Karyometric marker features in tissue adjacent to invasive cervical carcinomas

A companion study showed the existence of statistically significant changes in the value of karyometric "marker features" in the nuclei of histologically normal-appearing ectocervical epithelium adjacent to carcinomas in situ. In this second part of the study, the results obtained in patients with invasive cervical carcinomas were analyzed. Formalin-fixed, paraffin-embedded tissue sections were Feulgen stained and analyzed with a microTICAS video microphotometer. The results, demonstrated for the first time in histologic material, indicate that the marker features are clearly expressed in a majority of nuclei observed in the normal-appearing tissue adjacent to invasive lesions. This effect was statistically significant. The best marker features selected by the discriminant analysis were nuclear roundness, nuclear perimeter length, total optical density and a run length texture measure. These findings may reflect a subtle transformation of the apparently normal cervical epithelium adjacent to an invasive cervical carcinoma.     

Karyometric features in nuclei near colonic adenocarcinoma. Statistical analysis.

The normal mucosa adjacent to colonic adenocarcinoma (marginal or transitional mucosa) has been shown to have subtle alterations of architecture, surface glycoproteins and proliferative activity. To evaluate possible changes in nuclear configurations in this marginal mucosa, a large set of cytometric features was evaluated using a computer-assisted video analysis system. Preliminary statistical analysis of the measurements identified six nuclear features useful for discriminating marginal mucosa nuclei from normal (control) mucosa nuclei: total optical density (OD), nuclear area, chromatin texture (from gray value cooccurrence matrix), chromatin coarseness, average OD of nuclear staining and peripheral tendency of the chromatin in the nucleus. An analysis of variance revealed that both patient-to-patient and gland-to-gland variation would limit the usefulness of any one feature as a screening tool. As a group, however, these six features should be investigated further as markers of preneoplastic changes in histologically normal-appearing mucosa.     

Image cytometric evaluation of nuclear texture features and DNA content of the reticular form of oral lichen planus

OBJECTIVE: To analyze image cytometric chromatin changes reflected in nuclear texture features and DNA ploidy of oral lichen planus in relation to the normal buccal mucosa and buccal mucosa expressing malignancy-associated changes in cancer patients. STUDY DESIGN: Twenty-eight patients with the reticular form of oral lichen planus, with a follow-up period of 25 years, 50 healthy controls and 50 lung cancer patients were included in the study. Scrapings of buccal mucosa were suspended in transport medium. Monolayer filter preparations were Feulgen-thionin stained. Image cytometric analysis was performed by Cyto-Savant. RESULTS: All oral lichen planus specimens in our study were diploid. In univariate analysis, differences between the normal buccal mucosa and oral lichen planus were found in several nuclear texture features, which gave an 80% correct classification rate in multivariate analysis. In the second part of the study, the classifier that recognizes malignancy-associated changes on the buccal mucosa of patients with lung cancer correctly recognized > 80% of oral lichen planus samples as normal buccal mucosa. CONCLUSION: Our results indicate that chromatin changes in oral lichen planus exist compared to normal cells; however, the chromatin structure of the reticular form of oral lichen planus does not express malignancy-associated changes and is more similar to normal squamous cells.     

