Elsevier

Human Pathology

Volume 46, Issue 12, December 2015, Pages 1922-1934
Human Pathology

Original contribution
Loss of the retinoblastoma tumor suppressor correlates with improved outcome in patients with lung adenocarcinoma treated with surgery and chemotherapy,☆☆

https://doi.org/10.1016/j.humpath.2015.08.010Get rights and content

Summary

The retinoblastoma tumor suppressor pathway is frequently inactivated in human cancer, enabling unrestrained proliferation. Most cancers, however, maintain expression of a wild-type (WT) retinoblastoma tumor suppressor protein (pRB). It is generally in a hyperphosphorylated state (ppRB) because of mutations in upstream regulators such as p16 and cyclin D. Hyperphosphorylated ppRB is considered inactive, although data are emerging that suggest it can retain some function. To test the clinical relevance of pRB status, we obtained archival tissue sections from 91 cases of lung adenocarcinoma resected between 2003 and 2008. All cases received platinum doublet chemotherapy, and the median survival was 5.9 years. All cases were assessed for pRB and ppRB using immunohistochemistry and quantified based on intensity of signal and proportion of positive cells. pRB expression was lost in 15% of lung adenocarcinoma cases. In tumors that did not express pRB, the survival rate was significantly improved (hazard ratio, 0.21; 95% confidence interval, 0.06-0.69; P = .01) in comparison to tumors that express pRB. pRB status was found to be an independent predictor of overall survival on multivariate analysis (hazard ratio, 0.22; 95% confidence interval, 0.07-0.73; P = .01) along with increased stage and age. pRB status did not alter baseline levels of apoptotic or proliferative markers in these tumors, but the DNA damage response protein 53BP1 was higher in cancers with high levels of pRB. In summary, loss of pRB expression is associated with improved survival in patients treated with surgical resection and chemotherapy. This may be useful in classifying patients at greatest benefit for aggressive treatment regimes.

Introduction

Lung cancer is the leading cause of cancer-related death in North America [1]. The disease is separated into small cell carcinoma (SCLC) and non–small cell carcinoma (NSCLC). Adenocarcinoma is a subtype of NSCLC accounting for the majority of all lung cancer and is the least associated with smoking [2]. Outcomes for patients with NSCLC are suboptimal, with a median survival of approximately 5 years for patients with stage 1 disease, but only 4 months for patients with stage 4 disease [3]. Only patients with completely resectable disease or a select group of patients with more advanced disease treated with aggressive chemotherapy and radiation regimes have the potential for long-term survival or cure. For patients with stage II-III resectable disease, the addition of chemotherapy improves survival compared to surgery alone [4]. The current practice in lung oncology is rapidly changing to include the use of molecularly targeted agents [5]. The use of tyrosine kinase inhibitors in cancers with mutated epidermal growth factor receptor (EGFR) has been shown to dramatically increase survival [6]. The remainder of patients who do not have targetable driver mutations and patients that fail EGFR inhibition are treated with conventional therapies using cytotoxic agents.

One of the mainstays of chemotherapy for lung cancer is platinum doublets that consist of cisplatin or carboplatin, with a vinca alkaloid, typically vincristine or vinblastine [7]. This double-agent chemotherapy has been shown to provide a survival benefit when given in the adjuvant setting [7]. Most cases, however, do not have significantly prolonged survival in response to these agents, with a difference in survival at 1 year of 29% for patients treated with platinum doublet versus 20% for supportive care alone [7]. Given the harsh toxicities associated with these treatments, there is an important need for prognostic markers that may better define the potential response of patients to these treatments.

