Detection of a novel gene mutation, ERBB2 exon 20 insertion, in bronchial adenoma using next-generation sequencing and a review of the literature

Objectives: Ciliated muconodular papillary tumor (CMPT) is a rare peripheral lung tumor and is a subtype of bronchial adenoma (BA). Although recent studies have suggested that BA is a neoplastic disease, the complete histogenesis of BA is not fully understood and molecular data are limited. Methods: We examined the clinicopathological features of four patients with BA and performed immunohistochemical analysis and next-generation sequencing to characterize the molecular features of BA. A review of the previous literature was also undertaken to comprehensively conclude the molecular characteristics of this disease. Results: From previous studies and the present study, 99 BA /CMPT cases have been reported to date, with most of the patients from East Asia (77/99, 77.8%). The median age was 64 years old and the ages ranged from 19 to 84 years. The proportion of males and females was close, being approximately 1:1.3. From the computed tomography images, the BA /CMPT tumor usually presented as a peripheral solid mass, part-solid nodules, or ground-glass opaque with an irregular border and occasional central cavities. ERBB2, EGFR, BRAF, and AKT1 mutations were found on the computed tomography images of the BAs. To the best of our knowledge, this is the rst study to report about ERBB2 exon 20 insertion in BA. Conclusion: BA /CMPT is a rare pulmonary disease that mainly affects elderly Asian patients. Many abnormal molecular changes were found, which conrmed the neoplastic nature of BA /CMPT. However, it also added to the debate regarding the biological behavior of BA /CMPT.


Introduction
Ciliated muconodular papillary tumor (CMPT) is a newly recognized peripheral lung disorder, which was rst described in 2002 by Ishikawa et al. [[i]]. CMPT was once considered a very rare tumor, mainly affecting East Asian patients. However, in recent years, many cases have been reported with increasing recognition of this tumor . Kamata reported 10 cases in 2015 [6] and both Chang (Western country cohort) [18] and Zheng [19] reported more than 20 cases in 2018. To date, nearly 100 cases have been reported in the literature and most of these patients reside in East Asia countries. Thus, this tumor may not be as rare as once believed.
CMPTs often affect middle-aged to elderly adults and consist of ciliated columnar, mucous, and basal cells. The tumor is commonly surrounded by extracellular mucin pools in the peripheral lung. Some cases did not t all diagnostic criteria, such as the absence of papillary architecture or the lack of mucinous and/or ciliated cells; however, they shared the histological features of having a bilayered bronchiolar-type proliferation and continuous layer of basal cells, which have been termed "non-classic" CMPTs by Zheng et al. [19]. Both Zheng et al. [19] and Chang et al. [18] found that these lesions also shared genomic abnormalities. Chang et al. [18] proposed a new group of tumors called bronchial adenoma (BA) in 2018 and sub-categorized BA into two groups: proximal-type and distal-type based on morphological and immunohistochemistry (IHC) factors. Hence, classic CMPTs only represent a subset of proximal-type BA.So we refer to the tumor as BA in this study.
To date, accumulating evidence has shown that BA/CMPT is a neoplastic disease; however, its histogenesis and biological behavior remain unknown and its molecular data are limited. Recent studies have identi ed BRAF, ALK, EGFR, KRAS, HRAS, and AKT1 mutations in BA [7, 8, 13-16, 18, 19, 25, 26]. Among them, the most common driver mutations are BRAF V600E mutations.
Here, we report four cases with BA. For all cases, molecular analysis using next-generation sequencing (NGS) was performed and ERBB2, BRAF, AKT1, and EGFR gene mutations were identi ed. To the best of our knowledge, this is the rst study to report the existence of ERBB2 mutation in BA /CMPT.

Clinical samples
The four cases were diagnosed at the West China Hospital of Sichuan University between 2017 and 2019. Clinical data were extracted from the electronic medical records of the hospital. The samples were xed in 10% formalin, embedded in para n, and stained with hematoxylin and eosin. The clinical and pathological records were analyzed retrospectively and histological analysis was performed on the surgically resected specimens.

Isolation of genomic DNA
The slides were reviewed by two experienced pathologists and the proportion of tumor tissues was evaluated. Para nembedded tissue blocks corresponding to slides with a tumor proportion that was greater than 80% were chosen for DNA extraction and the genomic DNA was extracted using a QIAamp DNA FFPE kit (Qiagen; Germany). The concentration and quality were determined using a ScanDrop 200 spectrophotometer (Analytik Jena; Germany), with a minimum of 200 ng of DNA required for NGS library construction. The optical absorbance of wavelength 260 nm and wavelength ratio of wavelength 260-280 nm were calculated.

