation index (PI) of NPC was increased significantly as compared with that of the normal epithelium (P < 0.01). However there was no marked difference in PI. between NPC and paracarcinoma tissues. The rate of cervical metastasis in nondiploid NPC was significantly higher than that in diploid NPC (P < 0.025). But there was no correlation of DNA content between the stage of the tumor and the titer of VCA-IGA. These data show that FCM may prove useful in the diagnosis and predication of prognosis in NPC.     

喉癌及喉正常黏膜组织蛋白质组差异表达的初步研究

目的 研究喉癌及癌旁喉正常黏膜组织蛋白质组的差异表达。方法 用固相PH梯度双向凝胶电泳分离喉癌及癌旁喉正常黏膜组织总蛋白质，银染显色、PD(HYEST2DE软件分析所获得的双向凝胶电泳图谱，找出差异表达的蛋白质点。取差异表达的蛋白质点进行质谱鉴定。结果 获得分辨率和重复性均较好的双向凝胶电泳图谱，并鉴定出与喉癌发生密切相关的12种蛋白质。其中有10种蛋白质在喉癌组织中特异表达，分别是An-giopietin-2，sprouty homologues，Inhibitor of apoptosis-1ike protein2(ILP-2)，urokinase type plasminogen activator re-ceptor uPAR，Wnt-3a protein，癌基因Transforming protein N-Ras、P21以及c-Met．等.connexin31．1，原肌球蛋白Tropomyosin两种蛋白质在癌旁喉正常黏膜组织中特异表达。结论 本研究利用固相PH梯度双向凝胶电泳技术分离喉癌及癌旁喉正常黏膜组织总蛋白质，首次建立了重复性较强的喉癌及喉正常黏膜组织蛋白质组表达图谱，且两者蛋白质的表达明显不同，差异表达的蛋白质分别参与细胞的癌变、细胞间的信号传导、肿瘤血管生成，这对研究喉癌的癌变机制，寻找与喉癌诊断和治疗相关的肿瘤标志物相当重要。     

Measurement of nuclear DNA content of laryngeal carcinoma and determination of estrogen receptor (ER) level in laryngeal cancer cells

In 39 patients with laryngeal carcinoma, nuclear DNA content was measured, and the ER in cancer cells of 23 cases determined. The following results were obtained: 1. For all laryngeal carcinomas, there were two patterns-diploid and non-diploid carcinomas. The majority (27/39) belonged to the non-diploid pattern. 2. The laryngeal carcinomas of non-diploid pattern or with higher level of DNA content were easily metastasized to cervical nodes. This kind of cancers were more often seen in clinically advanced cases. Patients with these carcinomas, judged by the results of one-year's follow-up, had poor prognosis. 3. Carcinomas with diploid pattern or with lower level of DNA content had the tendency to become ER positive. The cervical node metastases was more easily taking place in ER negative carcinomas. Some indirect relationship between DNA content and ER in laryngeal cancers may exist.     

Nuclear DNA content and morphological parameters in laryngeal papillomas and carcinomas

The nuclear DNA content and morphological parameters were measured in 27 laryngeal papilloma (LP) and 64 laryngeal carcinoma (LC) specimens by image analysis technique. Results showed that the DNA contents between LC and control group (P less than 0.01) and that between LP and control group (P less than 0.05) were significantly different. The peak value of control group was 2C and that of LP 3C. The histogram of LC showed double peaks at 3C and 6C. The morphological parameters differed significantly among the above three groups (P less than 0.01). The data confirm that LP is an epithelial borderline tumor.     

Intratumoral DNA content heterogeneity in laryngeal squamous cell carcinoma.

Multiple tissue samples obtained from sections cut by Michaels and Gregor's method obtained from 21 consecutive total laryngectomies for squamous cell carcinoma were studied for intratumoral DNA content heterogeneity or homogeneity. Concordant DNA ploidy was manifested in all samples of five (23.8%) carcinomas (two diploid and three aneuploid), while 16 carcinomas (76.2%) demonstrated a variable DNA ploidy (diploid and aneuploid). Analysis of cellular proliferative activity demonstrated remarkable intratumoral stability in both concordant and discordant carcinomas. These data indicate there is a considerable heterogeneity of DNA ploidy but the proliferative rate is relatively stable within the carcinomas. Clinical implications of our findings are also presented.     