Surprisingly, loss of the retinoblastoma tumor suppressor protein (pRB) has been associated with improved survival rates, especially in cancers such as breast or ovarian [8], [9]. pRB is a tumor suppressor encoded by the RB1 gene, and disruption of its function leads to unrestrained proliferation, one of the central features of cancer [10]. In SCLC, RB1 expression is lost in more than 90% of cases [11]. In contrast, RB1 expression is maintained in the majority NSCLC cases including adenocarcinoma, as 80% to 95% have detectable RB protein [12], [13], [14], [15]. It is well established that SCLC is initially highly sensitive to chemotherapy, whereas NSCLCs tend to be resistant to standard chemotherapies [7], [16]. Intriguingly, pRB expression in NSCLC has been associated with improved survival [17], [18]. Despite these previous studies, there is little insight into the mechanism by which pRB may influence outcome or whether its expression is merely correlative.

pRB functions by binding to and regulating E2F transcription factors to control the G1-S phase of the cell cycle [10], [19]. Disruption of the pRB pathway occurs through loss of the RB1 gene or inactivation of pRB through phosphorylation. This can occur through up-regulation of cyclin-dependent kinase complexes containing CDK4 or CDK2 or by loss of inhibitor proteins such as p16 [10], [19]. In all of these scenarios, pRB is phosphorylated, preventing E2F regulation and proliferative control. Recently, the function of pRB has been extended beyond regulating E2F gene expression at the G1-S transition point. There are a number of scenarios where pRB apparently maintains function that may influence cancer cell survival and proliferation. pRB participates in cell survival through DNA damage signaling [20], the regulation of mitochondrial function [21], and maintaining chromosome stability [22]. pRB also represses pluripotency factors such as OCT4 in proliferating fibroblasts to oppose stem cell reprogramming [23] and influences cell fate decisions [24]. How phosphorylated pRB in proliferating cells is capable of these different activities remains an active area of research. Regardless of the explanation, each of these offers a potential function by which phosphorylated pRB could paradoxically support tumorigenesis.

This retrospective study sought to explore the relationship between pRB, its functional status, and their correlation with the overall survival (OS) of patients with lung cancer. The study used a cohort of patients with similar clinical case histories and was focused on resected cases of lung adenocarcinoma that received platinum doublet chemotherapy.

Section snippets

Patient selection

Pathology archives were searched for lung adenocarcinoma resection cases collected between 2003 and 2008. Histologic subtypes of adenocarcinoma were included but contributed to less than 10% of cases. Cases were reviewed for the use of chemotherapy and surgical resection. Only patients who received platinum doublet chemotherapy with platinum-based agent (cisplatin or carboplatin) and a vinca alkaloid (vinorelbine, vincristine, or vinblastine) were included. Patient demographics and outcome

Patient demographics

All patients included in this study were treated at the London Health Sciences Centre in Ontario, Canada. The baseline patient demographics are outlined in Table 1. The average age of participants was 63.2 with the majority of patients having stage I or stage II disease (71%) and an average tumor size of 4.6 cm. No clear correlation between histologic subtype and RB status was observed. Of the 34 stage I cases, most were stage Ib with only 6 stage Ia cases that received chemotherapy due to

Discussion

Expression of the retinoblastoma tumor suppressor protein was found to be lost in 15% of cases, and this was associated with an improved survival. Large-scale genomic efforts have suggested that RB1 is disrupted in 5% to 10% of cases, and this likely accounts for most cases that lose expression of pRB in our study [12], [13], [14]. The remainder of cases that lose pRB expression may do so through epigenetic mechanisms; this is supported by work in hepatocellular carcinoma where loss of pRB

Supplementary data

The following is the Supplementary data to this article.

. List of primary and secondary antibodies used in this study for immunohistochemistry on archival paraffin-embedded tissue

. Scoring of immunohistochemistry based on abundance and intensity. All IHC slides were blindly scored based on intensity and abundance. The total score was determined by a sum of the 2 individual scores

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    Competing interests: The authors have no conflicts of interest to report.

    ☆☆

    Funding/Support: M. J. C. was supported by a CIHR MD/PhD training award. M. J. C. and C. A. I. were supported by the Strategic Training Program in Cancer Research and Technology Transfer. F. A. D. is the Wolfe Senior Fellow in Tumor Suppressor Genes at Western University. This work was funded by the Canadian Institutes of Health Research (MOP-89765) Ottawa, Canada.

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