Tumor infiltrating neutrophils and gland formation predict overall survival and molecular subgroups in pancreatic ductal adenocarcinoma

Abstract

Background: RNA-sequencing-based classifiers can stratify pancreatic ductal adenocarcinoma (PDAC) into prognostically significant subgroups but are not practical for use in clinical workflows. Here, we assess whether histomorphological features may be used as surrogate markers for predicting molecular subgroup and overall survival in PDAC.

Methods: Ninety-six tissue samples from 50 patients with non-resectable PDAC were scored for gland formation, stromal maturity, mucin, necrosis, and neutrophil infiltration. Prognostic PDAC gene expression classifiers were run on all tumors using whole transcriptome sequencing data from the POG trial (NCT02155621). Findings were validated using digital TCGA slides (n = 50). Survival analysis used multivariate Cox proportional-hazards tests and log-rank tests.

Results: The combination of low gland formation and low neutrophil infiltration was significantly associated with the poor prognosis PDAC molecular subgroup (basal-like or squamous) and was an independent predictor of shorter overall survival, in both frozen section (n = 47) and formalin-fixed paraffin-embedded (n = 49) tissue samples from POG patients, and in the TCGA samples. This finding held true in the subgroup analysis of primary (n = 17) and metastatic samples (n = 79). The combination of high gland formation and high neutrophils had low sensitivity but high specificity for favorable prognosis subgroups.

Conclusions: The assessment of gland formation and neutrophil infiltration on routine histological sections can aid in prognostication and allow inferences to be made about molecular subtype, which may help guide patient management decisions and contribute to our understanding of heterogeneity in treatment response.

Keywords: molecular; pancreatic neoplasms; pathology; prognosis.

FIGURE 1

Representative images of FFPE in‐house samples with different neutrophil infiltration scores. Cases scoring ≤2 out of 4 for both stromal and luminal neutrophils were considered to have “low combined neutrophils.” H&E, X200.

FIGURE 2

When using the frozen section in‐house samples (n = 47), neutrophil infiltration and gland formation were significantly associated with basal‐like, quasi‐mesenchymal, and squamous subtypes. P‐values were calculated using the Wilcoxon–Mann–Whitney test to compare the poor prognosis subgroup (shown in bold) to the other subgroups. Boxes extend from the first to third quartile, with a line at the median. Points indicate scores for individual samples.

FIGURE 3

Correlation between percent gland formation, stromal neutrophil score, and luminal neutrophil score in the same sample, using frozen section in‐house validation samples. (A) Stromal and luminal neutrophil scores were well correlated, whereas (B,C) gland formation was poorly correlated with neutrophil scores. Bubble size is proportional to the number of case with a given score combination, ranging up to 8 for (A), up to 5 for (B), and up to 6 for (C). Spearman correlation (rho) and P‐values are shown.

FIGURE 4

The proportion of samples with low gland formation and low neutrophils (either [A] stromal or [B] luminal) in each subgroup of the gene expression‐based classifiers, scored using the frozen section in‐house samples (n = 47). Cases with both low gland formation and low neutrophils were significantly enriched in basal‐like, quasi‐mesenchymal, and squamous subtypes. Fisher's exact test P‐values are shown. Low gland formation was defined as ≤30% and low neutrophils as a score of ≤2 out of 4.

FIGURE 5

Kaplan–Meier curves for OS stratified by tumor gland formation and neutrophil infiltration, scored in frozen section in‐house samples. Low gland formation was defined as ≤30% and low neutrophils as a score of ≤2 out of 4. P‐values were calculated using log‐rank tests.

FIGURE 6

Subgroup and survival associations of low gland formation and low combined neutrophils in (A) pooled frozen and FFPE in‐house samples (n = 96) and (B) TCGA samples (n = 50). The top panels show the proportion of samples with low gland formation and low combined neutrophils in each subgroup of the gene expression‐based classifiers. Fisher's exact test P‐values are shown. The bottom panels show Kaplan–Meier curves for OS stratified by tumor gland formation and combined neutrophils. P‐values were calculated using log‐rank tests. Low gland formation was defined as ≤30%. Cases scoring ≤2 out of 4 for both stromal and luminal neutrophils were considered to have “low combined neutrophils.”