Introduction
Lung cancer ranks among the leading causes of cancer morbidity and mortality worldwide, with an estimated 2.2 million new cases diagnosed and 1.8 million deaths reported every year.1 The interpretation of lung biopsies and large resection specimens (wedge resection, segmentectomy, lobectomy, and pneumonectomy) can be challenging due to common confounding factors including the level of experience/subspecialty training of the pathologist, the quality and quantity of the specimens provided by the clinician, and the availability of clinical, imaging, and laboratory testing information.
Within the thoracic space, mesothelioma is the most common primary tumor of the pleura. Mesothelioma has been associated with occupation and environmental exposure to asbestos.2 The challenges in diagnosing malignant mesothelioma stem from the morphologic variants (deciduoid, clear cell, small cell, signet ring cell, pleomorphic, adenomatoid-like, lymphohistiocytoid, etc.), the availability of diagnostic markers at the local institution, and difficulty with interpreting ancillary studies, which are further exacerbated by the potential medicolegal consequences of this diagnosis.
The World Health Organization (WHO) Classification of Tumors published in 2015 expanded our knowledge of the diagnosis, classification, and genetics of lung tumors and pleural mesotheliomas. This provided us new information about novel immunohistochemical markers, improved understanding of underlying molecular biology, and refined diagnostic stratification of tumor types. However, the WHO published new guidelines on the Classification of Thoracic Tumors in 2021. The aim of this review is to provide a summary of this updated information regarding the new concepts, diagnostic criteria, and guidelines for practicing anatomic pathologists, cytopathologists, pathology trainees, and clinicians.
Highlights of New WHO Classification of Lung Tumors
Small diagnostic samples
Accurate pathologic diagnosis of small samples is essential for guiding clinical therapy of lung cancer. Providing a precise diagnosis based on small biopsy samples requires the pathologist to strike a balance between the clinical need for detailed classification and the limited tissue sample size, which can be further hampered by scant viable tumor cells and/or poorly differentiated tumor morphology. While the optimal approaches to obtaining diagnostic materials differ among institutions, routine methods include imaging guided biopsies and exfoliative specimens such as sputum, bronchial washings/secretions, bronchial brushings, and bronchoalveolar lavages.
Diagnoses of small cell carcinoma, squamous cell carcinoma, and adenocarcinoma with specific growth pattern(s) can be made based on morphological analysis of small biopsies. The use of certain terms for small samples of lung specimens is discouraged in the WHO 2021 guidelines.3 The term “non-small cell lung carcinoma-not other specified (NSCLC-NOS)” should be minimized, and NSCLC must be further classified as adenocarcinoma or squamous cell carcinoma whenever possible. The term “NSCLC-non-squamous cell carcinoma” should be avoided as a histopathologic diagnosis, as this entity is used by clinicians to describe a heterogeneous group of tumors for clinical trials.4–6 A few entities require a resection specimen to diagnose, including adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma; in these scenarios, the tumor should be diagnosed as adenocarcinoma with a lepidic growth pattern only if a noninvasive pattern is present, with a comment that definitive diagnosis is deferred to a larger specimen.
When evaluating a biopsy with malignant cells and no clear evidence of glandular formation, squamous differentiation, or neuroendocrine morphology, immunohistochemical analysis, including TTF1 and p40, could be helpful. A diagnosis of NSCLC, favoring adenocarcinoma, can be made based on positive TTF1 staining, and a diagnosis of NSCLC, favoring squamous cell carcinoma, would be supported by immunoreactivity with p40. Adenocarcinoma with a solid pattern should be ruled in/out with special stains, such as mucicarmine, Kreyberg, and PAS-D, to highlight cytoplasmic mucin in the neoplastic cells to be classified as adenocarcinoma. Large cell carcinoma cannot be diagnosed in small samples, as the tumor must be thoroughly sampled in a resection to exclude a different component and determine the proportion of each component. When both TTF1 and p40 staining are negative in the absence of definitive cytoplasmic mucin, NSCLC-NOS can be diagnosed, with a comment in the diagnostic report of the possibility of large cell carcinoma in the resection specimen. Pleomorphic carcinoma (more than 10% spindle or giant cell or combined components in the background of NSCLC), mixed mucinous and non-mucinous adenocarcinoma (more than 10% of each component), and adenosquamous cell carcinoma (more than 10% of each component) should not be diagnosed based on small biopsies, because the percentage of component cannot be precisely determined based on limited sampling. Of note, reporting the relative proportion of each pattern is discouraged due to possible misrepresentation of a larger tumor in a small biopsy/cytology specimen.
