Prognostic effect of intratumoral neutrophils across histological subtypes of non-small cell lung cancer

Abstract

Recent data indicate that tumor-associated neutrophils (TANs) serve a dual role in tumor progression and regression. CD66b is a neutrophil marker and has been associated with patient outcome in various cancers. However, its clinical impact in non-small cell lung cancer (NSCLC) remains controversial. 536 NSCLC patients, of which 172 harbored lymph node metastases, were included in this study. Tissue microarrays were constructed and multiplexed immunohistochemistry of CD66b, CD34 and pan-keratin was performed to evaluate the localization and quantity of CD66b+ TANs. High intratumoral CD66b+ TANs density in squamous cell carcinoma (SCC) subgroup was an independent positive prognosticator for disease-specific survival (P = 0.038). In contrast, high intratumoral TANs density was an independent negative prognostic factor in the adenocarcinoma (ADC) subgroup (P= 0.032). Likewise, in ADC patients with lymph node metastases, high level of intratumoral TANs was associated with poor prognosis (P = 0.003). Stromal CD66b+ TANs were not associated with outcome of NSCLC patients. In conclusion, CD66b+ TANs show diverging prognostic effect in NSCLC patients according to histological subgroups. The presence of CD66b+ TANs could prove pivotal for development of an immunoscore in ADC NSCLC patients.

Keywords: CD66b; NSCLC; TANs; lung cancer; neutrophils.

Figure 1

A. Scoring assessment guideline. Scoring of intratumoral (tumor nest) and stromal CD66b⁺ neutrophils was conducted utilizing the following exclusion- and inclusion-criteria. In intratumoral assessment, we have included only unaggregated CD66b⁺ cells completely surrounded by tumor epithelial cells. Stromal assessment only regarded extravascular CD66b⁺ cells. The excluded areas consisted of intratumoral and stromal aggregates of neutrophils, intravascular neutrophils and CD66b⁺ cells with granular background in stroma. Especially in SCC tissue, the central tumor zone often had dense granular CD66⁺ structures with some CD66b⁺ cells, considered a pre- or necrotic area, which were excluded from scoring. B. Multiplexed IHC analysis of TANs with CD66b/CD34/pan-CK panel, high versus low intratumoral densities. Brown, purple and yellow substrates were applied to visualize CD66b, CD34 and pan-CK respectively (magnification 10x, 20x).

Figure 2. Disease-specific survival curves for

A. Intratumoral CD66b in the overall cohort (OC) of primary tumors (PT); B. Intratumoral CD66b in squamous cell carcinomas (SCC) of PT; C. Intratumoral CD66b in adenocarcinomas (ADC) of PT; D. Stromal CD66b in the overall cohort of PT; E. Stromal CD66b in SCC of PT; F. Stromal CD66b in ADC of PT; G. Intratumoral CD66b in the overall cohort of LN+; H. Intratumoral CD66b in SCC of LN+; I. Intratumoral CD66b in ADC of LN+