下咽鳞状细胞癌组织claudin-1和MMP-9的表达及与临床病理特征及预后的关系研究

摘要 目的：研究下咽鳞状细胞癌组织紧密连接蛋白1（claudin-1）和基质金属蛋白酶-9（MMP-9）的表达及与临床病理特征及预后的关系。方法：选取2015年1月-2017年3月我院收集的75例下咽鳞状细胞癌组织标本进行研究,同期选取60例癌旁正常黏膜组织标本作为对照。采用免疫组织化学法检测claudin-1、MMP-9在下咽鳞状细胞癌组织与癌旁正常黏膜组织中的表达差异,分析claudin-1、MMP-9阳性表达与临床病理特征关系;Spearman相关性分析癌组织claudin-1与MMP-9的相关性。对患者进行5年随访,分析claudin-1、MMP-9表达与预后的关系。结果：下咽鳞状细胞癌组织claudin-1、MMP-9阳性表达率高于癌旁正常黏膜组织（P＜0.05）。claudin-1、MMP-9阳性表达与下咽鳞状细胞癌患者组织分化程度、淋巴结转移有关（P＜0.05）。经Spearman相关性分析显示,下咽鳞状细胞癌组织中claudin-1阳性表达与MMP-9阳性表达呈正相关（rs= 0.463,P＜0.05）。术后随访5年,2例失访,73例患者获得随访。Kaplan-Meier生存曲线显示,claudin-1阳性表达患者的5年生存率为35.71%,低于阴性表达患者的66.67%（P＜0.05）,MMP-9阳性表达患者的5年生存率为34.85%,低于阴性表达患者的57.14%（P＜0.05）。结论：下咽鳞状细胞癌组织中claudin-1、MMP-9阳性表达升高,其表达与组织分化程度、淋巴结转移和预后不良有关,两者呈正相关,可能发挥协同作用促进肿瘤发生发展。     

Immunohistochemical localization of glutathione S-transferase in preneoplastic and neoplastic lesions of the human uterine cervix

A mouse monoclonal antibody and a rabbit polyclonal antibody prepared against the placental form of the enzyme glutathione S-transferase (GST-pi) were used to immunohistochemically stain normal and neoplastic human uterine cervical tissues from 88 cases. Of 65 cases of preneoplastic squamous lesions and invasive carcinomas of the cervix, 94% stained with the monoclonal antibody and 100% with the polyclonal antibody. In the 23 benign tissues, staining of ectocervical squamous epithelium was generally not observed; however, areas of reserve-cell hyperplasia, immature squamous metaplasia and adjacent endocervical cells did show staining (68% with the monoclonal antibody and 95% with the polyclonal antibody). Many of the positive tissue types showed a variety of staining patterns and intensities. These findings do not support the concept that GST-pi staining can be used to distinguish preneoplastic lesions of the cervix from benign reactive or proliferative processes. These results are of interest in the investigation of cervical carcinogenesis since GST-pi may be involved in an early stage of neoplastic transformation of the cervical epithelium. The correlation of these findings with the results of human papillomavirus testing and DNA content analysis should be of interest in determining the relationship of this enzyme to cervical neoplasia.     

Rat colonic mucosal cell sialic acid metabolism in azoxymethane-induced tumours

Colonic tissue was examined from normal (control) rats and azoxymethane- (carcinogen-) treated animals. Tumour-bearing colons from azoxymethane-treated rats were divided into malignant and non-malignant areas. Mucosal cells were prepared from the three types of colonic tissue and then examined for DNA and protein content and for the activities of ten enzymes involved in sialic acid metabolism. Enzyme activities were related to either the protein or the DNA content of fractions. The DNA content of cell homogenates was significantly different between tumour and non-malignant tissue and between both these tissues and normal mucosa. The protein content of the 100000 X g membrane pellet and supernatant fraction did not vary significantly between normal and non-malignant material but both these tissues differed significantly from tumour tissue. Significant variation between normal control and tumour tissue was detected at all levels of sialic acid metabolism, including N-acetylhexosamine interconversion and phosphorylation, sialic acid formation and activation, CMP-NeuAc breakdown and transfer and sialic acid release from glycoconjugates. The results indicate that major changes at all levels of sialic acid metabolism are associated with malignancy in rat colonic mucosa. Some of these changes are apparent in non-malignant mucosa and may reflect a pre-malignant state.     

DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia in specimens obtained by punch biopsy

This study analyzed the feasibility of, and the strategy for, DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia by a computerized digital imaging system. Paraffin-embedded tissue provided satisfactory single-cell preparations for DNA ploidy analysis after enzyme digestion and additional procedures. Negative endocervical curettings and normal squamous mucosa were used as internal diploid controls. With suitable controls, 21 (81%) of the 26 aneuploid lesions were identified as such in the single-cell preparations. The remaining five lesions (not recognized as aneuploid in the single-cell preparations) had ploidy levels between 2.08n and 2.30n and required DNA measurements on 12-microns sections. Criteria for these DNA measurements were defined: specimens intended for DNA ploidy analysis should contain abnormal epithelium of at least 3 mm to 4 mm in greatest dimension and should be accompanied by diploid controls, such as endocervical curettings or normal ectocervical squamous mucosa. With a combination of single-cell preparations and 12-microns tissue sections, it was possible to accurately determine the DNA ploidy patterns of the cervical lesion specimens obtained by punch biopsies. Available evidence suggests that ploidy analysis can provide useful diagnostic and prognostic information.     

p53突变蛋白在胃癌组织中的表达及免疫电镜观察

作者应用抗ｐ５３单克隆抗体Ｐａｂ１８０１（Ａｂ２美国癌基因公司产品）对３８例手术切除的胃癌组织及癌旁胃粘膜的冰冻切片标本进行ｐ５３突变蛋白表达的检测,并进一步用胶体全免疫电镜技术对ｐ５３突变蛋白的分布特征进行观察。结果：３８例胃癌组织中,２４例有ｐ５３突变蛋白高表达,阳性率６３．２％。在对应的癌旁胃粘膜中１０例为ｐ５３的弱表达,正常组织无表达。伴有淋巴结转移的２３例胃癌标本中,１８例ｐ５３高表达（７８．３％）。免疫电镜结果表现,ｐ５３蛋白主要分布于核内染色质中,胞浆中有散在的阳性区,但以核膜周边为主,紧靠核膜。本研究结果提承胃癌的发生及其肿瘤的生物学行为与抑癌基因ｐ５３的突变密切相关,ｐ５３突变蛋白可能是通过对ＤＮＡ复制的影响而参与肿瘤的形成。     

Preneoplastic changes in ovarian tissues

OBJECTIVE: To test whether histologically normal epithelium within ovarian inclusion cysts and stroma exhibit changes in nuclear chromatin pattern that indicate the presence of occult ovarian lesions. STUDY DESIGN: Ovaries were collected from 10 low-risk women,from 7 high-risk women and from 3 women with ovarian cancer. Histologic sections were cut at 5 microm and hematoxylin and eosin stained. High-resolution images were recorded from the epithelium lining inclusion cysts and from the underlying stroma of ovaries from these 20 subjects. A total of 2860 epithelial nuclei and 3610 stromal nuclei were recorded. Karyometric features and nuclear abnormality were computed. Discriminant analyses and unsupervised learning algorithms defined deviations from normal that were designated "above threshold" and used to compute average nuclear abnormality of a second nuclear phenotype. RESULTS: Histologically normal epithelium from inclusion cysts of ovaries harboring a malignant lesion was shown to exhibit changes in the nuclear chromatin pattern that were statistically significant using quantitative image analysis procedures. Similar changes were seen in the inclusion cyst epithelia of high-risk ovaries. A subpopulation of cells representing a new phenotype was detected in the underlying stroma of women harboring a malignant ovarian lesion and in women at high risk of ovarian cancer. CONCLUSION: The karyometric changes observed in the epithelia lining inclusion cysts and in the underlying stroma of ovaries either with ovarian cancer or at high risk of ovarian cancer suggest the presence of preneoplastic changes in histologically normal tissue.     

Overexpression of prothymosin alpha predicts poor disease outcome in head and neck cancer

Background

In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC).

Methodology

Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients.

Results

Our immunohistochemical analysis demonstrated significant overexpression of nuclear PTMA in squamous cell hyperplasia (63.8%), dysplasia (50%) and HNSCC (61%) in comparison with oral normal mucosa (p_trend<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard''s ratio, HR = 5.2, 95% CI = 2.3–11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage.

Conclusions

Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease.     

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration.     