When sarcomatoid features (nuclear pleomorphism, malignant giant cells, or spindle cell morphology) are present in biopsies, the tumor should be classified based on the carcinomatous component (squamous cell carcinoma, adenocarcinoma, or NSCLC, favor adenocarcinoma/squamous cell carcinoma) with a comment describing the sarcomatoid features. In addition, immunohistochemical studies with neuroendocrine markers should be performed only in cases with sufficient neuroendocrine features based on histology/nuclear features (Fig. 1). Carcinoid tumor NOS is used when the tumor exhibits carcinoid morphology with <2 mitoses in a 2 mm2 region. Ki67 is especially useful in distinguishing carcinoid tumors from small and large cell neuroendocrine carcinoma, particularly in the presence of crush artifacts. Small and large cell neuroendocrine carcinoma should be considered if the tumor has neuroendocrine features with a Ki-67 proliferative index higher than 50%.
Precursor glandular lesions
“Precursor glandular lesions” is listed under epithelial tumors in the 5th edition of WHO, while “precursor lesions” is discussed under adenocarcinomas in the 4th edition of WHO. Precursor lesions of the lung include atypical adenomatous hyperplasia (AAH) and AIS, mucinous and non-mucinous types. Recent studies on tumorigenesis have suggested the step-wise progression from AAH to AIS, and subsequent development into minimally invasive adenocarcinoma and overt invasive adenocarcinomas.7 AIS is defined as a sole lepidic growth pattern, with atypical pneumocytes lining the preserved alveolar architectures (Fig. 2a). The size must be no greater than 3.0 cm, with no desmoplastic reaction or other invasive growth patterns such as acinar, papillary, micropapillary, solid, or complex glandular. AIS has no tumor necrosis, visceral pleural invasion, or lymphovascular invasion. AAH presents with atypical pneumocytes lining the alveolar septa with a cutoff size at 0.5 cm.
New high-grade patterns of lung adenocarcinoma
For staging purposes, the size of the invasive components (i.e. any growth patterns other than lepidic) must be documented, and the percentage of each growth pattern should be included. In general, invasive tumors with micropapillary (Fig. 2e) and solid (Fig. 2d) growth patterns are clinically more aggressive compared to those with acinar- (Fig. 2b) and papillary-predominant (Fig. 2c) growth.8,9 Studies have revealed that filigree and complex glandular patterns (Fig. 2h) are considered high-grade patterns and have significant impacts on clinical outcomes in patients with lung adenocarcinomas.8,10,11
Filigree pattern as a subtype of micropapillary growth pattern
The filigree micropapillary growth pattern has recently been defined as “tumor cells growing in delicate lace-like narrow stacks of cells without fibrovascular cores”.12 The filigree pattern differs from the classical micropapillary pattern by the absence of floret tufts (Fig. 3). Once this diagnostic criterion is incorporated, tumors conventionally recognized as lepidic, acinar, or papillary surrounded by filigree growth would have been reclassified as micropapillary, and these tumors have been shown to be associated with higher risk of recurrence, advanced stage, lymphovascular invasion, and solid tumor growth.12
Complex glandular pattern (CGP)
CGP is a newly recognized high-grade growth pattern in the 2021 WHO lung tumor classification and is defined as “fused glands (Fig. 2f) or single cells (Fig. 2g) infiltrating in a desmoplastic stroma”.3 While the cribriform pattern was initially included in the 2015 WHO classification system as a subtype of acinar growth pattern with no further discussion of CGP, there has been increasing evidence that CGP-predominant tumors are associated with worse clinical outcome compared to acinar-predominant tumors and frequently harbor ALK rearrangement and HER2 mutation.10 Histologically, high-grade features, including higher mitotic rate, more extensive tumor necrosis, and lymphovascular invasion, are seen in CGP-predominant tumors.13,14 In the 2021 WHO classification, CGP has been recognized as one of the high-grade patterns on par with solid and micropapillary growth.
