Patients

Fifty-four cases were included in the study. There were 26 B-SqCC (including 12 cases of PB-SqCC), 19 PD-SqCC and 9 SCLC cases. The mean age was 70 years (range = 53–79 years) in patients with B-SqCC, 69 years (range = 55–79 years) in PD-SqCC and 71 years (range = 54–88 years) in SCLC. Likewise, the gender distribution was 35% (n = 9), 42% (n = 8) and 56% female (n = 5), respectively.

Clinical outcome

Overall, mean follow-up was 22 months (range = 1–120 months). In patients with B-SqCC, there were 3 (12%) mortalities, 11 (42%) patients with disease recurrence or metastasis, and 12 (46%) with no event. Likewise, in patients with PD-SqCC, there was 1 (11%) mortality, 5 (26%) patients with disease recurrence or metastasis, and 13 (68%) with no event. Patients with PB-SqCC had the highest event rate. There was 1 (8%) mortality, 7 (58%) patients with recurrence or metastasis, and only 4 (33%) with no event. Controlling for stage, patients with B-SqCC demonstrated a trend towards poorer progression-free survival (HR = 2.16, 95%CI = 0.82–5.48, p = 0.13) compared to patients with PD-SqCC. Patients with PB-SqCC experienced significantly worse progression-free survival (HR = 3.12, 95%CI = 1.03–9.39, p = 0.043) adjusted for stage, compared to PD-SqCC (Suppl Fig. 1).

Histology and immunohistochemistry

Morphological features were assessed in each tumor type (Fig. 1). Nuclear molding and peripheral palisading occurred more frequently in B-SqCC compared to PD-SqCC (p = 0.021; p < 0.001 respectively) while nuclear pleomorphism and nucleoli were less prominent in B-SqCC than in PD-SqCC (p = 0.003; p = 0.001 respectively) (Table 1).

Table 1 Histological characteristics by tumor type

The pattern of neuroendocrine expression by IHC ranged from small discrete areas to full block positivity and stain intensity ranged from very weak to high intensity (Table 2). Overall, 65% (17/26) of B-SqCC and 67% (8/12) of PB-SqCC demonstrated > 10% NE reactivity with one NE marker compared to 21% (4/19 cases) of PD-SqCC (p = 0.006, p = 0.031, respectively). Positivity for more than one NE marker was seen in 27% (7/26) of B-SqCC cases, while no PD-SqCC cases were positive for more than one NE marker.

Table 2 IHC marker reactivity by tumor type

We also compared reactivity with other IHC markers routinely used in the diagnostic workup of SqCC. Interestingly, B-SqCC demonstrate lower expression of p40 with 19% of cases showing < 10% positivity, while 23% of B-SqCC cases showed > 10% positivity with TTF1 (Suppl Table 1) As stated in Methods Section Histology and immunohistochemical review, cases with features overlapping with small cell/large cell neuroendocrine carcinoma or thoracic SMARCA4-deficient undifferentiated tumor were excluded. Therefore, cases with TTF1 positivity were enrolled as B-SqCC if they had also high (> 70%) expression of p40 and CK5, while cases with low p40, were enrolled as B-SqCC if negative for TTF1, showed negative or low expression of NE markers (< 10%) and showed CK5 positivity (20 to 100% in our cohort).

Identification of differentially expressed genes (DEGs)

One hundred sixty four candidate genes, the expression of which is involved in neuroendocrine phenotype, were identified based on our literature review [12,13,14,15]. Of these, only 102 (62.2%) met the minimum read count. There were 6 paired samples, which contained both NE+ and NE- areas within the same tumor. They were composed of 4 (67%) PB-SqCC, 1 (16.6%) B-SqCC and 1 (16.6%) mixed B-SqCC/SCLC (index case).

Thirty genes were differentially expressed in paired samples (Table 3). Notably, downregulation of EP300, RBL2, PTEN and CREBBP and upregulation of TP73 and EEF1A2 was identified. These genes are known to play a role in the pathogenesis of SCLC 20. Downregulation of NOTCH2 and upregulation of DLK1, ASCL1, NOTCH3 and CHGB was also observed and are signs of NOTCH pathway inhibition (Fig. 2a).

