Heterogeneous components of lung adenocarcinomas confer distinct EGFR mutation and PD-L1 expression

Lung cancer is a most common cause of cancer-related deaths in the world. Lung adenocarcinoma (LAC) is a prevalent histological type in non-small cell lung cancer (NSCLC) [1]. The treatment of lung cancer is individualized, and thus relied on the results of molecular biology assays and each patient’s histology [2]. Individual responses are now suspected to tumor heterogeneity and challenge personalized medicine and biomarker development [3]. The development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) and immune check-point inhibitors have led a new era in lung cancer therapy. Meanwhile, The fact that EGFR-driven NSCLC inhibiting antitumor immunity through the activation of the PD-1/PD-L1 pathway has been demonstrated by preclinical studies. However, epidemiology studies suggest that EGFR mutant NSCLC is more likely to decrease PD-L1 expression. To palliate these controversies, intense studies focus on tumor heterogeneity, which tends to result in mixed responses (MR) to systemic EGFR-TKI and chemotherapy. However, the clinical significance and potential mechanisms remain to be testified. Physicians and oncologists turn on comprehensive therapies combined to targeted treatment and immune checkpoint inhibitors. Molecular discordances between primary and metastatic tumors differ among histological types [4]. A subset of patients with EGFR-mutant LACs fosters MRs to EGFR-TKIs. This uncertainty is suspected with intratumor heterogeneity (ITH). An MR may be an unfavorable prognostic factor and are suspected with tumor genetic heterogeneity [5]. Temporal/spatial heterogeneity between primary and metastatic tumors have only a limited forecast associated with markedly worsened outcomes [6]. The new classification has been reported as an independent predictor of overall survival [7]. LACs usually have mixed components (lepidic, acinar, papillary, solid and micropapillary) in tumor masses. Therefore, it is requisite to quantitatively evaluate histological components [1]. It have not been ascertained whether these outcomes reflect inappropriate use of targeted therapies or greater invasiveness of tumors with increased genomic instability results in generation of multiple subclones.

Lung adenocarcinomas frequently occur in ‘mixed pattern’ and percentages (up to 5%) of various histological components: acinar, papillary, micropapillary, lepidic and solid, are evaluated by semiquantitative assessment and should be reported according to the new WHO classification [1]. It is crucial to adopt a practical way to address tumors comprised of a complex histological constitution, since 70 to 90% of surgically resected lung cases were diagnosed invasive adenocarcinomas. Prominent diverse patterns in morphology and heterogeneity in biology among adenocarcinomas are paid more and more attention by pathologists upon the establishment of the new classification. We commenced our study after we reviewed all the sections and renewed diagnoses based on the new classification. Articles on the topic of micropapillary AC have reported patients in early-stage with a poor prognosis [10, 11]. It has recently been convinced that tumors classified as micropapillary also have a poor prognosis similar to adenocarcinomas with a predominant solid subtype [12].

This study discussed the association between LAC histological subtypes, PD-L1 expression levels and primary resistance to EGFR-TKIs. Our research demonstrated that PFS time of LAC patients exhibited a better prognosis among patients with TPS < 1%, than those with TPS > =50% (22.9 months versus 15.3 months). Since that LAC usually harbor more than one components in its parenchyma, we compared the PFS of different PD-L1 expression level with histological constitution. Patients harboring two histological components and with TPS < 1% had longer PFS time than those with TPS > =50% (25.5 months versus 13.6 months). However, prognosic significance was not evinced among LACs with single or > =three components. PD-L1 was not homogeneously expressed even in a tumor mass (Fig. 1b and Table 3). We also observed that heterogeneous PD-L1 expression, especially in the group of TPS 1–49%, was inclined to the tumor cells in the rim of tumor population against stroma (Fig. 1d). Given the heterogeneity of PD-L1 expression, it is important to understand the signals that induce the expression of PD-L1 on tumour cells. Two general mechanisms for the regulation of PD-L1 by tumour cells have emerged: innate immune resistance and adaptive immune resistance [13]. For this reason, we observed PD-L1 expression in different LAC subtypes and compared the effect on patients’ prognosis. Acinar LACs with TPS < 1% had longer PFS time compared with those with higher PD-L1 level. Likewise, solid LACs had a similar prognosis between the groups of TPS < 1% and TPS 1–49% even if it was not significant. K. Yoshimura, et al [14] reported that PD-L1 are also heterogeneously expressed in the same primary tumor tissue in a patchy pattern, as shown in our study (Fig. 1c and Fig. 5b). Histological subtypes were really associated with heterogeneous PD-L1 expression and patients’ prognosis.

