Ki-67 expression and prognosis for smokers with resected stage i Non–Small cell lung cancer

doi:10.1016/S0003-4975(03)00119-X

The Annals of Thoracic Surgery

Volume 75, Issue 6, June 2003, Pages 1727-1732

https://doi.org/10.1016/S0003-4975(03)00119-X Get rights and content

Abstract

Background

The cigarette smoking status of patients before surgery is an important prognostic factor in evaluation of stage I non–small cell lung cancer, and the proliferative activity of lung tumors is also related to the patient’s prognosis. This study evaluates relationships between various clinicopathologic factors, including tumor proliferative activity and smoking status, and the patient’s prognosis in stage I non–small cell lung cancer.

Methods

One hundred eighty-seven stage I adenocarcinoma and squamous cell carcinoma cases were evaluated. The patients underwent complete resection between 1988 and 1993 at Chiba University Hospital. Expression levels of Ki-67 nuclear antigen, p53 protein, and retinoblastoma protein were determined immunohistochemically, and postoperative survival rates for patients in the categories of clinicopathologic factors were estimated.

Results

The mean Ki-67 labeling index (LI) for all cases was 19.3%. Labeling index values were significantly higher in squamous cell carcinoma than in adenocarcinoma (p < 0.0001). Postoperative survival of adenocarcinoma patients was significantly related to the LI values and to the patient’s smoking status (p = 0.0164 and 0.0268, respectively). The LI values were also related to smoking status and the extent of histologic differentiation (p = 0.0112 and p < 0.0001, respectively). For nonsmoking adenocarcinoma patients, higher LI values were associated with abnormalities in p53 expression (p = 0.0048). Retinoblastoma protein abnormalities were not related to LI values.

Conclusions

In smokers with stage I pulmonary adenocarcinoma, tumor proliferative activity and smoking status before surgery were important prognostic determinants. The LI values were related to several clinicopathologic factors.

Section snippets

Tissue samples

One hundred eighty-seven patients (120 men and 67 women) were enrolled. The group included 122 patients with adenocarcinoma and 65 with squamous cell carcinoma. All of the patients had undergone complete resection, involving complete lobectomy or complete segmentectomy with standard lymph node dissection, at the Department of Thoracic Surgery, Chiba University Hospital, between 1988 and 1993. Patients were followed there for at least 5 years after their operations, and informed consents were

Clinicopathologic features and prognosis

According to the Kaplan-Meier method the postoperative overall 5-year survival rate for patients with adenocarcinoma was 81.7% and that for squamous cell carcinoma patients was 68.4%.

Table 1 shows the postoperative overall 5-year survival rates for the patients and their relationships to several clinicopathologic factors, as calculated using the Kaplan-Meier method. In the group of smokers with adenocarcinoma, the following factors affected survival rates: smoking status (p < 0.0001), tumor

Comment

Since we reported that the cigarette smoking status before surgery was one of the most important clinical prognostic factors in stage I NSCLC [3], we have tried to find the specific factors that can explain this result. In the current study, we elucidate the fact that smoking status before surgery is a prognostic factor for patients with stage I pulmonary adenocarcinoma, but not for patients with stage I squamous cell carcinoma. We also found that the Ki-67 LI was a significant pathologic

Acknowledgements

The authors thank Tamiyo Taniguchi, Michiko Hanazono, Ayaka Sato, and Kazuko Abe for their technical assistance.

