High Frequency of CTNNB1 Mutation in Low Grade Fetal Adenocarcinoma of the Lung: Two Case Series and Literature Review

Background Low-grade fetal adenocarcinoma of the lung (L-FLAC) is a rare pulmonary tumor resembling fetal lung histologically. Due to its rarity, there is limited information about L-FLAC pathogenesis and biological characteristics. Here, we describe two cases of L-FLAC treated at our hospital and summarize L-FLAC cases reported in the literature. We examined one woman and one man who were 30-years-old and 67-years-old, respectively. Histologically, tumor tissue from both cases had a complex glandular component with clear cuboidal and columnar cells that resembled histological features of fetal lung. In some areas, squamous morules were prominent. Immunohistochemically, nuclear/cytoplasmic expression of β-catenin was detected in both cases. Mutation analysis revealed a CTNNB1 mutation in both cases and a DICER1 mutation in 1 case. No mutations in EGFR, BRAF, KRAS, PIK3CA mutation were found. L-FLAC showed a high frequency of CTNNB1 mutation and low frequency of EGFR, KRAS, BRAF and PIK3CA mutation in our examined cases and in previous studies. This rare tumor has unique clinicopathological characteristics with specic genetic aberrations involving the Wnt pathway. These results provide a molecular basis for development of new therapies to treat these tumors. had no SALL4 expression, whereas case 1 had SALL4 expression, but no DICER1 mutation. Since the number of examined L-FLAC cases remains small, further examination will be needed to determine the incidence of DICER1 mutation in L-FLAC. In conclusion, L-FLAC showed a high frequency of CTNNB1 mutation and a low frequency of EGFR, KRAS, BRAF and PIK3CA mutation in our two examined cases and in previous studies. This rare tumor has unique clinicopathological characteristics with specic genetic aberrations involving the Wnt pathway. These results suggest that new therapies for L-FLAC could be developed based on these molecular features.


Introduction
Fetal lung adenocarcinoma (FLAC) is an extremely rare lung cancer that accounts for only 0.1-0.5% of all pulmonary neoplasms [1,2]. FLACs are divided into two subtypes: low-grade FLAC (L-FLAC) and high-grade FLAC (H-FLAC) [1,2]. These subtypes show distinct clinicopathological characteristics, biological behavior and outcomes. The incidence of L-FLAC is lower than that for H-FLAC. Zhang et al. reported that the prevalence of L-FLAC and H-FLAC was 0.32% and 0.54%, respectively, in Chinese patients [3]. Due to its rarity, information about the pathogenesis and biological characteristics of L-FLAC is limited. Here we describe two cases of L-FLAC.

Case Presentations
Case 1 was a 30-year-old female who visited our hospital for further examination of an abnormal chest shadow detected during a checkup. The patient had no medical history and never smoked. A computed tomography scan revealed a nodular lesion 30 mm in diameter in the peripheral area of the right upper lobe. On biopsy examination, the lesion was diagnosed as adenocarcinoma and right upper lobectomy with hilar and mediastinal lymph node dissection was performed. Case 2 was a 67-year-old male who came to ourhospital for a detailed examination of an abnormal chest shadow detected during a checkup. This individual smoked 20 cigarettes a day for 47 years. A nodular lesion in the peripheral area of the right lower lobe was detected by a chest CT scan. This lesion was diagnosed as adenocarcinoma by intraoperative pathological examination and right lower lobectomy with hilar and mediastinal lymph node dissection was performed.

Histopathological and immunohistochemical ndings
In both cases, tumor tissue was composed of complex glands and tubules with scant stroma at low magni cation (Figure 1a and 1b). At high power magni cation, cuboidal and columnar cells could be observed that were arranged in complex gland-like structures with supranuclear or subnuclear vacuoles. (Figure 1c and 1d). In some areas, squamous morules were prominent (Figure 1e and 1f). Diastase pretreatment periodic acid Schiff reaction demonstrated glycogen within the cytoplasm of the tumor cells. Immunohistochemical study was performed using an auto-

