A Histopathological Feature of EGFR-Mutated Lung Adenocarcinomas with Highly Malignant Potential – An Implication of Micropapillary Element -

The purpose of this study was to define histological features determining the malignant potential of EGFR-mutated lung adenocarcinoma (LADC). Surgically resected tumors (EGFR-mutated LADCs with (21) and without (79) lymph node metastasis and EGFR wild-type LADCs with (26) and without (108) lymph node metastasis) and biopsy samples from inoperably advanced tumors (EGFR-mutated LADCs (78) and EGFR wild-type LADCs (99)) were examined. In surgically resected tumors, the EGFR-mutated LADCs with lymph node metastasis had the micropapillary element in a significantly greater proportion than others (Mann-Whitney tests P ≤0.026). The proportion of micropapillary element was higher in the EGFR-mutated LADC at the advanced stage (stage II, III, or IV) than in the tumor at the early stage (stage I) (Mann-Whitney test, P<0.0001). In the biopsy samples from inoperably advanced LADCs (177), EGFR-mutated tumors also had micropapillary element at a higher frequency than EGFR-wild type tumors (53/78 (68%), versus 30/99 (30%), Pearson x2 test, P<0.0001). In stage I EGFR-mutated LADCs (84), the tumors with the micropapillary element (34) exhibited a significantly higher recurrence rate than tumors without micropapillary element (50) (5-year Recurrence-free survival 64.4% versus 93.3%, log-rank test P = 0.028). The micropapillary element may be an exclusive determinant of malignant potential in EGFR-mutated LADC. It is suggested that EGFR-mutated LADC may develop through a distinct histogenesis, in which the micropapillary element is important for promoting progression.


Introduction
Lung cancer is the leading cause of cancer-related death in the developed world, and lung adenocarcinoma (LADC) is the most common histological type of the disease. Recent research in molecular oncology has revealed that oncogenic mutations are required to promote tumor expansion, namely driver mutations, in LADC. These driver oncogenes include the EGFR, KRAS, ALK, RET, and ROS genes, mutations of which are mutually exclusive, and are crucial determinants indicating a favorable response to different molecular targeting agents [1] [2] [3] [4] [5] [6].
EGFR is the most common driver oncogene in LADCs, and mutations in this gene are seen in 20 to 50% of LADCs in Asians and 5 to 10% LADCs in Westerners [7] [8] [9]. EGFRmutated LADCs have several unique features. They predominantly occur in females and nonsmokers, and most cases are of the lepidic element-predominant histological subtype [7] [10] [11] [12] [13]. The lepidic element is a low-grade malignancy and is associated with a favorable outcome [14] [15] [16]. On the other hand, EGFR-mutated LADCs also include highly malignant tumors that are inoperably advanced. It remains unclear whether resectable tumors progress to become inoperable tumors or whether inoperable tumors develop independently through an exclusive carcinogenetic pathway. This is an important matter to be solved for better understanding of pathologic basis of EGFR-mutated LADC.
This study examined surgically resected tumors and biopsy samples from inoperably advanced tumors, and also defined the histopathological features associated with malignant potential in EGFR-mutated LADCs.

Materials and Methods Patients
Three hundred and thirty-six LADCs that had been surgically resected (clinicopathological characteristics are presented in Table 1) and 177 LADC biopsy samples from inoperably

Histopathological examination
Hematoxylin and eosin-stained sections were subjected to histological examination.

Results
Histological element that associates with malignant potential in EGFRmutated LADCs The study groups were assigned according to a flowchart described in figure 1 (Fig 1). Proportions of the histological elements (lepidic, acinar, papillary, micropapillary (mPAP), and solid elements) were described in 5% increments according to the World Health Organization . The proportions in the EGFR-mutated LADCs with lymph node metastasis were compared with those in the other three groups. The proportion of mPAP element was consistently and significantly greater in EGFR-mutated LADCs with lymph node metastasis than in any of the other groups (Table 3). Differences in proportions of the other elements were not consistent in comparisons between EGFR-mutated LADCs with lymph node metastasis and the other groups (Table 3). Representative appearances of the elements are shown in figure 2 (Fig 2).

The mPAP element and disease stage
In EGFR-mutated LADCs, the proportion of mPAP element in the tumor at the advanced stage (stage II, III, or IV) was significantly higher than that in the tumor at the early stage (stage I) (Mann-Whitney test, P<0.0001; Fig 3A). In EGFR wild-type LADCs, the proportion of mPAP element showed no significant differences between the early stage tumors and the advanced stage tumors (Mann-Whitney test, P = 0.085; Fig 3B). These results suggested that the mPAP element may participate exclusively in the progression of EGFR-mutated LADC.

