P14/ARF-Positive Malignant Pleural Mesothelioma: A Phenotype With Distinct Immune Microenvironment

Abstract

Introduction: The CDKN2A gene plays a central role in the pathogenesis of malignant pleural mesothelioma (MPM). The gene encodes for two tumor suppressor proteins, p16/INK4A and p14/ARF, frequently lost in MPM tumors. The exact role of p14/ARF in MPM and overall its correlation with the immune microenvironment is unknown. We aimed to determine whether there is a relationship between p14/ARF expression, tumor morphological features, and the inflammatory tumor microenvironment.

Methods: Diagnostic biopsies from 76 chemo-naive MPMs were evaluated. Pathological assessments of histotype, necrosis, inflammation, grading, and mitosis were performed. We evaluated p14/ARF, PD-L1 (tumor proportion score, TPS), and Ki-67 (percentage) by immunohistochemistry. Inflammatory cell components (CD3+, CD4+, CD8+ T lymphocytes; CD20+ B-lymphocytes; CD68+ and CD163+ macrophages) were quantified as percentages of positive cells, distinguishing between intratumoral and peritumoral areas. The expression of p14/ARF was associated with several clinical and pathological characteristics. A random forest-based machine-learning algorithm (Boruta) was implemented to identify which variables were associated with p14/ARF expression.

Results: p14/ARF was evaluated in 68 patients who had a sufficient number of tumor cells. Strong positivity was detected in 14 patients (21%) (11 epithelioid and 3 biphasic MPMs). At univariate analysis, p14/ARF-positive epithelioid mesotheliomas showed higher nuclear grade (G3) (p = 0.023) and higher PD-L1 expression (≥50%) (p = 0.042). The percentages of CD4 and CD163 in peritumoral areas were respectively higher and lower in p14/ARF positive tumors but did not reach statistical significance with our sample size (both p = 0.066). The Boruta algorithm confirmed the predictive value of PD-L1 percentage for p14/ARF expression in all histotypes.

Conclusions: p14/ARF-positive epithelioid mesotheliomas may mark a more aggressive pathological phenotype (higher nuclear grade and PD-L1 expression). Considering the results regarding the tumor immune microenvironment, p14/ARF-negative tumors seem to have an immune microenvironment less sensitive to immune checkpoint inhibitors, being associated with low PD-L1 and CD4 expression, and high CD163 percentage. The association between p14/ARF-positive MPMs and PD-L1 expression suggests a possible interaction of the two pathways. Confirmation of our preliminary results could be important for patient selection and recruitment in future clinical trials with anticancer immunotherapy.

Keywords: MPM; immune microenvironment; malignant pleural mesothelioma; p14/ARF; tumor microenvironment.

Figure 1

Kaplan Meier curves of p14/ARF positive and negative cases showing a lower survival rate in patients with p14/ARF expression. Censored patients are depicted as crosses intersecting the curve.

Figure 2

Pie chart of PD-L1 expression in p14/ARF-positive and negative samples. A higher percentage of PD-L1≥50% was noted in p14/ARF-positive samples than in p14/ARF-negative MPMs (A). Panel figures of two representative cases of p14/ARF-negative (B, D, F) and p14/ARF-positive MPM (C, E, G). (B) Histology showing trabecular pattern of MPM (hematoxylin and eosin, original magnification x 200). (D) Immunohistochemistry for PD-L1: TPS<1% (immunohistochemistry, original magnification x 200). (F) Immunohistochemistry for p14/ARF: negative (immunohistochemistry, original magnification x 200). (C) Histology showing prevalent solid pattern of MPM (hematoxylin and eosin, original magnification x 200). (E) Immunohistochemistry for PD-L1: TPS≥50% (80%) (immunohistochemistry, original magnification x 200). (G) Immunohistochemistry for p14/ARF: positive (immunohistochemistry, original magnification x 200).

Figure 3

Boruta feature selection showing predictive significance for PD-L1 expression in p14/ARF positivity. Unfilled circles indicating outliers.

Figure 4

CD4+ and CD163+ distribution in peritumoral areas. T helper lymphocytes and M2 macrophages were respectively higher and lower in p14/ARF-positive tumors than in negative samples (A). Panel figures of two representative cases of p14/ARF-negative (B–D) and p14/ARF-positive MPM (E–G). (B) Immunohistochemistry for p14/ARF: negative (immunohistochemistry, original magnification x 70). (C) Immunohistochemistry for CD163 showing a high percentage in peritumoral areas (immunohistochemistry, original magnification x 70). (D) Immunohistochemistry for CD4 showing a low percentage in peritumoral areas (immunohistochemistry, original magnification x 70). (E) Immunohistochemistry for p14/ARF: positive (immunohistochemistry, original magnification x 70). (F) Immunohistochemistry for CD163 showing a low percentage in peritumoral areas (immunohistochemistry, original magnification x 70). (G) Immunohistochemistry for CD4 showing a high percentage in peritumoral areas (immunohistochemistry, original magnification x 70).

Figure 5

Pie chart of nuclear grading in p14/ARF-positive and negative samples. p14/ARF-positive samples showed higher nuclear grade (G3) than p14/ARF-negative MPM (A). Panel figures of two representative cases of p14/ARF-negative (B, D, F) and p14/ARF positive MPM (C, E, G). (B) Histology showing an epithelioid MPM with low nuclear grading (G2 sec. Kadota et al.) (hematoxylin and eosin, original magnification x 200). (D) Immunohistochemistry for p14/ARF showing complete negative immunostaining (immunohistochemistry, original magnification x 200). (F) Immunohistochemistry for PD-L1: TPS<1% (immunohistochemistry, original magnification x 200). (C) Histology showing an epithelioid MPM with high nuclear grading (G3 sec. Kadota et al.) (hematoxylin and eosin, original magnification x 200). (E) Immunohistochemistry for p14/ARF showing strong nuclear and cytoplasmic immunostaining in most tumor cells (immunohistochemistry, original magnification x 200). (G) Immunohistochemistry for PD-L1: TPS≥50% (immunohistochemistry, original magnification x 200).

Figure 6

Boruta feature selection showing the highest importance of nuclear grade and PD-L1 expression among all variables in determining p14/ARF status. Unfilled circles indicating outliers.