ITH is attractive depending on recent technological advances in higher resolution and more rapid analysis on cancer genomes. Intratumoral heterogeneity is considered either the molecular features or the pathologic features of LAC. However, few studies have focused on the relationship between the two. In order to better address the current controversies and to conceive future directions, it seems apposite to investigate thoroughly the early motivations for targeting the PD-1/PD-L1 axis in EGFR-mutated NSCLC. This concept was in part recommended by retrospective studies prompting frequent PD-L1 expression in EGFR-mutated NSCLC [15, 16]. The co-occurrence of PD-L1-positivity and activating EGFR mutations in clinical NSCLC specimens was first reported based on 164 surgically resected samples [16]. Evidence to support this notion is that PD-L1 is a downstream target of EGFR signaling, and this is interceded through IL-6/JAK/STAT3, NFĸB and p-ERK1/2/p-c-Jun pathways [17‐19]. PD-L1 expression can be repressed by EGFR-TKI [18]. In contrast, one study found PD-L1 expression was increased following gefitinib treatment [20]. However, the conclusions from previous studies are drawn based on the analysis on tumor lesion rather than on each histological component. What potential impact of EGFR mutated cancer cells expressing PD-L1 in tumor cell population remains be illuminated. A striking finding of our study is the identification of intratumoral genetic heterogeneity in LAC that harbors EGFR heterogeneous alterations and differential PD-L1 expression. In spite of EGFR status, PD-L1 overexpression occurred in LACs with solid (47.6%) versus those without this pattern, whereas low level expression of PD-L1 in mucinous ACs. Zito Marino, F. et al reported that PD-L1 expression was more frequently in LACs with solid pattern [21]. In addition, PD-L1 expression was not homogeneous in a tumor parenchyma and more common in heterogeneous pattern. Therefore, we further investigated and analyzed the relevance of EGFR statuses and PD-L1 expression in the 18 LACs with sensitizing/T790M mutation. We noticed that PD-L1 expressed more frequently in those components without EGFR mutation, especially in acinar areas, but really overexpressed in EGFR-mutated components. We suspect that EGFR-mutation driven PD-L1 expression are activated through the pathways as reported before [22]. In our study, LACs with single components harboring EGFR mutation tended to be short of PD-L1 expression. It seemed to be paradoxical with previous study that the biological association is indirectly consolidated by coexistence of PD-L1 upregulation in EGFR-mutant NSCLC, as observed in retrospective studies [16, 19], but not supported in subsequent pooled analysis [22]. However, another cancer cell-centric mode of PD-L1 upregulation is considered as adaptive immune resistance [23], which is characterized by an increased expression of PD-L1 on tumor cells and immune cell subpopulation in reaction to robust CD8+ T-cell-mediated immunosurveillance. Adaptive PD-L1 upregulation relies on effective immunorecognition, which is promoted by an increased somatic mutational and neoantigen burden. However, for hitherto indefinite reasons, mutational burden seems to be lower in EGFR-driven tumors [24]. We consider that this dilemma results from the ITH of LACs. The fact that cancers with EGFR mutation partially expressed PD-L1 protein suggests that a tumor mass seems to exist as if intercoursed subpopulations of cancer cells present with different biological behavior, both in PD-L1 expression and EGFR mutation. These attributes enable LACs to represent a heterogeneous response to clinical therapy. It is also manifested that histological subtypes really prompt the possibility of potential resistance to both EGFR-TKI and PD-L1-related immunotherapy.