Mutation analysis
Mutation analysis was performed for CTNNB1, DICER1, KRAS, BRAF and PIK3CA. EGFR mutation testing carried out at the time of diagnosis detected no mutations. We conducted polymerase chain reaction (PCR) followed by Sanger sequencing and pyrosequencing of tissues samples from the two patients. Brie y, DNA from formalin-xed para n-embedded tissues was extracted using TaKaRa DEXPAT (Takara Bio Inc., Shiga, Japan). The tumor component on the slides was microdissected to increase the proportion of tumor cells. The PCR products were puri ed using a NucleoSpin Gel and PCR Clean-up, Mini kit (Marcherey-Nagel, Duren, Germany). Each puri ed product was directly sequenced using a forward primer and the BigDye Terminator version 3.1 cycle sequencing kit with an ABI 3730 instrument (Applied Biosystems Inc., Foster City, CA).

Discussion
FLAC is an adenocarcinoma resembling developing fetal lung in its pseudoglandular stage (8-16 weeks of gestation) [2] [4]. Histopathologically, FLAC comprises complex, branch-forming tubular glands lined by glycogen-rich, non-ciliated columnar or cuboidal cells. The cells have a clear cytoplasm; large vesicular nuclei are seen in addition to supranuclear or subnuclear vacuoles. The malignant glands are densely packed and situated within loose to moderate cellular broblastic stroma [1,2]. Due to their distinct clinicopathological characteristics, biological behavior and outcome, FLACs have been classi ed into L-FLAC and H-FLAC. L-FLAC shows low nuclear atypia, prominent morule organization and pure histological pattern [1][2][3]. In contrast, H-FLAC displays prominent nuclear atypia and nucleoli, high mitotic rate and necrosis [2]. H-FLAC includes fetal morphology ranging between 51% and 100% and is often accompanied by other conventional types of lung adenocarcinoma [4,5]. Our two cases showed typical L-FLAC morphology.
In addition to distinct histopathological patterns, L-FLAC and H-FLAC demonstrate different immunohistochemical staining and genetic alterations. To date, aberrant β-catenin nuclear expression/cytoplasmic staining and/or CTNNB1 mutation have been frequently observed in L-FLAC. Meanwhile, p53 protein overexpression is not observed in L-FLAC and mutations in EGFR, KRAS, BRAF and PIK3CA occur at very low frequency relative to both H-FLAC and conventional lung adenocarcinoma [1, 6-8]. Our two cases also showed the same features. Studies reporting clinicopathological characteristics of L-FLAC with genetic analysis are summarized in Table 1 [6-12]. To date, 21 L-FLACs, including the two in our study, have been reported. All showed β-catenin nuclear/cytoplasmic staining. CTNNB1 mutation was detected in 17/21 (80.9%) L-FLACs. This result supports a close relationship between β-catenin nuclear/cytoplasmic staining and CTNNB1 mutation. β-catenin is a key protein in the Wnt signaling pathway, which plays important roles during embryogenesis. Altered Wnt signaling activity can lead to tumor formation [3].  [15]. Therefore, the high prevalence of CTNNB1 mutation is a hallmark of L-FLAC and could serve as a diagnostic marker. Next generation sequencing for L-FLAC cases allowed the detection of DICER1 mutations [8, 10,11]. DICER1 encodes an RNase III family endoribonuclease that plays an essential role in microRNA production [16]. Germline inactivation of DICER1 is associated with familial DICER1 syndrome [17]. DICER1 mutation has been observed in different kinds of tumors and could contribute to the development of various cancers [12]. To our knowledge, nine L-FLAC cases, including one of our two cases, have been reported to carry a DICER1 mutation  [11]. However, case 2 in our study that had a DICER1 mutation had no SALL4 expression, whereas case 1 had SALL4 expression, but no DICER1 mutation. Since the number of examined L-FLAC cases remains small, further examination will be needed to determine the incidence of DICER1 mutation in L-FLAC.
In conclusion, L-FLAC showed a high frequency of CTNNB1 mutation and a low frequency of EGFR, KRAS, BRAF and PIK3CA mutation in our two examined cases and in previous studies. This rare tumor has unique clinicopathological characteristics with speci c genetic aberrations involving the Wnt pathway. These results suggest that new therapies for L-FLAC could be developed based on these molecular features.