Background
Low-dose chest computed tomography (CT) for lung cancer screening has increased the detection of solitary pulmonary nodules (SPN) not visualized on chest radiography, and has contributed to a reduction in lung cancer mortality [
1]. Some of these visualized nodules are nodular ground-glass opacities (nGGOs). nGGOs on chest CT are defined as hazy, increased attenuation of the lung with preservation of bronchial and vascular margins, and are classified as pure and mixed GGOs, which contain a solid component [
2].
Nodular GGOs can be found in eosinophilic lung disease, pulmonary lymphoproliferative disorder, and interstitial fibrosis, with a persistent nGGO being a possible sign of early lung cancer [
3]. The natural development of nGGO follows a stepwise progression from atypical adenomatous hyperplasia (AAH) to adenocarcinoma
in situ (AIS: formerly bronchioloadenocarcinoma), to microinvasive adenocarcinoma (MIA), and finally to invasive adenocarcinoma (IA) [
4]. However, some adenocarcinomas do not follow this pathway, manifesting as consolidation and/or solid mass, with different genetic profiles. Therefore, lung adenocarcinoma exhibits heterogeneity in pathogenesis and progression [
5].
Several driver mutations have been identified in lung cancer, such as
epidermal growth factor receptor (EGFR) and
K-ras mutations and
anaplastic lymphoma kinase (
ALK) rearrangement. Lung cancers expressing
EGFR mutations respond well to the EGFR tyrosine kinase inhibitors [
6‐
8]. The fusion of echinoderm microtubule-associated protein-like 4 (
EML4) and
ALK gene by rearrangement in non-small cell lung cancer was identified [
9] and developed as a target of the ALK tyrosine kinase inhibitor, crizotinib [
10,
11]. These biomarkers predict response to these molecular targeting agents and testing for these markers is recommended in lung cancer patients [
12,
13], enabling personalized medicine for patients harboring
EGFR mutations or
ALK gene rearrangements. It is therefore very important to investigate the frequencies and clinical implications of these driver mutations in nGGOs, a specific type of lung adenocarcinoma.
Many studies have reported that
EGFR mutations are frequent in lung cancer with nGGOs, even in precancerous lesions such as AAH [
14‐
17]; however, the role of
ALK rearrangement in nGGOs remains unknown. We analyzed patients with lung cancer with nodular GGOs to investigate the correlation between biomarker status and clinicopathological and radiologic characteristics and to determine the roles of
ALK rearrangements and
EGFR mutations in nGGOs.
Discussion
Lung cancer, in its early stage, can present as nGGOs on chest CT. Lung adenocarcinoma with growth patterns involving the alveolar septum and a relative lack of acinar filling shows GGOs on chest CT, and a high GGO proportion is correlated with good prognosis [
27]. Pathology of GGO nodules has shown that the proportion of GGO in nodular adenocarcinomas decreases through the AAH-AIS-MIA-IA pattern of progression [
28], and that GGO nodules must undergo
in situ changes, since AIS (formerly called BAC) and precancerous lesions such as AAH correspond to pure GGO [
15].
The clinicopathologic, radiologic, and molecular biological characteristics of nGGOs are important for our understanding of the mechanism of carcinogenesis and for predicting the chemotherapeutic response. Since the introduction of molecular targeting agents, many groups have studied the EGFR mutation status of nGGOs, but there is little data on ALK rearrangements in nGGOs. EGFR mutations are frequently found in the early stages of nGGO, such as in AAH and AIS, and play an important role in the pathogenesis of adenocarcinoma with GGO patterns. However, the role of ALK rearrangement, another potent driver mutation in adenocarcinoma, has not been described in GGO nodules.
In this study, we investigated the frequencies and clinicopathological characteristics of driver mutations, focusing on ALK rearrangement in resected adenocarcinoma with GGO patterns. To our knowledge, this is the largest comprehensive analysis of lung cancer presenting as GGO nodules. We included lung cancer nodules exhibiting any amount of GGO regardless of its size, thereby investigating the molecular biomarker status of lung cancer at early stages.
Adenocarcinoma with
ALK rearrangement is usually found in younger, female patients who have light to no smoking history, and has been reported to have acinar, papillary, cribriform, and signet-ring patterns. The radiological characteristics of lung cancer with
ALK rearrangement have hardly been studied, and there is a lack of data concerning the role of
ALK rearrangement in nGGO lesions. In one study, Fukui et al. reported that no GGO nodules were found in patients with
ALK rearrangement while 50% of adenocarcinomas that did not have
ALK rearrangement also had GGO nodules and also
EML4-ALK-positive tumors mainly exhibited a solid pattern on CT [
29].
In this study, the proportion of
ALK-positive nGGO lesions was significantly lower (2.8%) than that obtained in previous studies of a large cohort of adenocarcinomas (3.9-20.4%) (Table
6) [
23,
25,
26,
29‐
32], and was significantly lower than the 6.8% of 395 resected adenocarcinoma patients in our previous study, which included all types of curatively resected adenocarcinoma [
23]. This could be indirect evidence of the lower incidence of
ALK rearrangements in adenocarcinomas with GGO patterns compared to adenocarcinomas of all types.
It is well known that
ALK-positive adenocarcinoma is likely to present a signet-ring cell or cribriform pattern and abundant mucin production on histological analysis [
33,
34]:
ALK-positive lesions are observed as a solid, rather than a GGO, nodule [
29,
35,
36]. This explains the low proportion of
ALK-positive patients in this study, which focuses on nGGOs. Fukui et al. studied the radiologic characteristics of 28
ALK-positive adenocarcinomas and revealed no GGO portion [
29] and another report on CT characteristics of ALK rearranged advanced NSCLC from Japan also report low frequency of ALK rearrangement (one among 36 cases) [
36], consistent with our findings.