A striking finding of this study is the identification of intratumoral genetic heterogeneity in LACs harboring driver coalterations. 17 patients was identified with co-occurrence of EGFR sensitizing and resistant mutations providing an incidence rate of 6.5%. Because it is unclear whether sensitizing and resistant mutations coexist in same or different tumor cells, we used LCM to capture pure tumor cells within both the same and different growth patterns. With this method, we found that EGFR mutation types did not concomitantly coexist in all tumor cells. Interestingly, EGFR mutations by ddPCR assay showed that abundance of these mutations were different in the same cell population. Therefore, we determined a difference in the driver status among spatially separated tumor areas. In particular, the relative abundances of the two altered genes were different in the same tumor areas, which suggests that oncogenic genetic profile may not be the same in all tumor cells within a same primary tumor (Fig. 5c-e). To fully recognize morphologic heterogeneity, we observed that the abundances of sensitizing and T790M mutation were not concordant in micropapillary and papillary components. We also observed similar results by using ARMS according to cycle threshold value. Intratumoral heterogeneity of EGFR mutations is demonstrated associated with the distribution of histological components in mixed LACs [25]. We also revealed that intratumoral heterogeneity of EGFR mutations really existed in the same histological subtypes of LAC. Therefore, we speculate that clone evolution, not only histological heterogeneity, may be mainly responsible for molecular intratumoral heterogeneity of LAC. The findings of intratumoral heterogeneity in EGFR sensitizing/T790M coaltered LAC may be hypothesized by Darwinian-like clonal evolutionary dynamics and the resulting complex clonal architecture of LAC as reported by Cai et al [26]. In this study, tumor cells with dual altered driver genes occurring mainly in micropapillary and papillary components prompt that these components most likely harbor heterogeneous clone. Previous studies have shown that a substantial proportion of malignant tumors have a multiclonal signature [27]. The fact that T790M clones were selected by the TKI therapy support this hypothesis, because T790M occurs in 60% patients upon EGFR-TKI therapy [28, 29]. Differential expression of PD-L1 in a tumor mass seems to testify this speculation as well. Discordant PD-L1 expression in a tumor mass is thereof suspected to reactive/adaptive expression owing that it usually occurs among the peripheral cancer cells against stroma. Resistance to TKIs is considered one of the unknowns of cancer; therapy selection may make tumors become more heterogeneous, which may be the major reason for resistance to TKIs [30]. The complex dynamics of clonal evolution could produce unique and unpredictable patterns of clonal architecture that are spatially and temporally heterogeneous [31, 32]. Clonal evolution underlying tumor progression probably proceeds in a branching, rather than in a linear manner, which might result in substantial clonal diversity that accordingly contributes to genetic heterogeneity within tumors [32]. Importantly, our findings may provide a rationale more reasonable to treat patients with dual mutations with Osimertinib. In addition, any pathologic diagnosis based on a whole mass does not adequately determine the oncological heterogeneity. Analyses on the correlation of histological subtypes and EGFR mutation show that EGFR mutation were significantly more common in LACs with micropapillary, papillary and lepidic pattern and less common in those with mucinous pattern. However, considering the potential impact of genetic intratumoral heterogeneity on histological feature, especially in LAC with more than one histological component, the situation may be more intricate than it looks.

Our study provided additional evidence for the notion that concomitant EGFR sensitizing/TKI-resistant mutation and PD-L1 expression could limit the clinical prognosis in LAC patients received anti-PD-L/EGFR TKI-based therapies. However, it should be emphasized that our data, together with previous studies, facilitate the potential to promote the combination of EGFR-TKI and immune checkpoint inhibitor in some specific LAC subtypes, such as those with solid pattern. The findings from this study that patients of T790M coaltered with EGFR sensitizing mutation should have a better response to the third-generation EGFR-TKI (Osimertinib) combined with immune checkpoint inhibitor. However, the algorithm of therapeutics to LACs should be defined relying on more studies on the components of LACs. Intratumoral heterogeneity of oncogenic drivers in LACs should be taken seriously, because they can hamper precise molecular diagnosis and selection of the most appropriate treatment in clinical practice. These findings should be viewed with caution in clinical practice, suggesting that the use of gefitinib should be limited to patients with micropapillary or papillary, but Osimertinib will be a better candidate for EGFR-mutated LACs. The explorations in clinical setting are urgent and promising.