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Locoregional recurrence of non-small cell lung cancer (NSCLC) occurs even among patients with stage I disease, as a result of tumor proliferative activity. The aim of this study was to evaluate the clinical reliability of a new rapid immunohistochemistry (IHC) technique for assessing malignant potential through detection of tumoral Ki-67 expression.
The rapid IHC method uses non-contact alternating current (AC) mixing to achieve more rapid/stable staining within 20 min during surgery. First, to investigate the association between clinical outcomes and tumoral Ki-67 labeling with rapid IHC, 21 pairs of surgical patients treated between 2012 and 2020 for pStage IA1-3 NSCLC with/without recurrence were retrospectively reviewed. Second, 40 frozen section (FS) samples in patients with NSCLC for whom radical surgery was planned between April 2021 and February 2022 were deemed eligible for comparison of the clinical performance of conventional IHC and intraoperative rapid Ki-67 IHC with FS.
Detection of tumoral Ki-67 expression using rapid IHC with formalin-fixed, paraffin-embedded (FFPE) blocks was significantly associated with clinical outcomes in R0 pStage IA NSCLC surgical patients, including overall and recurrence-free survival (P = 0.0043 and P < 0.0001, respectively). Levels of Ki-67 expression among resectable NSCLC patients detected using rapid IHC with FS significantly correlated with those detected using conventional FFPE-IHC (p < 0.001). An intraoperative cut-off of > 7.5 % tumor cell Ki-67 positivity accurately predicted pathological stage more advanced than IA3 [P = 0.0185, Odds ratio = 20.477, 95 % confidence interval (CI): 1.660–252.55].
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On the basis of the inclusion criteria, a total of 103 studies were retrieved from the electronic databases PubMed, Cochrane Library, and Web of Science. After careful review, fifteen studies [14–28] including 1931 stage I NSCLC patients were eventually selected (Fig. 1). The characteristics of these studies are listed in Table 1.
There is growing interest in exploring the prognostic value of Ki-67 in non-small-cell lung cancer (NSCLC). However, whether Ki-67 can be regarded as a routine biomarker in clinical practice is still under debate. The present meta-analysis investigated the relationship between Ki-67 and the overall survival (OS) or disease-free survival (DFS) of patients suffering from stage I NSCLC. We searched the Web of Science, Cochrane, and PubMed databases to extract eligible articles. In total, 15 studies involving 1931 patients were included. Pooled hazard ratio (HR) analysis revealed that patients with high Ki-67 labeling index (LI) had poorer OS (HR = 1.95, 95% confidence interval (CI) = 1.43–2.66, P < 0.0001) and DFS (HR = 3.12, 95% CI = 2.17–4.48, P < 0.00001) than those with low Ki-67 LI. In subgroup analysis, high Ki-67 LI was significantly associated with poor prognoses in stage I adenocarcinoma. In future studies, a consensus for the optimal cutoff value for high Ki-67 LI needs to be explored and demonstrated in stage I NSCLC patients.
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Our study aimed to investigate the biological role of FOXP3 expression in human lung adenocarcinoma (LAD) tissues and evaluate its involvement in cell proliferation and chemosensitivity to cisplatin in LAD cells. Paraffin-embedded tissues from 50 LAD patients were collected to detect FOXP3 and Ki-67 expression using immunohistochemistry (IHC). Downregulation of FOXP3 in A549 cells was performed using siRNA transfection. Real-time PCR or western blot assay was performed to analyze FOXP3 expression in A549 cells. Cell proliferation and cisplatin cytotoxicity test were assessed by CCK-8 assay. The expression of FOXP3 was significantly associated with lymph node metastasis and TNM stage of LAD patients. The FOXP3 expression was positively correlated with Ki-67 labelling index(LI)in LAD tissues. The downregulated expression of FOXP3 by siRNA transfection significantly inhibited cell proliferation and enhanced chemosensitivity to cisplatin in A549 cells. The expression of FOXP3 was significantly upregulated following cisplatin treatment in A549 cells. Our study indicates that FOXP3 may potentially be a novel molecular target in combating drug resistance in the chemotherapy of LAD.
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Another potentially important result of our study is that the simultaneous presence of KRAS mutations and high Ki67 expression is related to a worse response to CRT. Ki67 is a reliable marker/parameter for measuring the proliferative activity of the cell and could be a useful prognostic marker in some cancer types [43,44]. A previous study of Jakob et al. [45] has demonstrated that low levels of Ki67 expression are associated with good response to CRT in rectal cancer, and our results are in line with this.
