Ciliated muconodular papillary tumors of the lung with KRAS/BRAF/AKT1 mutation

Background Ciliated muconodular papillary tumors (CMPTs) are newly recognized rare peripheral lung nodules that are histologically characterized by ciliated columnar, goblet, and basal cells. Although recent studies have shown that CMPTs constitute a neoplastic disease, the complete histogenesis of CMPTs is not fully understood and molecular data are limited. Methods We reviewed four cases of CMPT and performed immunohistochemical and genomic analyses to establish CMPT profiles. Results All cases were positive for hepatocyte nuclear factor-4α and mucin 5B and negative for programmed death ligand 1 expression, as determined by immunohistochemistry. The genetic analysis revealed three pathogenic mutations (BRAF V600E, AKT1 E17K, and KRAS G12D), with the KRAS mutation reported here for the first time. Conclusion Histological and genetic profiles indicate that CMPTs are likely neoplastic and exhibit features similar to mucinous adenocarcinoma. This suggests that some CMPTs may be a precursor lesion of mucinous adenocarcinoma. Electronic supplementary material The online version of this article (doi:10.1186/s13000-017-0651-2) contains supplementary material, which is available to authorized users.


Background
Ciliated muconodular papillary tumors (CMPTs) are a newly recognized small-size papillary tumor of the peripheral lung that contain columnar cells, occasional basal cells, and mucus-producing cells as well as extracellular mucin pools of various sizes. Although CMPTs were first described based on certain pathological features that suggested a malignant potential, similar diseases such as extremely well-differentiated papillary adenocarcinoma with prominent cilia formation have been reported under different names [1][2][3]. Due to its complex histology and presence of inflammation and fibrosis, the metaplastic nature of CMPTs has been debated. However, recent reports have revealed the frequent presence of driver gene mutations, and CMPT is now recognized as a neoplasia [4][5][6]. The histogenesis and molecular characteristics of CMPTs are not well understood owing to the rarity of the disease. To address this issue, we reviewed our case archive and characterized CMPTs by immunohistochemistry and nextgeneration sequencing (NGS).

Patient selection and tissue preparation
Cases of surgically resected CMPT from 2012 and 2016 were searched in the pathology archive of Nagasaki University Hospital. Hematoxylin and eosin-stained slides of each case were reviewed by two pathologists specializing in thoracic medicine. We identified four CMPT cases; two of these had been originally diagnosed as CMPT, whereas the other cases had been diagnosed as glandular papilloma and mucinous adenocarcinoma. Clinical data were extracted from the hospital's electronic medical records. Sections with a thickness of 4 μm from formalinfixed, paraffin-embedded (FFPE) tissue samples were examined by immunohistochemistry, and 15 sections with a thickness of 5 μm were subjected to NGS of 50 cancer-related genes using the Ion Torrent PGM system (Life Technologies, Carlsbad, CA), and10 μm thick section on the conventional glass slide were submitted for 29 genes analysis using MiSeq (Illumina, San Diego, CA, USA).

Isolation of genomic DNA
Genomic DNA was extracted from FFPE tumor samples using the QIAamp DNA FFPE kit (Qiagen, Valencia, CA, USA), and the concentration was determined using the Quant-iT PicoGreen dsDNA Assay kit (Life Technologies).
Targeted deep sequencing of mutational hotspots in 50 cancer-related genes Genomic DNA was subjected to whole-exome sequencing using the Ion AmpliSeq Cancer Hotspot Panel v.2 (Life Technologies) according to the manufacturer's instructions. The purified library was sequenced on an Ion PGM instrument using Ion PGM Hi-Q Sequencing kit and Ion 318 Chip Kit v.2 (all from Life Technologies). DNA sequencing data were accessed with the Torrent Suite v.5.0 program (Life Technologies). The coverage analysis was performed using the coverage analysis plugin v5.0. Reads were aligned with the hg19 human reference genome, and potential mutations were identified using Variant Call Format v.5.0. Raw variant calls were annotated with CLC Genomics Workbench software (CLC bio, Aarhus, Denmark). Variants were manually verified using the integrative genomics viewer (Broad Institute, Cambridge, MA, USA). Known single nucleotide polymorphisms were identified using the Human Genetic Variation Database (http://www.hgvd.genome.med.kyoto-u.ac.jp/) [7] and were excluded.

