A comprehensive study on the oncogenic mutation and molecular pathology in Chinese lung adenocarcinoma patients

Background Several genetic driver alterations have been identified in micropapillary lung adenocarcinoma (MPA). However, the frequency of co-alteration of ROS1, EGFR, and EML4-ALK is yet unclear. Herein, we investigated the relationship between clinicopathologic characteristics and well-identified driver mutations of MPA compared with non-micropapillary lung adenocarcinoma (LA). Methods Formalin-fixed paraffin-embedded (FFPE) sections derived from lung adenocarcinoma patients who never received adjuvant chemotherapy or radiation therapy prior to surgical resection were collected from October 2016 to June 2019. EGFR mutations, ROS1 rearrangements, and EML4-ALK fusion were identified in a set of 131 MPA and LA cases by using the amplification refractory mutation system (ARMS). The response rate and duration of response were assessed using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Results EGFR mutations had occurred in 42 (76.4%) MPA patients and 42 (55.3%) LA patients. Interestingly, ROS1 rearrangements were highly enriched only in the MPA cases (6/55, 10.9%) but rarely in the LA cases (1/76, 1.3%). Furthermore, 7.3% (4/55) MPA samples had double gene mutations, while only 1.3% (1/76) LA cases had double gene alterations. Of 5 patients with harboring two driver oncogene mutations, four patients (80%) obtained partial response, and one patient (20%) suffered recurrence. Conclusions A higher prevalence of ROS1 rearrangement or combined mutations of ROS1, EGFR, and EML4-ALK may play a critical role in the tumorigenesis of MPA. These findings provide a novel therapeutic strategy for patients with malignant MPA through combining TKIs than one TKI.


Introduction
Lung cancer remains to be the leading cause of cancerrelated death worldwide, and the most frequent histological subtype is lung adenocarcinoma [1]. Lung adenocarcinoma usually includes various histological subtypes, including solid, lepidic, acinar, papillary, and micropapillary patterns [2] according to the International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/ European Respiratory Society (ERS) [3,4]. Numerous studies have reported that lung adenocarcinoma with a micropapillary pattern (MPA) shows more aggressive behaviors and worse survival than other histological subtypes of lung adenocarcinoma (LA) [5][6][7].
Several oncogenic drivers have been identified in lung adenocarcinoma, including mutations in the epidermal growth factor receptor (EGFR) [8], fusions of anaplastic lymphoma kinase (ALK) [9], and rearrangements of ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) [10]. Accumulating evidence demonstrated that mutations of EGFR were identified in 15-30% of lung adenocarcinomas in Caucasians [11] and 40-60% in East Asians [12][13][14], indicating that the frequency of activated mutations of EGFR depends on ethnicity. Besides, ALK fusions were firstly identified in 2007 and occurred in approximately 3-7% of all lung adenocarcinoma patients, and the most common form was echinoderm microtubule-associated protein-like 4/anaplastic lymphoma kinase (EML4-ALK) [15]. In the same year, an additional novel oncogenic fusion gene, ROS1, was identified, which accounted for 1-2% of all lung adenocarcinoma patients. Of special interest, this ratio increased to 5-7% for lung adenocarcinoma patients without EGFR/KRAS/BRAF/ALK mutations [16]. With the development of tyrosine kinase inhibitors (TKIs), TKIs served as the first-line option for patients harboring EGFR-sensitive mutations, ALK fusions, or ROS1 rearrangements. Therefore, the discovery of TKIs against EGFR gene activation mutations (for example, gefitinib and erlotinib) [17] and ALK or ROS1 gene rearrangements (for example, crizotinib) [18,19] has significantly improved the outcomes of patients. For detection of ROS1 and EML4-ALK fusions, immunohistochemistry (IHC), next-generation sequencing (NGS), ARMSpolymerase chain reaction (ARMS-PCR), and fluorescence in situ hybridization (FISH) have been widely used [20]. Although FISH is the gold standard test, it is expensive and time-consuming. By contrast, ARMS-PCR is a more sensitive and feasible approach compared to FISH and IHC [21].
There is growing evidence that EGFR gene mutations are more common in MPA than in LA, while ROS1 gene rearrangement has not been clearly demonstrated in MPA patients [22][23][24]. Moreover, the co-existence of EGFR gene mutations, ALK gene fusions, and ROS1 gene rearrangements has been reported in a few lung adenocarcinoma cases [25][26][27], but the co-alteration of ROS1, EGFR, and EML4-ALK in MPA remains unclear.
The molecular features of MPA may differ from other histopathological subtypes of lung adenocarcinoma [28]; however, the determinate information is not available. In the present study, we investigated the relationship between the most common driver mutations and the pathology features in Chinese lung adenocarcinoma patients.