In this study, we investigated the mutation status of KRAS gene in pretherapeutic and preoperative biopsies in 63 specimens of locally advanced rectal cancers in order to evaluate its potential predictive and/or prognostic role. Regions of interest of KRAS exon 2 were amplified and visualized on 2% agarose gel. Obtained PCR products were subjected to direct sequencing. KRAS mutations were detected in 35% of patients, 91% of which were located in codon 12 and 9% in codon 13. In general, KRAS mutation status did not affect the response to neoadjuvant chemoradiotherapy (CRT). However, patients harboring mutated KRAS gene, simultaneously with high vascular endothelial growth factor (VEGF) expression, exhibited a worse response to CRT (p = 0.030), a more frequent appearance of local recurrences and distant metastasis (p = 0.003), and shorter overall survival (p = 0.001) compared to all others. On the contrary, patients with GGT>GCT KRAS mutation exhibited a significantly better response to CRT than those with any other type of KRAS mutation (p = 0.017). Moreover, the presence of GGT>GCT mutation was associated with low VEGF and Ki67 expression (p = 0.012 in both cases), parameters related to less aggressiveness of the disease. Our results suggest that KRAS mutation status could have some predictive and prognostic importance in rectal cancer when analyzed together with other parameters, such as VEGF and Ki67 expression. In addition, it seems that not only the presence but the type of KRAS mutation is important for examining its impact on CRT response.
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For the development of new treatment strategies against cancer, understanding signaling networks and their changes upon drug response is a promising approach to identify new drug targets and biomarker profiles. Pre-requisites are tumor models with multiple read-out options that accurately reflect the clinical situation. Tissue engineering technologies offer the integration of components of the tumor microenvironment which are known to impair drug response of cancer cells. We established three-dimensional (3D) lung carcinoma models on a decellularized tissue matrix, providing a complex microenvironment for cell growth. For model generation, we used two cell lines with (HCC827) or without (A549) an activating mutation of the epidermal growth factor receptor (EGFR), exhibiting different sensitivities to the EGFR inhibitor gefitinib. EGFR activation in HCC827 was inhibited by gefitinib, resulting in a significant reduction of proliferation (Ki-67 proliferation index) and in the induction of apoptosis (TUNEL staining, M30-ELISA). No significant effect was observed in conventional cell culture. Results from the 3D model correlated with the results of an in silico model that integrates the EGFR signaling network according to clinical data. The application of TGFβ1 induced tumor cell invasion, accompanied by epithelial–mesenchymal transition (EMT) both in vitro and in silico. This was confirmed in the 3D model by acquisition of mesenchymal cell morphology and modified expression of fibronectin, E-cadherin, β-catenin and mucin-1. Quantitative read-outs for proliferation, apoptosis and invasion were established in the complex 3D tumor model. The combined in vitro and in silico model represents a powerful tool for systems analysis.
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The rationale for the choice of cut-off values varied between the including papers. Thirteen papers did not explain how the cut-value was decided [31,33,34,42,44,45,47,49,53–55,57,58], three papers defined the median ki-67 labeling index value as cut-off point [32,36,41] and one study calculated and used the H-score median [52]. Seven studies referred to cut-off values used in previous articles [35,37–40,46,51], and one study used the best discriminatory value [48].
The ki-67 index is a marker of proliferation in malignant tumors. Studies from the period 2000 to 2012 on the prognostic and predictive value of ki-67 labeling index (LI) in non-small cell cancer (NSCLC) are reviewed. Twenty-eight studies reported on the prognostic value of ki-67 index with various endpoints. No consensus on the prognostic value of ki-67 LI was found among the published studies neither according to disease stage nor histological subtype. Comparison of studies is hampered by differences in patient populations, methodologies and cut-off values. Five studies explored the predictive value of ki-67 to chemotherapy and none revealed significant influence. Ki-67 index seems to be of prognostic influence in NSCLC although largely variable cut-off levels have been used in the various studies and standardization of methodology is required. The relative importance of ki-67 compared to newer biomarkers has not been explored. It is likely that a signature of several biomarkers in combination may be necessary to more sufficiently stratify patients to various treatment options than is currently possible, especially when it comes to the question of the optimal use of classical chemotherapy. A predictive impact of ki-67 to treatment in NSCLC remains unclear.

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Original article: general thoracicKi-67 expression and prognosis for smokers with resected stage i Non–Small cell lung cancer

Abstract

Background

Methods

Results

Conclusions

Section snippets

Tissue samples

Clinicopathologic features and prognosis

Comment

Acknowledgements

Chest

J Thorac Cardiovasc Surg

Cell

J Immunol Methods

Ann Thorac Surg

Adv Cancer Res

Hum Pathol

Ann Thorac Surg

Eur J Cancer

Prognostic factors for surgically treated lung adenocarcinoma patients, with special reference to smoking habit

Jpn J Cancer Res

Smoking before surgery predicts poor long-term survival in patients with stage I non-small cell lung carcinomas

J Clin Oncol

A risk-stratification model of non-small cell lung cancers using cyclin E, Ki-67, and ras p21different roles of G1 cyclins in cell proliferation and prognosis

Cancer Res

Cancer cell cycles

Science

Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation

Int J Cancer

Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67

J Immunol

Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67

Am J Pathol

Original article: general thoracic
Ki-67 expression and prognosis for smokers with resected stage i Non–Small cell lung cancer