Table 3 Genes associated with neuroendocrine differentiation that showed differential expression in neuroendocrine positive areas in basaloid squamous cell carcinoma samples
Fig. 2
figure 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

Evidence of NOTCH pathway inhibition was identified in 83.3% (5/6) of paired B-SqCC samples, as evidenced by either upregulation of ASCL1 or DLK1, or downregulation of NOTCH2 or HEY2. In all samples with evidence of NOTCH pathway inhibition, at least 1 downstream marker of NE differentiation (NCAM1, CHGA, CHGB, SYP, INSM1) was upregulated. In 60% of samples (3/5), 3 or more downstream markers of NE differentiation were upregulated (Fig. 2b).

Validation of differentially expressed genes (DEGs)

There were 9 unpaired B-SqCC samples, entirely composed of NE+ or NE- cells, included in the study. Of those, 4 (44%) were NE+ and 5 (56%) were NE-. Gene expression analysis of unpaired B-SqCC samples reproduced many of the findings identified in the paired sample analysis, such as decreased gene expression in KIF1A and increased gene expression in CEACAM5, EEF1A2, CHGB, CALML3 and TP73 in NE+ samples (Supple Table 2).

Hierarchical clustering, using the 30 DEGs identified in the paired analysis, was performed on the unpaired B-SqCC samples. Hierarchical clustering distinguished cases by their corresponding immunohistochemical NE status (p = 0.048) (Fig. 3a). This result provides further molecular support for NE differentiation in NE+ B-SqCC cases suggested by IHC. The gene expression profile of NE+ cases demonstrated upregulation of NOTCH3, CHGB, CEACAM5 and EEF1A2 as well as downregulation of RBL2, PTEN, EP300, ADNP and KIF1A. NCAM1 was removed from the analysis, as it was used as an IHC marker to determine the NE status of the cases.

Fig. 3
figure 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)

Hierarchical clustering of TCGA samples

Hierarchical clustering, using the same DEGs as above, was performed on TCGA samples. In total, 15 B-SqCC and 30 random SqCC TCGA samples were included. Hierarchical clustering of TCGA samples using the NE-implicated DEGs distinguished cases by tumor type (B-SqCC versus SqCC) (p = 0.0094) (Fig. 3b). Interestingly, higher expression of ASCL1, DLK1, CHGB and NCAM1 was observed in a subset of cases in the B-SqCC enriched cluster. Heightened expression of these 4 genes is associated with NOTCH pathway inhibition.

The index case: a diagnostic dilemma of a single tumor with both B-SqCC and SCLC features

The index case is a 2.8 cm, pT2N2 tumor separated into three regions based on morphological and immunohistochemical characteristics: (1) PB-SqCC without NE differentiation, (2) PB-SqCC with NE differentiation and (3) SCLC (Fig. 4). On histology, the tumor consists of nests and clusters of basaloid-looking cells with intermediate-sized pleomorphic nuclei, a small rim of cytoplasm and shows peripheral palisading, molding, and focal rosetting. Zonal necrosis was not present, however, there was abundant individual cell apoptosis. Parts of the tumor demonstrated p40 and CK5 positivity, together with negative to patchy neuroendocrine positivity (60% CD56, 30% chromogranin and 50% reactivity with synaptophysin). These areas merged in a continuous fashion with another part of the tumor showing no p40/CK5 positivity as well as diffuse and strong CD56, Chromogranin and synaptophysin reactivity. Correlating with IHC, the histological features were quite similar in the basaloid-like area compared to the SCLC-like area except for the fact that SCLC clusters were very compact with marked nuclear molding and no discernable cytoplasm.

Fig. 4
figure 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

Gene expression analysis was performed on each of the three regions. SCLC and NOTCH pathway genes such as ASCL1, DLK1, CEACAM5, SST, CHGB, NCAM1, TP73, EEF1A2, TMSB15A, INA and HEY1 showed lowest expression in the B-SqCC NE- region, intermediate expression in the PB-SqCC NE+ region and highest expression in the SCLC region. PTEN and NOTCH2 showed the opposite pattern with highest expression in the PB-SqCC NE- region and lowest expression in the SCLC region.

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