Clinicopathological features of HNF4α-positive adenocarcinomas

We conducted an immunohistochemical analysis of HNF4α using 241 primary lung adenocarcinoma samples surgically resected at Jichi Medical University Hospital and found that 33 samples (14%) were positive for HNF4α. Table 1 shows the relationships between HNF4α expression and the clinicopathological features of 238 patients (241 samples). A total of 6 lung adenocarcinoma samples from the 3 patients with double primary lung adenocarcinomas were all positive for TTF-1 and negative for HNF4α. All samples of mucinous (15/15, 100%), enteric (2/2, 100%), and colloid (1/1, 100%) adenocarcinoma exhibited HNF4α expression. HNF4α expression was detected in a proportion of acinar (4/24, 17%), papillary (6/123, 5%), and solid (5/43, 12%) adenocarcinomas. Representative figures of HNF4α-positive lung adenocarcinomas are shown in Fig. 1. None of the HNF4α-positive lung adenocarcinomas showed hepatoid differentiation. None of the in-situ non-mucinous, minimally invasive, or lepidic adenocarcinoma samples (WHO grade 1), representing TRU-type adenocarcinomas, exhibited HNF4α expression.

Table 1 Relationships among HNF4α expression and clinicopathologic factors, including expression patterns of TTF-1 and SMARCA4, and genetic status of EGFR, KRAS, ALK, HER2, MET, BRAF, RET, and ROS1 in 241 primary lung adenocarcinomasFig. 1

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

Table 1 also shows the correlations among HNF4α expression levels and driver mutations, clinicopathological factors and immunohistochemical patterns. In HNF4α-positive samples, the frequency of KRAS mutations was significantly high (20/33, 61%) (p < 0.001), and the frequency of EGFR mutations was significantly low (3/33, 9%) (p < 0.001), whereas no common drive mutations other than KRAS and EGFR (e.g., ALK, HER2, MET, BRAF, RET, or ROS1) were found. EGFR and KRAS mutations were mutually exclusive.

HNF4α expression was correlated with the advanced pT stage (pT2-pT4) (p = 0.001) and STAS (p = 0.001), but not correlated with pleural invasion, lymphatic or vessel invasion, intrapulmonary metastasis, or nodal involvement.

Immunohistochemically, HNF4α expression was correlated with a loss of SMARCA4 (p = 0.035) and MUC5AC expression (p < 0.001), and inversely correlated with the expression of TTF-1 (p < 0.001) (Table 1), but seven samples were double-positive for TTF-1 and HNF4α, including six papillary adenocarcinomas and one solid adenocarcinoma. Although the loss of SMARCA2 was not significantly more frequent in HNF4α-positive adenocarcinomas, two of the four HNF4α-positive Grade 3 adenocarcinomas that expressed SMARCA4 showed the loss of SMARCA2.

TTF-1 and SMARCA4 expression and gene mutation patterns differed in HNF4α-positive lung adenocarcinomas according to histology

Based on the 2021 WHO classification of thoracic tumors [39], we divided HNF4α-positive adenocarcinoma cases (n = 33) into two groups: the variant group (mucinous, enteric, and colloid adenocarcinomas) (n = 18) and the conventional non-mucinous group (acinar, papillary, and solid adenocarcinomas) (n = 15) (Fig. 2a). All variant group cases were diffusely HNF4α-positive and completely TTF-1-negative. None of them harbored EGFR mutations, but more than half of the cases harbored the KRAS mutation (10/18, 55.6%). In contrast, almost half of the cases in the non-mucinous group were double-positive for TTF-1 and HNF4α (7/15, 46.7%), and their expression patterns were heterogenous and mutually exclusive within the same tumor (Online Resource 5a).

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)

The three EGFR-mutated cases in the non-mucinous group were all double-positive for TTF-1 and HNF4α. Given the high frequency of EGFR mutations in these double-positive cases (3/7, 43%), we speculated that the double-positive adenocarcinomas were of the TRU-type and that TTF-1-positive TRU-type adenocarcinomas were induced to express HNF4α through the local loss of TTF-1 (e.g., by epigenetic silencing). In addition, all cases in the variant group retained SMARCA4 expression, but in the non-mucinous group, loss of SMARCA4 was detected in 3 of the 15 cases (20%), much more frequently than in HNF4α-negative non-mucinous adenocarcinomas (3/208, 1.4%) (Fig. 2a and Table 1). Figure 2b shows histological images of two representative cases of HNF4α-positive non-mucinous adenocarcinoma with the loss of SMARCA4.

The loss of SMARCA2, a paralog of SMARCA4, did not correlate with the expression of HNF4α (Table 1) and was detected among HNFα-positive cases in both the variant group (5.6%, 1/18) and conventional non-mucinous group (13.3%, 2/15) (Fig. 2a and Online Resource 5b). MUC5AC expression was frequently positive in HNF4α-positive cases (in both the variant and conventional groups), but was almost negative in TTF-1-positive cases (6/7, 85.7%) (Fig. 2a and Online Resource 5b).

