Computational protocol: Association between Argyrophilic Proteins of Nucleolar Organizer Regions, Clinicomorphological Parameters, and Survival in Non-Small-Cell Lung Cancer

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Protocol publication

[…] The study was performed on 243 surgical specimens of non-small-cell cancer excised in 2007–2009 at Altay Regional Oncologic Dispensary (cases of M1 and multiple tumors were excluded from the research). The mean age of patients was 59 years (35 to 75 years); the study population included 209 men and 34 women. Lobectomy was performed in 172 (70.8%) patients and pulmonectomy in 71 (29.2%) patients. Preoperative chemoradiotherapy was not used. Postoperative chemotherapy was performed in 27 of the cases (11.1%); cisplatin and etoposide were most often used. Postoperative radiation therapy was performed in 72 of the cases (29.6%), dose of 50–60 Gr most often used. Histopathological characteristics of tumors were determined according to the WHO criteria []. The greatest tumor dimension was measured (in cm). Fragments of pathologically intact lung tissue obtained from distant regions were used as controls in 10 cases.Tissue fragments were fixed for 18–24 h in 10% neutral buffered formalin. After standard preparation of the surgical specimens 4 μm histological slices were prepared. Specimens were stained with haematoxylin and eosin, Schiff reagent/Alcian blue, and Kreyberg stain. Cytokeratins 7 (clone SP52) and 20 (clone SP33) and high molecular weight (clone 34βE12) were determined immunohistochemically in an automated staining instrument Ventana XT.In order to study AgNOR slices were stained with silver nitrate (AgNO3) according to a “one-step” method []. Before staining slices were autoclaved at 120°C for 20 minutes in a 0.01 M citrate buffer (pH = 6.0) [, ]. Nucleus counterstaining was not performed; slices were immersed into Canadian balm. In each case the area of AgNOR (in μm2) was determined in nuclei of 100–120 randomly selected cells from 10–15 digital images obtained from the corresponding microscopic fields at original magnification ×1000 (lens ×100, 1.25, oil). Computer analysis of images was performed using software ImageJ 1.42. To avoid a measurement error granules of <0.1 μm2 in size were excluded from analysis. As an internal staining standard the area of AgNOR in small lymphocyte nuclei was used []. AgNOR area index (AI AgNOR) was calculated as a quotient of AgNOR areas in the tumor cell and in small lymphocyte, and in each case the mean value and CV AgNOR (percent) were calculated.Statistical analysis of data was performed using STATISTICA 6.0 software. The data obtained in the samples corresponded to the normal distribution test (Shapiro-Wilk test W = 0.99, P > 0.05); the measure of central trend in the groups was presented as the mean (M), while the measure of dispersion was presented as a standard deviation (SD). To check statistical hypotheses nonparametric methods were used, including Mann-Whitney U test (M-W) and Spearman's rank correlation coefficient (r). The total adjusted survival of patients for five years after surgery (expressed in percent) was determined using Kaplan-Meyer method, log-rank test, and Cox regression model. The reliability of the criteria obtained was assessed at P < 0.05. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Carcinoma, Non-Small-Cell Lung, Lung Neoplasms, Neoplasms