Elsevier

Lung Cancer

Volume 135, September 2019, Pages 151-160
Lung Cancer

Stromal markers of activated tumor associated fibroblasts predict poor survival and are associated with necrosis in non-small cell lung cancer

https://doi.org/10.1016/j.lungcan.2019.07.020Get rights and content

Highlights

  • The prognostic value of fibrotic markers α-SMA and fibrillar collagens is unclear in NSCLC.

  • Fibrotic markers α-SMA and fibrillar collagens were associated with poor survival.

  • Prognostic value of α-SMA and fibrillar collagens was independent of TNM staging.

  • Fibrotic markers were associated with necrosis, which is often indicative of hypoxia.

  • Patients with desmoplastic tumors are at higher risk, and could be considered for adjuvant therapy.

Abstract

Objectives

Tumor associated fibroblasts (TAFs) are essential contributors of the progression of non-small cell lung cancer (NSCLC). Most lung TAFs exhibit an activated phenotype characterized by the expression of α-SMA and fibrillar collagens. However, the prognostic value of these activation markers in NSCLC remains unclear.

Material and Methods

We conducted a quantitative image analysis of α-SMA immunostaining and picrosirius red staining of fibrillar collagens imaged by bright-field and polarized microscopy, respectively, using tissue microarrays with samples from 220 surgical patients, which elicited a percentage of positive staining area for each marker and patient.

Results

Kaplan-Meier curves showed that all TAF activation markers were significantly associated with poor survival, and their prognostic value was independent of TNM staging as revealed by multivariate analysis, which elicited an adjusted increased risk of death after 3 years of 129% and 94% for fibrillar collagens imaged with bright-field (p = 0.004) and polarized light (p = 0.003), respectively, and of 89% for α-SMA (p = 0.009). We also found a significant association between all TAF activation markers and tumor necrosis, which is often indicative of hypoxia, supporting a pathologic link between tumor desmoplasia and necrosis/hypoxia.

Conclusions

Our findings identify patients with large histologic coverage of fibrillar collagens and α-SMA + TAFs to be at higher risk of recurrence and death, supporting that they could be considered for adjuvant therapy.

Introduction

Lung cancer is the leading cause of cancer-related death in both men and women worldwide, with an overall 5 year survival rate of 18% [1,2]. This leading position is partly associated with the fact that most lung cancer patients remain undiagnosed until the disease is symptomatic and has reached an advanced stage [3]. Surgically-treated patients tend to exhibit better prognosis, particularly when diagnosed at early stages, but still have a suboptimal potential cure of only ˜30-50% [1].

Histologically, non-small cell lung cancer (NSCLC) is diagnosed in up to 90% of lung cancer patients, and adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are the major subtypes [1]. Because these lung cancer subtypes are epithelial in origin, most previous studies have focused on the pathologic features of lung carcinoma cells. However, it is increasingly acknowledged the prominent role of the stiff desmoplastic tumor stroma that surrounds carcinoma cells in the progression of lung cancer and other solid tumors [4]. This desmoplastic stroma is rich in activated fibroblasts (referred to as cancer- or tumor-associated fibroblasts (TAFs)), infiltrated immune cells and other less frequent cell types, in the background of an abundant deposition of fibrillar collagens and other fibrotic extracellular matrix components [4,5]. Of note, TAFs are largely responsible for the aberrant stromal deposition of fibrillar collagens within the tumor stroma [6], and are receiving increasing interest as a therapeutic target [7], as illustrated by the recent approval of the antiangiogenic and antifibrotic drug nintedanib to treat lung ADC patients in combination with docetaxel [8,9].

In addition to their therapeutic relevance, studies in vitro have reported the prognostic value of epigenetic and transcriptional signatures associated with pulmonary TAFs [10,11]. In contrast, little is known on the clinical relevance of standard markers of activated fibroblasts in histologic samples from NSCLC patients. Thus very few studies have examined the prognostic value of stromal alpha-smooth muscle actin (α-SMA) in lung cancer, which is the gold standard marker of fibroblast activation [12]; moreover these studies have reported contradictory results [13,14], underscoring that the prognostic value of α-SMA in NSCLC remains unclear. In addition to α-SMA, the deposition of fibrillar (type I and III) collagens is another common marker of activated fibroblasts; however, to our knowledge their prognostic value in NSCLC has not been determined. To address this gap of knowledge we conducted a retrospective multicenter study of the prognostic value of standard markers of activated fibroblasts in using tissue microarrays (TMAs) containing samples from a cohort of early stage surgically-treated NSCLC patients gathered from multiple hospitals in Spain [15]. For this purpose we performed a quantitative image analysis of both α-SMA immunostaining and picrosirius red (PSR) staining of fibrillar collagens imaged with bright field and polarized microscopy, respectively, and combined these data with clinical information including survival gathered within a 3 year follow-up.

Section snippets

Patients and tissue samples

This study involved the retrospective analysis of tissue samples from surgical patients collected by multiple Spanish hospitals belonging to the Bronchogenic Carcinoma Cooperative Group of the Spanish Society of Pneumology and Thoracic Surgery (GCCB-S) [15], as part of their contribution to the 8th edition of the IASLC staging project [16]. The initial cohort included 220 patients that were observed during a minimum of 3 years. Eighty-three patients died within the three years after surgery

Description of clinicopathologic data

We analyzed retrospectively TMAs containing samples from a cohort of 220 surgical patients gathered from multiple hospitals in Spain, which were observed during a minimum of 3 years. TMAs included samples from 3 representative tumor regions for each patient. The clinicopathologic features of our cohort and their overall survival (OS%) 3 years after surgery are shown in Table 1. The median patient age at the day of surgery was 66.2 years, with a range of 31.5–85.0 years, and the OS% was 60.3%.

Discussion

A hallmark of NSCLC and other solid tumors is the presence of a stiff desmoplastic stroma rich in activated TAFs in the background of an excessive deposition of fibrillar collagens [5]. Stromal TAFs have been implicated in virtually all steps of tumor progression [7]. Therefore it is unsurprising that stromal proteins, including those related to TAFs, are receiving increasing interest as biomarkers or therapeutic targets [9,30]. However, the prognostic value of standard markers of activated

Conclusions

In summary this study clarifies the prognostic value of α-SMA in NSCLC, and identifies the histologic coverage of fibrillar collagens assessed through PSR staining, particularly when imaged with polarized microscopy, as a novel and independent stromal biomarker associated with adverse prognosis in resected patients. Our analysis also identifies patients with large fibrillar collagen content to exhibit higher risk of recurrence and death, and reveal that they may benefit from a closer follow-up

Declaration of competing interest

None.

Acknowledgements

We thank Adriana Velásquez, Concepción Fernández (UB) and Victoria Stanley (UCSD) for technical support, and Daniel Navajas and Ramon Farré (UB) for support. This work was further supported by grants from the Ministerio de Economía y Competitividad (MINECO/FEDER, UE) (PI13/02368 and SAF2016-79527-R to JA, FIS 12/02040 to EM and FIS 12/02534 to EB), Fundació Privada Cellex (to JA), Generalitat de Catalunya AGAUR (SGR 661 to JA, SGR 801 to EM), Junta Provincial de Barcelona de l’Associació

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