Markers for malignancy in the nuclear texture of histologically normal tissue from patients with thyroid tumors

The chromatin of nuclei from histologically normal-appearing glands in tissue sections from patients with follicular adenoma, papillary carcinoma and follicular carcinoma of the thyroid exhibited changes that were statistically significant when compared to the nuclear chromatin in tissue sections of thyroid from normal individuals. Certain chromatin texture features assumed values approximating those found in tumor cell nuclei. Staining was affected only slightly, and morphometric features, such as nuclear area, roundness and ellipticity, were statistically not different from those in normal controls. In patients with Hürthle-cell tumors, such marker features could not be found. Results from measurements on 30 patients (approximately 1,000 nuclei) are reported.     

Comparison of morphonuclear features in normal, benign and neoplastic thyroid tissue by digital cell image analysis.

We analyzed the morphonuclear features of thyroid cell nuclei from 10 normal tissues (10 multinodular goiters), 10 benign tissues (10 adenomas) and 10 neoplastic tissues (10 carcinomas--2 follicular, 2 medullary and 6 papillary). These morphonuclear features quantitatively described the nuclear size, DNA content and chromatin texture of 200-400 Feulgen-stained cell nuclei that were digitized by means of a cell image processor. Nuclear DNA estimations revealed that the benign thyroid tumors showed an aneuploidy level that was not different from that of the neoplastic tissues. Morphometric measurements showed a significant and positive correlation between an increase in nuclear area and the development of thyroid pathology--i.e., from normal to a neoplastic stage. We also observed a significant decrease in chromatin condensation and heterogeneity from normal to neoplastic thyroid tissue. In the specific case of thyroid tumors, such criteria were not found sufficient per se for a diagnosis of malignancy. More detailed data banks will be necessary for this purpose.     

口腔鳞状细胞癌线粒体DNA的HVRⅢ区突变的研究（英文）

目的：检测口腔鳞状细胞癌患者线粒体DNA复制控制区（mtDNA D-loop）高变Ⅲ区（hypervariable regionⅢ,HVRⅢ）的突变情况,并探讨其意义。方法：以口腔鳞状细胞癌患者癌旁组织及正常组织作为对照,对7例口腔鳞状细胞癌组织样本的mtDNA D-loop HVRⅢ区进行PCR扩增和测序分析。结果：在7例患者的癌组织、癌旁组织、正常组织样本中共发现72个（56种）核苷酸改变,其中51个（26种）为核苷酸多态性改变;3个肿瘤组织样本中共发现21个突变,其中16个位于HVRⅢ区范围内;癌旁组织及正常组织未发现突变;口腔鳞状细胞癌的mtDNA D-loop HVRⅢ区突变率为42.9%（3/7）。结论：mtDNA D-loop HVRⅢ区的变异可能与口腔鳞状细胞癌的易感性有一定的联系;本研究为寻找新的肿瘤基因诊断和肿瘤遗传易感性的标志物提供了依据。     

Identification of Tumor-Associated Proteins in Well Differentiated Laryngeal Squamous Cell Carcinoma by Proteomics

Introduction

This study established two-dimensional gel electrophoresis (2-DE) profiles for human well-differentiated laryngeal squamous cell carcinoma tissue and paired normal mucosa epithelia tissue and identified proteins with different expressions. Well-resolved and reproducible 2-DE patterns of well-differentiated laryngeal squamous cell carcinoma and adjacent normal mucosa were obtained.

Results

Thirteen proteins were preliminarily identified, among which ten proteins including cofilin-1, nuclear body protein SP140, GRP94, HSP 90, GSTP1-1, superoxide dismutase [Mn], cyclophilin A, proteasome activator complex subunit 2, apolipoprotein A-I precursor, and CaM-like protein were upregulated and three proteins including fatty acid-binding protein (E-FABP), calgranulin A, and calgranulin B were downregulated in laryngeal cancer tissue. The different expressions of cyclophilin A and MRP8 were confirmed by Western blotting.

Discussion

We first identified 13 proteins that might be associated with the tumorigenesis of the laryngeal squamous cell carcinoma. Some proteins were the products of oncogenes and apoptosis and others were related to signal transduction and immune defense. These extensive protein variations indicated that multiple protein molecules were simultaneously involved in the oncogenesis of laryngeal cancer, which in turn is a basis for the rational designs of diagnostic and therapeutic methods.     

