Bronchiolar Adenoma with ERBB2 exon 20 insertion: report of four cases and review of the literature


 Objectives: Ciliated muconodular papillary tumor (CMPT) is a rare peripheral lung tumor and is considered to be a subtype of Bronchial Adenoma (BA). Although recent studies have suggested that BA belongs to a neoplastic disease, the complete histogenesis of BA is not fully understood and molecular data is limited. Methods: We demonstrated clinicopathologic features of four cases of BA and performed immunohistochemical and next generation sequencing to characterize its molecular features. Besides, a review of previous literature was done to comprehensively conclude the molecular characteristic of this disease.Results: We found ERBB2, EGFR, BRAF and AKT1 mutations in BAs. It’s the first time that ERBB2 exon 20 insertion has been reported in BA. Conclusion: BA is a rare pulmonary disease mainly affected elderly Asian patients. More and more abnormal molecular changes were observed, which confirmed the neoplastic nature of BA. However, it also raise the debate about BA’s biological behavior.


Introduction
Ciliated muconodular papillary tumor (CMPT) is newly recognized peripheral lung disorders, which was first described in 2002 by Ishikawa et al []. CMPT was once considered as a very rare tumor, mainly affected the East Asia patients. But in recent years, many cases have been reported with the increasing recognition of this entity . Kamata reported 10 cases in 2015 [6], and then both Chang (Western country cohort) [18] and Zheng [19] reported more than 20 cases in 2018. So far, nearly 100 cases have been reported in the English literatures and most of these patients are in East Asia countries. So this tumor may not be as rare as they are once believed.
CMPTs often affect middle-aged to elderly adults, and consist of ciliated columnar cells, mucous cells and basal cells. The entity is commonly surrounded by extracellular mucin pools in the peripheral lung. Some cases did not fit all diagnostic criteria, such as absent papillary architecture or lack of mucinous and/or ciliated cells, but shared the histological feature of bilayered bronchiolar-type proliferation and continuous layer of basal cells, which were termed as "non-classic" CMPTs by Zheng 3 et al. [19]. Moreover, both Zheng et al. [19] and Chang et al. [18] have found these lesions also shared genomic abnormalities. Therefore, Chang et al. [18] proposed a concept of Bronchiolar Adenoma (BA) in 2018. In addition, they sub-categorized BA into 2 groups: proximal-type and distal-type, according to morphologic and immunohistochemistry (IHC) grounds. Hence, classic CMPT only represent a subset of proximal-type BA.
Here we report four cases of BA. All the cases were performed molecular analysis by next-generation sequencing (NGS) and identified ERBB2, BRAF, AKT1 and EGFR gene mutations. To our knowledge, this is the first time that ERBB2 mutation has been found.

Clinical samples
The four cases were diagnosed in West China Hospital of Sichuan University between 2017 and 2019. Clinical data were extracted from the hospital's electronic medical records. The samples were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). The clinical and pathological records were analyzed retrospectively, and histological analysis was performed on the surgically-resected specimens.

Isolation of genomic DNA
Genomic DNA was extracted from paraffin-embedded tissue blocks by using the QIAamp DNA FFPE kit (Qiagen, German). The concentration and quality was determined using the ScanDrop 200 spectrophotometer (Analytik Jena, Germany), and the optical absorbance of wavelength 260nm and wavelength ratio of wavelength 260-280nm were calculated.

Clinical characteristics
Three of the four patients were female and one was male, the age range from 32 to 65 years. The male patient had a history of smoking, while all the female patients hadn't. All the four patients were found to have pulmonary nodules by physical examination. Computed tomography (CT) imaging shows solid or ground-glass nodules in the peripheral lung. The volume of all of our cases was less than 1 cm in diameter.
All of them received wedge resections either in West China Hospital of Sichuan University or other hospitals. The detailed clinicopathological features are shown in table 1. Some cases were misdiagnosed at first. Case 2 was originally diagnosed as atypical alveolar epithelial hyperplasia and suspected to adenocarcinoma in 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 (MIA) of the lung.

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

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

Molecular analysis
All cases were performed NGS sequencing to profile the molecular abnormalities (Table 1)

Discussion
BA is a newly entity defined by Chang et al. [18], which is a more extensive terminology than CMPT. It has not been well recognized by pathologists and could not be classified according to 2015 WHO classification system [28]. So far, 26 English literatures about this family of neoplasms are searched, plus our cases, there are 99 cases have been reported. The clinicopathologic features of these cases were summarized in Table 2.
BA often affect middle-aged to elderly adults from East Asia (77/99, 77.8%), with the median age of 64 years old (range from 19 to 84 years old). Expect one patient is 19 years' old [10], the remaining are range from 31 to 84 years old. The incidence rate of male and female was similar, close to 1:1.3.
Grossly, the tumor is a pale mucinous nodule with irregular border and sometimes a central cavity in But for distal-type BAs, TTF1 and Napsin A showed diffuse positivity. Some cases revealed discontinuous (skipping) growth pattern which is resembling Tumor Spread Through Air Spaces (STAS) and micropapillary tufts detached into the alveolar cavity which is similar to the micropapillary adenocarcinoma. Chang et al. hypothesized that these cells being interconnected with each other in 3-dimensional spaces [18], because these skip lesions do not extend away for more than a few alveoli. Anyway, the basal cells were always present. However, these microscopic features of BA may be an extremely diagnostic challenge for pathologists, especially for intraoperative frozen sections.
Again, it's the first time that ERBB2 exon 20 insertion has been reported in BA. Human epidermal growth factor receptor 2 (HER2 /ERBB2) is a receptor tyrosine kinase of the ERBB family, which plays a significant role in cancer development and progression, especially in breast, ovarian and gastric cancers. Overexpression of HER2 protein associated with poor prognosis. Recent research shows that HER2 mutations is a distinct subset of lung adenocarcinomas. ERBB2 mutations are exclusive to 8 EGFR/KRAS/ALK mutations, representing 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 were more frequent among non-smokers [29]. It is obvious that there is some overlap on genetic changes between BA and NSCLC. But of all the patients, there are 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.
Of all the 99 patients, twenty-three were diagnosed with adenocarcinoma originally (23.0%) [6,[17][18][19]5,21,23,24], two were suspected of malignant tumors (2.0%) [6]. Furthermore, some cases were just pathologically descriptive diagnosis. So it's important for pathologist to distinguish BA from malignant tumors, especially in intraoperative diagnosis. The well-differentiated mucinous adenocarcinoma could have ciliated columnar cells especially when adenocarcinoma infiltrates into bronchioles, but never present basal cells. Evidence support to malignancy should be carefully ruled out, and immunohistochemistry highlighted basal cells with p63 and/or CK5/6 is helpful. Otherwise, solitary peripheral ciliated glandular papilloma, mixed squamous cell and glandular papilloma and mucoepidermoid carcinoma needs to be considered as differential diagnosis.
In conclusion, we reported four cases of BAs and detected the mutation of ERBB2 exon 20 insertion for the first time. BA is likely a rare peripheral tumor, and exhibit characteristics similar to adenocarcinoma, including morphological and genetic changes. At present, BA shows benign biological feature might due to the limited number of cases. The pathogenesis and biological behavior of BA need to be further clarified, requiring more cases and longer follow-up.

Availability Of Data And Materials
The data used and/or analysed during the current study are available from the corresponding author on reasonable request.

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