Patient selection
A total of 131 lung adenocarcinoma patients were enrolled in the First People' Hospital of Huzhou from January 2016 to June 2019. Of them, 55 cases harbored at least 5% micropapillary component [22], who were represented as MPA, and the remaining cases (43 solid, 20 acinar, and 13 lepidic) were defined as LA. All of them were initially diagnosed with lung adenocarcinoma and had not received neoadjuvant or adjuvant chemotherapy or radiation therapy prior to surgical resection. The pathological diagnosis was confirmed and classified using hematoxylin and eosin staining by two certified pathologists (Qilin Shi and Xiaolan Zhang from the First People's Hospital of Huzhou) based on the IASLC/ATS/ ERS multidisciplinary classification system [3]. All specimens contained 60% of tumor cells and sufficient tissues for further mutational analysis. Clinical information collected includes age, gender, tumor differentiation, tumor size, smoking history, lymphatic invasion, pleural invasion, tumor node metastasis (TNM) stage, micropapillary pattern, and prognostic data. This study was undertaken with the agreement of our hospital ethics committee, and the informed consent signature was provided by all patients.

Evaluations of treatment
After surgical resection, 5 patients were treated with 3 months chemotherapy following with targeted therapy (TKIs). Tumors were evaluated during the treatment with chemotherapy, EGFR-TKIs or ROS1/EML4-ALK inhibitors every 6 weeks. Efficacy was obtained using CT scan according to the Response Evaluation Criteria in Solid Tumors version 1.1(RECIST 1.1) [29]. Objective response rate (ORR) included complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD).

Statistical analysis
Comparisons between two or more categorical variables were conducted using the Chi-square test and Fisher's exact test. Data were statistically performed on SPSS22.0 (Chicago, IL, USA). The results were considered statistically significant at p < 0.05.

Clinicopathologic characteristics
Among MPA cases, 36 patients fell into stage I, 11 into stage II, 6 into stage III, and 2 into stage IV. In LA, 39 (16 solid, 16 acinar, and 7 lepidic) fell into stage I, 12 (10 solid, 1 acinar, and 1 lepidic) into stage II, 8 (4 solid, 1 acinar, and 3 lepidic) into stage III, and 17 (13 solid and 4 lepidic) into stage IV. Seventy-two people had no smoking history, and fifty-nine were smokers, including former smokers and current smokers. The histological images of MPA and LA are presented in Fig. 1.
The MPA group consisted of 24 (43.6%) women and 31 (56.4%) men with an age at diagnosis ranging from  (Table 1). However, the micropapillary pattern was not significantly associated with gender, smoking history, tumor size, and tumor differentiation (Table 1).