Gene mutation analysis
The sequence data were analyzed as previously described [27]. Sequence data were mapped to the reference human genome (hg19) using a Burrows-Wheeler Aligner. Alignment and variant calling were performed using a Genome Analysis Tool Kit and VarScan. Variants were ltered using the VarScan fp lter pipeline, loci with a depth less than 100 were ltered out. Single nucleotide variants (SNVs) and short insertions/deletions (indels) were identi ed using VarScan2, with a minimum variant allele frequency threshold set at 0.01 and a p-value threshold for calling variants set at 0.05 to generate variant call format les. All SNVs/indels were annotated using ANNOVAR and each SNV/indel was manually checked on an Integrative Genomics Viewer.

Clinical characteristics
Three of the four patients were female and one was male, with an age range from 32 to 65 years. The male patient had a history of smoking, whereas all female patients did not. All four patients were found to have pulmonary nodules by physical examination. Computed tomography (CT) imaging showed solid or ground-glass nodules in the peripheral lung. The volume of all tumors in the studied cases was less than 1 cm in diameter.
All cases received wedge resections either at the West China Hospital of Sichuan University or other hospitals. The detailed clinicopathological features are shown in Table 1. Some cases were misdiagnosed at rst. Case 2 was originally diagnosed as atypical alveolar epithelial hyperplasia and suspected adenocarcinoma from the frozen section. Case 3 was misdiagnosed as mucinous adenocarcinoma. Two patients had combined diseases: case 1 had metastatic invasive ductal carcinoma of the breast and adenocarcinoma in situ (AIS) of the lung, whereas case 3 had a minimally invasive adenocarcinoma of the lung.

Pathological features
Gross examination of the tumors in the four cases showed a well-demarcated gray-white solid mass. Microscopically, our cohort consisted of two proximal-types and two distal-types of BA. The tumors were composed of various proportions of ciliated columnar, mucus, and continuous basal cells with surrounding mucous lakes (Fig. 1A). The nuclear atypia was mild and mitosis and necrosis were not found. Muscular arteries were often seen in the center of the tumor (Fig. 1A), indicating that the tumor was localized around the bronchioles.
One proximal-type case showed predominantly papillary architectural patterns (Fig. 1B) and the other showed predominantly adherence architectural patterns with an occasional papillary structure ( Fig. 1A & C). The ciliated columnar cells were easily found in this subtype (Fig. 1C). The distal-type cases revealed adherence and glandular architecture, the papillary structure was not obvious, and there was a lack of ciliated columnar cells (Fig. 1D-F).
Both the proximal and distal growth patterns revealed a skipping growth pattern (Fig. 1A, D) at the edge of the tumor and the micropapillary tufts were detached into the alveolar cavities (Fig. 1C, F). In addition, we found brous tissue hyperplasia and lymphocytes and plasma cells in ltrating in the focal areas (Fig. 1E).

Immunohistochemistry
All tumor cells were positive for CK7 ( Fig. 2A); however, they were negative for CK20 (Fig. 2B), whereas the basal cells were positive for P63 (Fig. 2C). Consistently, the Ki67 index was less than 5% (Fig. 2D), indicating a relatively indolent biological behavior. For the proximal-type BAs, a few ciliary and basal cells were weakly positive for TTF-1 (Fig. 2E), whereas the distal-type BAs were strongly positive for it (Fig. 2F).