HNF4α-positive non-mucinous adenocarcinomas with high-grade morphology (WHO grade 3) showed the worst prognosis

The three-tiered grading system is the common prognostic indicator of non-mucinous lung adenocarcinomas [39]. In the present study, the 5-year survival rates of grade 1 (n = 29), grade 2 (n = 128), and grade 3 (n = 56) groups were 100%, 86.0%, and 61.4% respectively, and the survival rates differed significantly (grade 1 vs. grade 2: p = 0.032, grade 2 vs. grade 3: p = 0.002) (Fig. 3a). Next, for survival analysis, we re-classified non-mucinous adenocarcinoma cases of each grade group into HNF4α-positive and HNF4α-negative groups: HNF4α-positive grade 3 group (n = 6), HNF4α-negative grade 3 group (n = 50), HNF4α-positive grade 2 group (n = 9), HNF4α-negative grade 2 group (n = 119), and HNF4α-negative grade 1 group (n = 29), as well as the variant group (n = 17). Notably, the HNF4α-positive grade 3 group showed worse prognosis than the HNF4α-negative grade 3 group (3-year survival rates of 51.4% and 69.3%, respectively) (p = 0.024), showing the worst prognosis among the six groups (Fig. 3b).

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

We found that in grade 3 non-mucinous adenocarcinomas (n = 56), sex, pleural invasion, pStage, HNF4α expression and MUC5AC expressions, were poor prognostic factors (Online Resource 6a). We performed a multivariate analysis, excluding the expression of MUC5AC, which correlated with the expression of HNF4α, and found that the expression of HNF4α and the pStage remained significant in the multivariate analysis (HR, 3.318; CI, 1.344–8.188 for HNF4α expression and HR, 9.019; CI, 4.107–19.804 for pStage) (Online Resource 6b). Although HNF4α-positive grade 3 non-mucinous adenocarcinomas frequently showed the loss of SMARCA4 (2/6, 33%), it was not identified as a poor prognostic factor (Online Resource 6a).

We also compared clinicopathological factors among the six groups (Online Resource 7) and found that advanced pT factor, advanced pStage, lymph node metastasis, vessel invasion, pleural invasion, and pulmonary metastasis were most frequently observed in the HNF4α-positive grade 3 group, indicating that this group was the aggressive phenotype.

Xenograft tumors of HNF4α-positive lung adenocarcinoma cell lines showed high-grade, non-mucinous morphology

Finally, we examined whether HNF4α-positive grade 3 adenocarcinoma cell lines were present among the 39 non-squamous non-small cell lung cancer cell lines. Online Resource 8 shows the gene-level expressions of HNF4A and TTF-1 in the 39 cell lines. The four cell lines with the highest expression of HNF4A were A549, H2405, Calu-3, and H1651, in that order. Online Resource 8 also shows the common driver mutations of the 39 cell lines, and among the four HNF4A-high cell lines, SMARCA4 and KRAS mutations were found in A549, HER2 amplification was found in Calu-3, and no common driver mutations were found in H2405 or H1651.

Figure 4a summarizes (i) the genetic status of EGFR, MET, HER2, KRAS, and SMARCA4 (upper panel), (ii) gene-level expressions of HNF4A, TTF-1, and SMARCA4 (middle panel), and (iii) protein-level expressions of HNF4α, TTF-1, SMARCA4, and ACTB (lower panel) for the four HNF4A-high cell lines (A549, H2405, H1651, and Calu3), compared with the four representative TRU-type cell lines with TTF-1-high expressions (HCC827, PC3, H1648, and H2009). The four HNF4A-high cell lines showed high HNF4α expression and low level of TTF-1, except for H1651, at both the gene and protein levels. A marked decrease in the expression level of SMARCA4 was only observed in SMARCA4-mutated A549, whereas the other three HNF4A-high cell lines exhibited SMARCA4 expression. An aberrant band of SMARCA4 was detected in H2405 by western blot analysis (Fig. 4a).

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

Next, using xenograft tumors of the four HNF4A-high cell lines, we examined the histological growth patterns in HE staining and performed immunohistochemical analysis for HNF4α, TTF-1, and SMARCA4 (Fig. 4b). A549 and H1651 showed solid growth patterns, H2405 showed solid growth patterns with focal cribriform patterns, and Calu-3 showed fused glandular and papillary growth patterns (Fig. 4b, the top row). All of these growth patterns are features of grade 3 primary lung adenocarcinoma, and notably, none of the cell lines showed morphological features of mucinous adenocarcinoma. Immunohistochemically, all of the four HNF4A-high cell lines were HNF4α-positive and TTF-1-negative in the nucleus (Fig. 4b, the second and third row), but H1651, which showed high TTF-1 expression at both the gene and protein levels, exhibited intracytoplasmic TTF-1 expression. SMARCA4 expression was diffusely lost in the A549 xenograft tumor but retained in the other three cell lines (Fig. 4b, the bottom row).