MHC molecule expression in laryngeal epithelium of patients with laryngopharyngeal reflux compared to normal subjects

Objectives. We recently demonstrated an increase in the MHC‐associated molecule, β‐2‐microglobulin in laryngeal epithelium from patients with laryngopharyngeal reflux (LPR). This could not be accounted for by an increase in MHC Class I, particularly in the superficial layers where Class I is weakly expressed. We hypothesised that this reflected an increase in CD1d, a non‐classical MHC molecule. Methods. Posterior laryngeal biopsies were obtained from 10 controls and 11 LPR patients. Multicolour immunofluorescence microscopy was used to measure expression of CD1d, CD161 (NK/NKT marker), CD4/8 T‐cells, macrophage, neutrophil and eosinophil antigens. Two‐way analysis of variance and unpaired T‐tests were performed on transformed data. Results. Expression of CD1d increased from basal to superficial layers, and was marginally raised in LPR (P = 0.067), whilst CD161 increased significantly (P = 0.02). While CD161(+) cells were distributed throughout the epithelium, CD8(+) T‐cells were localised to the deep, MHC class I‐expressing layers. EMBP was significantly reduced in LPR (P = 0.01). Conclusions. CD1d and MHC class I exhibit reciprocal expression patterns, which are reflected by the distribution of cells expressing their respective ligands (NKT cells and CD8(+) T‐cells). An increase in CD1d in LPR may be responsible for the increase in β‐2‐microglobulin observed, and is associated with a significant increase in NKT‐cells. The CD1d‐NKT cell axis may be a crucial part of maintaining tolerance in the laryngeal mucosa and changes in this system may be responsible for the persistence of inflammation in LPR.     

Flow cytometric analysis of DNA content in paraffin-embedded tissue in head and neck cancer (the second report)--primary lesions of laryngeal cancer

Flow cytometric analysis of DNA content using paraffin-embedded materials has become an important diagnostic, as well as prognostic, method for clinical pathology and investigative oncology. DNA content as measured in paraffin-embedded materials is closely related with that obtained from fresh specimens. This method also permits retrospective analysis on a lot of cases and studying different specimens of a tumor for intratumor heterogeneity. Flow cytometry was used to investigation of the DNA distribution in biopsy specimens from 30 patients with squamous cell carcinoma of the larynx. These patients had initially treated in our institute in the period from 1986 to 1988 and followed for two to four years. A modification of Hedley's method was used to prepare paraffin-embedded materials. DNA histogram were assessed in terms of DNA index (DI). The coefficient of variation for determination of DI ranged 4.2 to 13.7% (mean 7.8 +/- 2.4%). Using infiltrated lymphocytes and mesenchymal cells as an internal standards, aneuploid DNA histogram were found in 13 of 30 specimens (43.3%). DI ranged from 1.05 to 1.22. There was no significant correlation between DNA content and sex, age, tumor size, staging or treatment. The aneuploidy was found in 2 of 17 cases with well differentiated tumor (11.8%), 10 of 12 with moderately differentiated tumor (83.3%), one with poorly differentiated tumor (100%). 11.8% of patients with diploid tumor cells had recurrence during the interval of observation, compared to 38.5% of those with aneuploid tumor cells (p less than 0.05). In T1 lesions of glottic cancer aneuploid group had a significantly lower local control rate than diploid group (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear DNA cytofluorometry of normal human laryngeal epithelia and squamous cell carcinoma

Normal human laryngeal epithelia and laryngeal squamous cell carcinoma were assayed by Feulgen DNA cytofluorometry using free cell nuclei isolated from carnoy-fixed, paraffin-embedded specimens. In all of the 12 normal specimens, the epithelium showed typical diploid cell clones with low proliferative activity. Polyploid cells were seen in only two specimens from subjects aged 61 and 69 years respectively, and the number of polyploid cells seen in these two specimens was only two. Fifteen cancer cases were divided into three groups: an untreated group (5 cases), a chemotherapy group (5 cases) and a group of cases with recurrence after radiation therapy (5 cases). Among these three groups the DNA ploidy patterns were compared. In the untreated group, all cases showed a two-peak diploid pattern and a high proliferative activity, and polyploid cells were present. In the chemotherapy group, a wide one-peak histogram extending from 2C to about 5C was noted in 4 cases, and an aneuploid pattern in one case. Thus, the DNA ploidy pattern in the chemotherapy group differed from that in the untreated group. Of the 5 cases with recurrence after radiation therapy, one had a tetraploid pattern, but the remaining 4, a two-peak diploid pattern similar to that seen in the untreated group. Polyploid cells were observed in all these cancer cases. However, because they were also seen in some normal subjects, the finding of polyploid cells is not considered to be conclusive of cancer diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroendocrine cells in the cat laryngeal epithelium