We revealed that maximal diameters and the solid portion of nGGOs with ALK rearrangement were significantly larger than were those without ALK rearrangement. All nGGOs with ALK rearrangement were IA (invasive adenocarcinoma) with acinar predominant subtypes (n = 3) and three with cribriform pattern. Patients with ALK-positive lesions showed more advanced pathologic stages than those with EGFR-positive GGOs. Therefore, we suggest ALK rearrangement is associated with cellular and histological type as well as clinical aggressiveness.
Several studies have revealed that adenocarcinomas with
ALK rearrangement have more lymph node metastases [
23,
25]. Combined with the radiological characteristics discussed above, the
ALK-positive adenocarcinoma seems not to follow the stepwise carcinogenesis pattern of AAH-AIS-MIA-IA, but to grow rapidly and bypass the phase of lepidic growth. This assumption is consistent with the histological analysis of
ALK-positive adenocarcinomas showing lower frequencies of lepidic growth and AAH/BAC (AIS) in the background of
ALK-positive lung adenocarcinomas [
35].
Distinct subsets of adenocarcinoma with morphologic differentiation to type II pneumocytes, Clara cells, or non-ciliated bronchioles are thought to originate from the terminal respiratory unit (TRU), and
EGFR mutation is involved with early-stage carcinogenesis of TRU-type adenocarcinoma [
5,
37]; nGGOs appear to be another marker of TRU-type adenocarcinoma [
5].
Thyroid transcription factor-1 (TTF-1) is a marker of TRU-type adenocarcinoma [
37,
38], and two studies concerning 11 and 12
ALK-positive patients each revealed TTF-1 positivity in all
ALK-positive adenocarcinomas [
26,
39]. This finding suggests that this subtype of adenocarcinoma may have TRU-origin histogenesis [
39]. However, the low proportion of GGO with
ALK rearrangement and the advanced stage in ALK-positive nGGOs found in this study indicates that it is still possible that this subtype may not follow a process of TRU origin. Further pathologic analysis of morphological characteristics is required.
Because the prevalence of adenocarcinoma with
ALK rearrangement is low compared to
EGFR mutation, studies investigating various characteristics of
ALK-positive lung cancer do not gather enough participants to yield consistent results. Previous studies on a large, unselected population of adenocarcinoma with
ALK rearrangement reported that patients with
ALK-positive lung cancer were younger [
23,
29,
30,
32], female [
23,
25,
40], and light or non-smokers [
23,
25,
29,
30,
32,
40,
41]. We previously reported that
ALK-rearranged lung adenocarcinomas of all radiologic types showed higher stage at diagnosis and more solid pattern, were more cribriform, and had a closer relationship with adjacent bronchioles [
42] and more frequently positive bronchoscopic findings than
EGFR-positive lung adenocarcinoma [
43], which suggested more proximal origin of ALK rearranged lung adenocarcinoma than EGFR-positive adenocarcinoma. These findings were consistent with low frequency of ALK rearrangement in nGGOs which presented in peripheral location.
We found no correlation between age, sex, smoking status, and ALK positivity, probably due to the small number of ALK-positive patients and the weak representation of adenocarcinoma, since we enrolled only patients with nGGOs.
We found that
EGFR mutation was associated with female, never/light smokers, as expected [
44]. The frequency of
EGFR mutation in nGGOs in this study was 54.8%, which was relatively high in comparison to other, large cohorts of adenocarcinoma [
25,
45‐
50] (Table
6). However, we could not predict
EGFR mutation status by the GGO proportion of nodules or tumor size.
EGFR mutation status was not associated with pathologic stage, nodal involvement, or histologic invasiveness.
It is interesting that after stratifying
EGFR mutations in exons 19, 20, and 21, only the mutation in exon 21 (mostly L858R) correlated with female gender and never/light smoking status. This result is consistent with other studies of the characteristics of adenocarcinoma and
EGFR mutation type [
51,
52]. The association between
EGFR and female non- or light smoker may be limited to
EGFR mutation in exon 21.
According to large cohort studies, EGFR mutations and
ALK rearrangements are mutually exclusive. However, several cases of co-incident
EGFR mutation and
ALK rearrangement have been reported, most of which demonstrated good response to EGFR tyrosine kinase inhibitors [
32]. In our study, which recruited participants at the early stage of adenocarcinoma, these molecular biomarkers were mutually exclusive. It is thought that they act through different mechanisms in early carcinogenesis.
The major strength of study is that it is the largest cohort concerning lung cancer with nGGOs. All nodules were resected by curative surgery, which reinforced the accuracy of pathologic and molecular diagnoses of the surgical specimens. Although we collected enough GGO nodules with EGFR mutations in exons 19 and 21, we could not collect sufficient numbers of samples with ALK rearrangement due to the inherent limitation that adenocarcinoma with ALK rearrangement tends to present as solid nodules in chest CT.
Authors’ contributions
SJK and CTL had full access to data, writing, and responsibility for the manuscript. YJL, JSP, YJC, HIY, and JHL assisted with recruitment and critical reading of the manuscript. JHC examined the pathology and analyzed EGFR and ALK status. HK reviewed the pathologic specimen. TJK and KWL analyzed radiological characteristics of nGGOs. KK and SJ performed surgical resection of nGGOs. All authors read and approved the final manuscript.