Mutational status of classic oncogenes
MPA and LA groups included 42 (76.4%) and 42 (55.3%) EGFR mutations, respectively. Interestingly, ROS1 rearrangements were highly enriched only in the MPA group (6/55) but rarely in the LA group (1/76) (p = 0.041, Table 1). Furthermore, we also discovered that different genetic driver alterations often co-existed in the MPA group, for instance, EGFR combined with ROS1 (n = 2) and EML4-ALK combined with ROS1 (n = 2), while only one LA case harbored EGFR combined with ROS1, suggesting that co-existent alterations of EGFR, ROS1, and EML4-ALK were more frequent in MPA than in LA (p = 0.043, Table 2). These results indicate the potential combined treatments of MPA with two different TKIs targeted to EGFR and ROS1.
To further explore the association between clinicopathologic characteristics and genetic driver alterations, we analyzed the general information and therapeutic outcomes in 5 lung adenocarcinoma patients with combined mutations. After surgical resection, all patients were treated with 3 months chemotherapy following with targeted therapy (TKIs). Follow-up data were accessible to all 5 patients after postoperative ranging from 0 to 12 months (median 10.8 months). All patients survived to the last day of follow-up. The results showed that four patients partially responded, and one patient suffered a recurrence according to RECIST 1.1 [29] ( Table 3).

Discussion and conclusions
Accumulating evidence indicates that the co-existence of classic oncogenes, involving EGFR, ALK, ROS1, and MET, was identified in lung adenocarcinoma patients, especially in younger and women patients without a smoking history. However, few studies have focused on the frequency of two-driver alterations of EGFR, ROS1,  or EML4-ALK in MPA and LA. Therefore, we investigated the relationship between the most common oncogenic mutations and molecular pathological characteristics in Chinese lung adenocarcinoma patients. Consistent with previous reports [30,31], we here discovered that MPA has positive lymph node metastasis, positive pleural invasion, and earlier disease staging compared with LA (Table 1). Increasing studies have shown that a micropapillary component was associated with lymph node metastasis, pleural invasion, and an early recurrence in stage I patients, suggesting MPA had a poorer prognosis compared with those without micropapillary component or other histological subtypes [32][33][34][35][36][37][38][39]. Our results further implied that the higher prevalence of lymph node metastasis and pleural invasion may be a valuable poor prognostic marker for MPA.
According to previous reports, patients with coalterations of EGFR, ALK, ROS1, and other oncogenic drivers showed distinctive clinical responses to TKIs in lung adenocarcinoma [44,[46][47][48]. Yang et al. demonstrated that the median progression-free survival of gefitinib was 11.2 months in patients with concomitant EGFR and ALK alteration [49]. Mao et al. indicated that the median progression-free survival of EGFR-TKIs and/ or ALK/ROS1 inhibitor was 6.6 months in patients with concomitant EGFR and ALK alteration [44]. However, 75% of patients with crizotinib treatment obtained disease control [44]. In the present study, all patients undertook the operation and chemotherapy initially and undertook subsequently targeted therapy. In addition, Watanabe et al. showed that the micropapillary component was associated with an early recurrence in stage I patients but not in advanced-stage patients, indicating MPA retained a high risk of early recurrence after 1-year surgery [39]. In the present study, among five patients with concomitant alterations of EGFR, ROS1, and EML4-ALK, four patients partially responded and one patient suffered a recurrence during 1 year follow-up. Our study provided evidence that lung adenocarcinoma patients with co-alterations of EGFR, ROS1, or EML4-ALK may benefit from TKIs treatment.
So far, there is little progress in digging the pathogenic mechanism of MPA or the treatment of this subtype by TKIs. Therefore, based on our findings, we will focus on elucidating the function of ROS1 rearrangement and EGFR mutations in MPA by establishing the cell and animal models both in vitro and in vivo in the future. In addition, we will verify the efficacy of one targeted TKI or combined TKIs for MPA and provide the potential treatment strategy. In summary, we report for the first time the relationship between the most common oncogenic mutations and pathological characteristics in Chinese lung adenocarcinoma patients. We discover the higher incidence of ROS1 rearrangements and the coexistence of genetic alterations involving EGFR, ROS1, and EML4-ALK in MPA cases, indicating that targeting of ROS1 combined with EGFR mutations may provide a novel therapeutic strategy for these patients. However, these results still should be supported by further studies with larger cases and in multi-centers.