The structure and distribution of neuroendocrine cells in the feline laryngeal epithelium were examined using immunohistochemical techniques. Neuroendocrine cells were often spindle shaped, with cytoplasmic processes directed towards the lumen and basement membrane. The apical portion of the cells usually reached the laryngeal lumen with microvillous projections. The cytoplasm always contained variable numbers of electrondense cored vesicles. The number of neuroendocrine cells decreased in the following order: subglottis, posterior glottis, supraglottis, anterior glottis. Neuroendocrine cells contained calcitonin gene-related peptide, substance P and/or 5-hydroxytryptamine. They also showed protein gene product 9.5 or neuron-specific enolase immunoreactivity. These observations suggest that neuroendocrine cells play a part in the regulatory function of the cat larynx by releasing various peptides. These substances may contribute to allergic reactions or control mucus secretion by acting via the endocrine or paracrine pathways and/or neurosecretory pathways.     

蛋白激酶CK2在喉正常组织及喉癌前病变和喉癌中的表达及意义

目的:探讨蛋白激酶CK2在喉癌的发生、发展、浸润和转移过程中的作用。方法:采用免疫组织化学(SP法)染色技术检测18例正常黏膜、14例声带息肉、11例喉乳头状瘤及50例喉鳞状细胞癌(LSCC)标本中蛋白激酶CK2的表达情况,并用RT-PCR技术检测CK2α及CK2β在50例LSCC中mRNA的表达水平,分析LSCC中CK2α及CK2β表达的相关关系。结果:免疫组织化学显示CK2α及CK2β在正常喉黏膜、声带息肉、癌旁1.0cm组织、颈部非转移性淋巴结→喉乳头状瘤、癌旁0.5cm组织→LSCC组织、颈部转移性淋巴结组织中的阳性表达率逐渐升高,它们之间的差异具有统计学意义,组内各病变间表达的差异无统计学意义。在LSCC组内,CK2α的表达与年龄、颈部淋巴结转移及T分期无关,而与病理分级有关。CK2β的表达与年龄及T分期无关,而与病理分级及淋巴结转移有关,这与RT-PCR检测的其在蛋白质水平的表达一致。在LSCC组织中,CK2α与CK2β在mRNA水平的表达呈正相关。结论:蛋白激酶CK2过表达可能对LSCC的发生和发展有一定的促进作用,检测CK2可作为判定LSCC恶性程度的潜在指标之一,抗CK2可能为肿瘤治疗提供一种新途径。     

Magnesium content of laryngeal precancerous conditions and laryngeal cancers

The content of magnesium in tissues, erythrocytes and serum was analysed by atomic absorption spectrometry. A higher concentration of magnesium was found in malignant laryngeal tissue and in lymph nodes of the neck compared to homologous pre-cancerous tissue. In pre-cancerous lesions of the larynx the magnesium concentration of the erythrocytes and serum lay at the lower limit of normal. In patients with cancer of the larynx, the magnesium concentration was considerably below the normal levels and continued to decline as the disease progressed.     

喉角化症中EGFR的表达及与恶变的关系

目的观察表皮生长因子受体(EGFR)在喉角化症中的表达,探讨喉角化症的转归和预后。方法利用免疫组化ABC法检测32例喉角化症患者组织中EGFR的表达情况。结果在轻、中、重度非典型增生中,EGFR表达分别为12.5%、22.5%和47.8%。重度非典型增生的EGFR表达与轻、中度非典型增生比较有显著性差异(P＜0.05)。结论EGFR在喉角化症的转归和预后方面是一个有价值的指标。     

Significance of keratosis and dyskeratosis for classifying hyperplastic aberrations of laryngeal mucosa

Retrospective study of 325 tissue samples of hyperplastic aberrations of the laryngeal mucosa by means of light and electron microscopy and immunohistochemical methods has demonstrated that superficial keratinization is not important for determining changes into precancerosis or "risky epithelium" or even for the progress from hyperplastic aberrations to carcinoma. The degree of keratinization of the epithelial surface in all three forms of hyperplastic aberrations is almost the same. In grouping the hyperplastic aberrations into "precanceroses" or "risky epithelium," the following morphologic changes have to be considered as extremely important factors: the occurrence of dyskeratotic cells, the basalification of the epithelium, and the response of the organism in the form of infiltration of immunocompetent cells in the subepithelial stroma.