Clinical summary
Clinical data for the four CMPT patients are summarized in Table 1. The patients were all female with a median age of 67 years old. None had a history of smoking. Tumors ranged in size from 8 to 25 mm (median: 11 mm). Lobectomy was performed in three cases, and segmentectomy was carried out in one case.

Histological findings
The histological features of the four CMPT cases were consistent with those previously reported [1,8,9]. That is, the tumors typically exhibited a mixture of acinar and papillary growth patterns without clear evidence of invasion, and were surrounded by abundant mucus pools in the alveolar spaces (Fig. 1a). The tumors were composed of two basal cell layers and surface epithelia. The latter consisted of an uneven mosaic of ciliated columnar, goblet, and mucin-producing epithelial cells of the gastric type (Fig. 1b). Few tumor cells showed nuclear atypia, and no mitosis or necrosis was observed.

Immunohistochemistry
All four cases were positive for nuclear HNF4α and TTF-1. Two cases showed diffuse and strong staining for HNF4α, as seen in mucinous adenocarcinoma or colorectal carcinoma (Fig. 1c). The Ki-67 index was low (2.5%-10%), consistent with a less aggressive nature (Fig.  1d). In contrast to mucinous adenocarcinoma, all cases examined in this study showed diffuse TTF-1 positivity and one case showed possible overexpression. There was sparse cytoplasmic expression of MUC5AC (Fig. 1e) while diffuse MUC5B-which is very rare in the normal lung parenchyma-was detected in all cases (Fig. 1f ). Nuclear expression of p53 was sporadic in one case and more frequent in two cases, and in one case p53 was clearly overexpressed. There were no or only a few cells that were positive for CDX2 and CK20. p63 expression highlighted the basal layers in all cases, although the area of coverage was focal in two cases and broad in the two others (Table 2). One case with BRAF V600E mutation identified by NGS showed diffuse and strong BRAF V600E staining, while the other three cases showed none (Fig. 1g). There was no ALK and EGFR mutations identified in our cases. ALK status was also confirmed by immunohistochemistry.
All cases exhibited <1% PD-L1 positivity in tumor cell membranes (Fig. 1h). These findings suggest that CMPTs are tumors that originated from a terminal respiratory unit and are showed features of gastric-type glands.

Genetic analysis
Gene mutations were detected in two of the four cases (50%) ( Table 1). One case harbored BRAF V600E and AKT1 E17K mutations-which was interestingly identical as what have been recently reported [4,5]-and another had a KRAS G12D mutation. The detected mutation status was identical on both Ion PGM instrument system and Illumina MiSeq system. A KRAS G12C mutation was found in one of the two other cases; however, the significance of this mutation is unclear due to its low frequency.

Discussion
We carried out immunohistochemical and molecular analyses of four cases of CMPT and identified a previously unreported KRAS mutation in addition to known BRAF and AKT1 mutations. The findings of BRAF and AKT1 mutations were identical to those reported in recent studies [4,5]. However, we could not identify either EGFR or ALK mutation in our cases [4,6,10].
Interestingly, the case with a KRAS G12D mutation detected by Ion PGM and Illumina method showed fewer basal cell layers, as confirmed by p63 immunostaining, while another case with reduced basal cell layer coverage also had a KRAS mutation (G12C). We did not take this mutation into account due to its low frequency by Ion PGM method and negative result by Illumina method. This KRAS status could be due to intratumoral heterogeneity-i.e., the few cells harboring this mutation may have been overshadowed by wild-type cells, which constituted the majority of the tumor cells.
Our immunostaining results were consistent with previous reports [6,8,[10][11][12][13][14][15][16]. However, we showed for the first time that CMPTs were positive for HNF-4α and MUC5B. Although these tumors show similarities to mucinous adenocarcinoma, there are significant differences between them such as the presence of cilia in columnar epithelia and basal cell intervention in the latter. In addition, invasive mucinous adenocarcinoma is usually TTF-1-negative and has distinct malignant features [17].

Conclusion
This is the first report of CMPTs of the lung harboring a KRAS mutation. Our findings suggest that CMPT cases