Prostaglandins in squamous cell carcinoma of the larynx: tumor and peritumor synthesis

Prostaglandin (PG) E2, 6ketoPGF1 alpha and Thromboxane B2 (TxB2) production by the tumor, peritumor and control tissue were investigated in specimens from patients (n = 11) with squamous cell carcinoma of the larynx, in relation to the extension and infiltration of the neoplasm and to the presence of inflammation, fibrosis and necrosis. In all specimens detectable amounts of 6ketoPGF1+ and TxB2 were found, but the predominant metabolite was PGE2. No differences in the levels of TxB2 and 6ketoPGF1 alpha were observed, but the only patient with lymphnodal involvement showed the lowest levels of 6ketoPGF1 alpha both in tumor and peritumor tissue. Higher amounts (p less than 0.05) of PGE2 were synthesized by peritumor tissues in comparison to control mucosa and tumor tissue independently of the occurrence of reactive infiltration. PGs synthesis did not correlate with inflammation, fibrosis, necrosis or staging of the neoplasm. However the two cases in stage T4 showed PGE2 generation at the highest levels both in neoplastic and perineoplastic tissue. These findings indicate that in squamous cell carcinoma of the larynx an increased production of PGE2 occurs, stemming not only from inflammatory cells but at least in part from neoplastic cells. This suggests that the study of arachidonic acid metabolism may contribute to characterization of the primary cancer and lead to better understanding of the mechanisms of tumor growth and diffusion.     

Influence of Genetic Background and Tissue Types on Global DNA Methylation Patterns

Recent studies have shown a genetic influence on gene expression variation, chromatin, and DNA methylation. However, the effects of genetic background and tissue types on DNA methylation at the genome-wide level have not been characterized extensively. To study the effect of genetic background and tissue types on global DNA methylation, we performed DNA methylation analysis using the Affymetrix 500K SNP array on tumor, adjacent normal tissue, and blood DNA from 30 patients with esophageal squamous cell carcinoma (ESCC). The use of multiple tissues from 30 individuals allowed us to evaluate variation of DNA methylation states across tissues and individuals. Our results demonstrate that blood and esophageal tissues shared similar DNA methylation patterns within the same individual, suggesting an influence of genetic background on DNA methylation. Furthermore, we showed that tissue types are important contributors of DNA methylation states.     

LaminB1蛋白在食管鳞癌组织中表达的形态学观察

目的探讨laminB1蛋白在食管鳞癌患者的癌组织及上切缘正常粘膜上皮中表达的形态变化。方法制备组织切片原位核基质,应用免疫组化的方法检测核基质制备前后正常粘膜上皮及癌组织中laminB1的表达;同时提取组织核基质蛋白,应用Westen blot检测核基质蛋白中laminB1的表达。结果正常食管粘膜上皮及食管鳞癌组织核基质制备前后laiminB1表达的阳性率分别为:正常粘膜上皮93.3%、正常粘膜上皮核基质86.7%、癌组织96.7%、癌核基质86.7%。正常粘膜上皮laminB1表达阳性细胞从基底层至颗粒层逐渐减少,制备核基质后正常粘膜上皮核基质laminB1表达阳性细胞数目减少、强度明显减弱,阳性细胞集中于基底层,多数细胞整个胞核着色。癌组织laminB1表达阳性细胞散在分布,无规律;癌核基质laminB1表达阳性强度减弱,阳性颗粒在核周较集中。癌核基质laminB1表达的阳性强度比正常粘膜上皮核基质高,差异有显著性(x2=5.042,P<0.05)。Western blot检测显示正常粘膜核基质的laminB1条带比癌核基质弱。结论laminB1蛋白在食管正常粘膜上皮及食管鳞癌组织中广泛存在。制备核基质后,laminB1蛋白在癌核基质的表达比正常粘膜上皮核基质强;且癌核基质中laminB1的分布与正常粘膜上皮核基质存在差异。