Abstract
In this study, we developed an image analysis algorithm for quantification of two potential apoptotic biomarkers in non-small-cell lung cancer (NSCLC): FLIP and procaspase-8. Immunohistochemical expression of FLIP and procaspase-8 in 184 NSCLC tumors were assessed. Individual patient cores were segmented and classified as tumor and stroma using the Definiens Tissue Studio. Subsequently, chromogenic expression of each biomarker was measured separately in the nucleus and cytoplasm and reported as a quantitative histological score. The software package pROC was applied to define biomarker thresholds. Cox proportional hazards analysis was applied to generate hazard ratios (HRs) and associated 95% CI for survival. High cytoplasmic expression of tumoral (but not stromal) FLIP was associated with a 2.5-fold increased risk of death in lung adenocarcinoma patients, even when adjusted for known confounders (HR 2.47, 95% CI 1.14–5.35). Neither nuclear nor cytoplasmic tumoral procaspase-8 expression was associated with overall survival in lung adenocarcinoma patients; however, there was a significant trend (P for trend=0.03) for patients with adenocarcinomas with both high cytoplasmic FLIP and high cytoplasmic procaspase-8 to have a multiplicative increased risk of death. Notably, high stromal nuclear procaspase-8 expression was associated with a reduced risk of death in lung adenocarcinoma patients (adjusted HR 0.31, 95% CI 0.15–0.66). On further examination, the cells with high nuclear procaspase-8 were found to be of lymphoid origin, suggesting that the better prognosis of patients with tumors with high stromal nuclear procaspase-8 is related to immune infiltration, a known favorable prognostic factor. No significant associations were detected in analysis of lung squamous cell carcinoma patients. Our results suggest that cytoplasmic expression of FLIP in the tumor and nuclear expression of procaspase-8 in the stroma are prognostically relevant in non-small-cell adenocarcinomas but not in squamous cell carcinomas of the lung.
Highlights
Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer diagnoses and is the most common cause of death from cancer globally.[1,2,3] The AJCC/UICC-TNM system remains the gold standard for assessing patient prognosis; it gives no prediction for treatment benefit
Our findings indicate that high cytoplasmic FLIP intensity and hematoxylin intensity thresholds were determined, expression in tumor cells is an important determinant of patient and we used these to segment and identify both positive and prognosis for non-small-cell adenocarcinoma but not for squa- negative nuclei within the tumor and positive and negative nuclei mous carcinoma
Procaspase-8 and its endogenous inhibitor FLIP are key regulators of the extrinsic apoptotic pathway activated by cell surface death receptors and are key regulators of cell death induced by cytoplasmic complexes, such as the ripoptosome.[12,22]
Summary
Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer diagnoses and is the most common cause of death from cancer globally.[1,2,3] The AJCC/UICC-TNM system remains the gold standard for assessing patient prognosis; it gives no prediction for treatment benefit. From the segmented and classified tumoral and stromal cells, the expression was quantified in each compartment, and using brown chromogen intensity and hematoxylin intensity thresholds, a H-score was calculated for nuclear and cytoplasmic FLIP and procaspase-8 using the formula: (1 × percentage of low brown chromogen intensity-positive cells)+(2 × percentage of medium brown chromogen intensity-positive cells)+(3 × percentage of high brown chromogen intensity-positive cells) based on the quantitative data generated from the image analysis algorithm.
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