Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features

Abstract

Introduction: HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear.

Materials and methods: HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression.

Result: HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors.

Conclusion: HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.

Keywords: A549; HNF4α; KRAS; Lung adenocarcinoma; SMARCA4; TTF-1.

Fig. 1

HE (top) and HNF4α staining (bottom) sections from five representative samples of HNF4α-positive lung adenocarcinomas. Scale bar: 50 μm

Fig. 2

a The histological subtypes (mucinous, enteric, colloid, papillary, acinar, and solid adenocarcinomas), histological grades, immunohistochemical expression of HNF4α, TTF-1, SMARCA4, SMARCA2 and MUC5AC and genetic mutations of EGFR and KRAS in 33 HNF4α-positive lung adenocarcinoma cases, with division into the variant and non-mucinous groups. b HE, SMARCA4, HNF4α, and TTF-1 staining of representative cases of HNF4α-positive non-mucinous adenocarcinomas with loss of SMARCA4 (Cases 22 and 26). Both cases were grade 3 adenocarcinomas, SMARCA4 lost, HNF4α-positive, and TTF-1-negative. Note that lymphoid cells within the tumor were SMARCA4-positive (100 × magnification, Scale bar: 100 μm)

Fig. 2

a The histological subtypes (mucinous, enteric, colloid, papillary, acinar, and solid adenocarcinomas), histological grades, immunohistochemical expression of HNF4α, TTF-1, SMARCA4, SMARCA2 and MUC5AC and genetic mutations of EGFR and KRAS in 33 HNF4α-positive lung adenocarcinoma cases, with division into the variant and non-mucinous groups. b HE, SMARCA4, HNF4α, and TTF-1 staining of representative cases of HNF4α-positive non-mucinous adenocarcinomas with loss of SMARCA4 (Cases 22 and 26). Both cases were grade 3 adenocarcinomas, SMARCA4 lost, HNF4α-positive, and TTF-1-negative. Note that lymphoid cells within the tumor were SMARCA4-positive (100 × magnification, Scale bar: 100 μm)

Fig. 3

a Overall survival among 213 cases of non-mucinous adenocarcinomas categorized according to the WHO grading system. b The prognoses of 230 lung adenocarcinomas were analyzed in 6 groups; HNF4α + G3: HNF4α-positive grade 3 (n = 6), HNF4α-G3: HNF4α-negative grade 3 (n = 50), HNF4α + G2: HNF4α-positive grade 2 (n = 9), HNF4α-G2: HNF4α-negative grade 2 (n = 119), HNF4α-G1:HNF4α-negative grade 1 (n = 29), and the variant group (n = 17). The samples with unknown prognoses (n = 5) and double carcinoma cases (n = 3) were excluded

Fig. 4

a Genetic status of EGFR, MET, HER2, KRAS, and SMARCA4 (upper panel), gene-level expressions of HNF4A, TTF-1, and SMARCA4 (middle panel), and protein expression levels of HNF4α, TTF-1, SMARCA4, and ACTB (lower panel) for 8 cell lines, including the four cell lines that highly express HNF4A (A549, H2405, H1651, and Calu-3) and the four cell lines that highly express TTF-1 (HCC827, PC3, H1648, and H2009). In the upper panel, the gray box indicates the presence of genetic abnormalities and the white box indicates the absence of genetic abnormalities. In the middle lane, red means more than or equal to the average of each gene expression, orange means under the average but more than or equal to one-quarter of the average, and green means under one-quarter of the average. b The histological features and immunohistochemical expression patterns of HNF4α, TTF-1, and SMARCA4 for the xenograft tumors of A549, H2405, H1651, and Calu-3