The mPAP element in inoperably advanced LADCs
Biopsy samples from inoperably advanced LADCs were also examined. Representative histological appearances of the biopsy specimens are shown in figure 4 (Fig 4). The mPAP element was detected at a significantly higher frequency in EGFR-mutated LADCs than in the EGFR wild-type LADCs (53/78 (68%), versus (vs) 30/99 (30%), Pearson x2 test, P<0.0001). This result supports the idea that the mPAP element may participate exclusively in the progression of EGFR-mutated LADC.

The mPAP element and postoperative recurrence
The association between the proportion of mPAP element and postoperative recurrence was analyzed in surgically resected stage I EGFR-mutated LADCs. The median follow-up period was 57 months (range: 1-159 months). Seventeen patients had recurrent disease and 15 patients died during follow-up. The recurrence-free survival (RFS) of EGFR-mutated LADCs that contained the mPAP element was worse than that of the EGFR-mutated LADCs that did not contain the mPAP element ( Fig 5A). The difference was statistically significant when the mPAP element proportion cut-off value was set at 5% (5-year RFS 64.4% vs 93.3%, P = 0.028) or 10% (5-year RFS 57.1% vs 87.6%, P = 0.005) (Fig 5A and 5B), although no significant difference was found when the cut-off value was set at 20% (5-year RFS 40.0% vs 84.0%, P = 0.102) ( Fig 5C). Number of tumors with mPAP element proportions of !20% may be too small for analysis. It was confirmed that the mPAP element could be a determinant of the malignant potential in EGFR-mutated LADCs.

The potential prognostic impact of mPAP element for EGFR-mutated LADCs
We additionally evaluated a prognostic impact of mPAP element for EGFR-mutated LADCs, as we considered an absolute volume of mPAP element may be more closely correlated with the malignant potential of the tumor than mPAP proportion. We defined the mPAP estimated Table 3.  Fig 6B). Table 4 summarizes the univariate association between clinicopathologiacal factors and RFS. Lymphatic canal invasion (P<0.001), vascular invasion (P = 0.011) and mPAP EV (cut-off value: 15, P<0.001) were associated with worse RFS. Multivariate analysis revealed that the mPAP EV (P = 0.004) and lymphatic canal invasion (P = 0.009) were independent predictors of disease recurrence (Table 5). These results    The mPAP element and types of EGFR mutations No significant difference in types of EGFR mutations (major or minor mutations) between tumors with mPAP and those without mPAP was found (Table 6).

Discussion
The     [29]. Taken together with these findings, EGFR-mutated LADC may develop through a unique carcinogenetic pathway in which the low-grade lepidic subtype progresses to the high-grade mPAP subtype (Schema shows the virtual carcinogenetic pathways of the EGFR-mutated and the EGFR wild-type LADCs; Fig 7). On the other hand, it is noteworthy that the papillary element as well as mPAP element was also detected at a higher frequency in EGFR-mutated LADCs. This finding agrees with the notion that the papillary element may be a precursor for the mPAP element [30]. Papillary and mPAP are also occasionally found in EGFR wild-type LADCs, although these elements were rarely detected and their association with the malignancy grade was not statistically significant. Undefined mutations having potential biological activity equivalent to that of EGFR mutations (mutations of EGFR family members) may occur in EGFR wild-type LADCs with mPAP elements [31].
The present study also proposed that the mPAP EV may be a useful prognostic marker for predicting the recurrence of EGFR-mutated LADCs. Although patients with EGFR-mutated LADC generally exhibit favorable postoperative outcomes, a considerable proportion still dies of recurrent disease [12] [32]. Clinical trials of postoperative adjuvant EGFR-TKI therapy for patients with EGFR-mutated LADCs are currently in progress (WJOG6410L study, CTONG1104 study, ALCHEMIST study) [33] [34] [35]. The identification of tumors that are at high risk of recurrence and the adjuvant use of appropriate molecular targeting agents may be one way of improving postoperative survival. The mPAP EV parameter proposed here can be used to aid the identification of tumors that are at high risk of recurrence.
In summary, EGFR-mutated LADC may develop through a distinct carcinogenetic pathway, in which the mPAP element may play an important role in promoting progression. The mPAP element also has prognostic value. We hope that our efforts will increase current knowledge about the carcinogenesis of EGFR-mutated LADC and lead to improvements in the therapeutic strategies for such tumors.