Neuroendocrine differentiation distinguishes basaloid variant of lung squamous cell carcinoma

Abstract

Background: Neuroendocrine (NE) differentiation is widely studied in non-small cell lung carcinomas (NSCLC) however, its significance remains unclear in basaloid squamous cell carcinomas (B-SqCC). This study aims to assess the extent of NE differentiation in B-SqCC and characterize the underlying molecular process.

Methods: This study evaluated resected B-SqCC, small cell lung cancer (SCLC) and poorly differentiated SqCC (PD-SqCC) from 2005 to 2020 at the Ottawa Hospital. Samples were subject to pathological review, immunohistochemistry (IHC) and survival analysis. Gene expression analysis was performed on B-SqCC samples exhibiting NE+ and NE- regions (paired samples) to identify differentially expressed genes (DEGs). These DEGs were subsequently validated in unpaired B-SqCC and TCGA samples.

Results: B-SqCC cases were more likely to exhibit nuclear molding, resetting and peripheral palisading than PD-SqCC. B-SqCC were also more likely to demonstrate NE differentiation compared to PD-SqCC (p = 0.006). Pure basaloid squamous cell carcinoma (PB-SqCC) experienced poorer disease-free survival (HR = 3.12, p = 0.043) adjusted for stage. Molecular characterization of paired B-SqCC samples demonstrated DEGs implicated in NOTCH signaling, SCLC and pulmonary neuroendocrine differentiation. Hierarchical clustering using discovered DEGs in unpaired B-SqCC samples distinguished tumors based on NE status (p = 0.048). Likewise, clustering The Cancer Genome Atlas (TCGA) samples with DEGs distinguished B-SqCC from SqCC samples (p = 0.0094).

Conclusion: This study provides IHC and molecular evidence of significant NE-differentiation in B-SqCC and demonstrates their aggressive clinical behavior. These findings suggest that B-SqCC are biologically distinct from SqCC and share characteristics with SCLC.

Keywords: Basaloid squamous cell carcinoma; Gene expression; Immunohistochemistry; NOTCH; Neuroendocrine differentiation.

Fig. 1

The photomicrograph shows morphological features of A small cell lung carcinoma, B pure-basaloid squamous cell carcinoma, C basaloid squamous cell carcinoma (focal <50% squamous cell differentiation) and D poorly differentiated squamous cell carcinoma

Fig. 2

A A Overview of NOTCH and TP73 signaling. Arrows indicate activation, while blunt ends indicate inhibition. Note, upregulation of DLK1 inhibits NOTCH activity, while ASCL1 is inhibited by NOTCH. TP73 is upregulated in areas of neuroendocrine positivity and contributes to oncogenesis through an alternative pathway. B Heat map depicting differences in gene expression (log₂ fold-change) of genes in the NOTCH pathway in 6 paired B-SqCC samples. Red signifies lower expression, while green signifies heightened expression in neuroendocrine regions. In 5/6 samples, upstream inactivation (low expression of NOTCH2 or high expression of ASCL1, DLK1 or NOTCH3) with corresponding upregulation downstream neuroendocrine markers (CHGA, CHGB, NCAM1, INSM1 and SYP) is observed of genes in the NOTCH pathway. Red signifies lower expression, while green signifies heightened expression in neuroendocrine regions. In 5/6 samples, upstream inactivation (low expression of NOTCH2 or high expression of ASCL1, DLK1 or NOTCH3) with corresponding upregulation downstream neuroendocrine markers (CHGA, CHGB, NCAM1, INSM1 and SYP) is observed

Fig. 3

Hierarchical clustering using differentially expressed genes. Red indicates decreased expression, while green indicates heightened expression. A Hierarchical clustering of unpaired basaloid squamous cell carcinoma samples distinguishes tumors based on immunohistochemical NE status. Cluster 1 is enriched for NE negative (by IHC) samples (0/4), while Cluster 2 is enriched in NE positive tumors (4/5) (p = 0.048). B Hierarchical clustering of TCGA samples distinguished cases based on tumor type. Cluster 1 is enriched with non-basaloid squamous cell carcinomas (2/19 are basaloid type), while Cluster 2 is enriched with basaloid squamous cell carcinomas (13/26 are basaloid type) (p = 0.0094). A subset of cases from Cluster 2 (Sub-cluster 1) demonstrate higher expression of NOTCH pathway genes including DLK1 (log₂ FC = 2.8), ASCL1 (log₂ FC = 1.8), CHGB (log₂ FC = 1.0) and NCAM1 (log₂ FC = 2.9)

Fig. 4

The photomicrographs show the morphological features and immunohistochemical profile of a tumor exhibiting small cell lung carcinoma (SCLC) morphology transitioning to basaloid carcinoma (B-SqCC) features. A The SCLC characterized by hyperchromatic densely packed cells with scant cytoplasm on the left (*) and pure B-SqCC with hyperchromatic nuclei surrounded by moderate eosinophilic cytoplasm on the right (**). The immunohistochemical stains highlight the transitioning spatial differentiation with B TTF-1 positive staining in SCLC and negative staining in pure B-SqCC and conversely C p40 negative staining in SCLC and strong positive in pure B-SqCC. On the other hand neuroendocrine stains including D Chromogranin, E Synaptophysin and F CD56 are positive in SCLC while pure B-SqCC is either focally positive or is negative