Molecular analysis
All cases underwent NGS to pro le the molecular abnormalities (Table 1). Case 1 harbored ERBB2 exon 20 insertion in the BA. Case 2 contained detected EGFR mutations. Case 3 was negative and case 4 harbored BRAF and AKT1 mutations. The detailed information of these detected mutations are shown in Table 2.
NGS was performed on the BA sample, as well as on the metastatic breast cancer and AIS of the lung tissues for case 1.
Consequently, multiple gene mutations were detected in the breast cancer metastatic focus including KRAS-ex2-G12A (48.82%), PIK3CA-ex21-H1047R (45.53%), TP53-ex5-V173L (42.76%), and ATM-ex18-C907F (27.64%), and there was an increase in the KRAS copy number (copy number = 3.51). However, no ERBB2 mutation was found; therefore, the ERBB2 exon 20 insertion was unique for BA. Unfortunately, DNA could not be extracted from the AIS tissue because of the limited sample amount. Discussion BA is a newly described tumor de ned by Chang et al. [18], which is a more extensive terminology than that of CMPT. It has not been well recognized by pathologists and cannot be classi ed according to the 2015 WHO classi cation system [28]. A total of 26 previous studies about this family of neoplasms were searched in the literature, plus the cases in the present study, from which 99 cases of BA /CMPT were reported. The clinicopathological features of these cases are summarized in Table 3.
BA often affects middle-aged to elderly adults from East Asia (77/99, 77.8%), with the median age being 64 years old (range from 19 to 84 years old). The incidence rate of male and female is similar, being close to 1:1.3. BA occurs almost exclusively in the peripheral lungs. Chest CT images showed peripheral solid mass, part-solid nodules, or ground-glass opacity with an irregular border, and some of the patients showed a central cavity [3,6,9,10,16,17,19,21,23,24]. The median diameter was 0.9 cm (range 0.2 to 4.5 cm), mostly between 0.2 and 2 cm. Only two cases were 3.5 cm and 4.5 cm in diameter [11].
Typically, the tumor is a pale mucinous nodule with an irregular border and sometimes a central cavity is found in the resected specimens. Histologically, BA /CMPT displays diverse morphological patterns including adherence, glandular, papillary, and micropapillary architecture, with abundant mucin around the tumor and the mucinous pool spreading into the adjacent alveolar spaces. The tumor is mainly composed of mucous and basal cells, while ciliated columnar cells may be present or absent. A few cells showed apical cytoplasmic snouts similar to club (Clara) cells [18]. The tumor cells lacked nuclear atypia, mitosis, and necrosis. Chang et al. [18] reported 25 lesions from 21 patients, with most of the lesions being at and only seven lesions containing focal papillary architecture. They categorized the lesions into two groups: proximaltype and distal-type based on the morphological and IHC similarities of the bronchiolar structures. The classical CMPT belongs to the proximal-type BA and the TTF1 staining is negative or weakly positive. However, for distal-type BAs, the TTF1 and Napsin A show diffuse positivity.
Some cases revealed discontinuous (skipping) growth patterns, which resembled the spread of tumors through air spaces and micropapillary tufts detached in the alveolar cavity, which is similar to micropapillary adenocarcinoma. Chang et al. [18] hypothesized that these cells were interconnected with each other in 3-dimensional spaces because these skip lesions do not extend away for more than a few alveoli and the basal cells were always present. However, these microscopic features of BA /CMPT may be an extreme diagnostic challenge for pathologists, especially when studying intraoperative frozen sections.
Human epidermal growth factor receptor 2 (HER2/ERBB2) is a receptor tyrosine kinase of the ERBB family, which plays a signi cant role in cancer development and progression, especially in breast, ovarian, and gastric cancers. The overexpression of HER2 protein is associated with poor prognosis. Recent studies have shown that the HER2 mutation is a distinct subset of lung adenocarcinomas. ERBB2 mutations are exclusive to EGFR/KRAS/ALK mutations and represent 6% of EGFR/KRAS/ALK negative specimens of non-small cell lung cancers (NSCLC). In NSCLCs, the most common mutations of ERBB2 are in-frame insertions in exon 20 and are more frequent among non-smokers [29]. There is some overlap on genetic changes between BA and NSCLC. However, for all patients, there was no recurrence or metastasis after 2-120 months of follow-up. However, this type of disease often leads to misdiagnosis because they may morphologically mimic mucinous adenocarcinoma.
The well-differentiated mucinous adenocarcinoma can have ciliated columnar cells especially when adenocarcinoma in ltrates into the bronchioles; however, basal cells are never present. Evidence support of malignancy should be carefully ruled out and immunohistochemical analysis highlighting basal cells with p63 and/or CK5/6 is helpful. Otherwise, solitary peripheral ciliated glandular papilloma, mixed squamous cells, and glandular papilloma and mucoepidermoid carcinoma must be considered as differential diagnoses.
In conclusion, we reported four cases of BAs and detected the mutation of ERBB2 exon 20 insertion for the rst time. BA /CMPT is a rare peripheral tumor that exhibits characteristics similar to those of adenocarcinoma, including morphological and genetic changes. At present, BA shows benign biological features that might be due to the limited number of cases. The pathogenesis and biological behavior of BA /CMPT must be examined further in future research and requires the study of more cases and longer follow-up times.