More recent studies suggest a further refined stratification scheme. Specifically, cribriform (Fig. 2h) as a predominant pattern has been shown to be associated with poor prognosis akin to solid and micropapillary patterns, whereas the 5-year survival for patients with fused gland-predominant adenocarcinomas ranked between papillary- and micropapillary-predominant tumors.11 Histologically, cribriform-predominant adenocarcinoma is associated with larger tumor size, more frequent pleural involvement, lymphovascular invasion, and spread through airspaces (STAS), and recurrent tumors with cribriform pattern may have therapeutic implications with tyrosine kinase inhibitors in a small subset of patients.15 These data suggest that the cribriform pattern could be recognized as a stand-alone aggressive subtype, and such classification would further stratify the prognostic values of the complex glandular pattern and potentially provide guidance for targeted therapy.
Updates on grading
The conventional grading system for nonmucinous adenocarcinomas consistently correlates with prognosis and predicts tumor response to adjuvant chemotherapy. In general, lepidic predominant tumors are considered low grade; acinar or papillary predominant adenocarcinomas are more aggressive and thus considered intermediate grade, while micro-papillary and solid-predominant tumors have the worst prognosis and are classified as high grade. However, a specific drawback on the prognostic value, based solely on the “predominant growth pattern,” is the omission of the “non-predominant”, or secondary growth pattern, which can be associated with worse prognosis, especially with regards to the micropapillary pattern. To account for the more aggressive minor growth patterns, a formal three-tier grading system has been developed to incorporate secondary high-grade patterns (Table 13,16). Micropapillary, solid, cribriform, and complex glandular patterns (Fig. 2) must be included based on the current grading scheme if they comprise at least 20% of the tumor in resected specimens.16
Table 1Grading of lung adenocarcinomas based on growth patterns3
| Grade | Predominant Pattern | Secondary Pattern |
|---|
| Grade 1: Well differentiated | Lepidic | <20% high-grade pattern |
| Grade 2: Moderately differentiated | Acinar, Papillary | <20% high-grade pattern |
| Grade 3: Poorly differentiated | High grade pattern ≥20%; Micropapillary, solid, cribriform, complex glandular |
Tumor cell STAS is defined as the spread of lung cancer cells into air spaces in the lung parenchyma beyond the edge of the main tumor. STAS encompasses three histologic patterns, namely micropapillary, solid nests/tumor islands, and discohesive single cells.17 STAS tumor cells can be distinguished from alveolar macrophages and detached reactive type pneumocytes by the lack of cytoplasmic pigment or foamy cytoplasm, higher degree of nuclear atypia and hyperchromasia, and more prominent nucleoli. Artifacts constitute another potential confounding phenomenon, which are featured by (1) the presence of tumor cell clusters randomly scattered in tissue distant from the main tumor, lacking evidence of continuous spread of tumor; (2) tumor cell clusters at the edge of a resection specimen; (3) jagged edges of tumor cell clusters (tumor fragmentation, or carry-over from a knife during grossing); and (4) linear strips of tumor cells lifted off alveolar walls. STAS is associated with a more aggressive clinical course, especially in patients undergoing limited resection compared with lobectomy. STAS should not be included in the tumor size for staging or the percentage of growth patterns, as it is considered a feature of tumor spread rather than a component of the invasive tumor.
Molecular alterations in large cell neuroendocrine carcinoma (LCNEC)
Recent genetic and molecular studies have advanced our knowledge about the etiology and pathophysiology of neuroendocrine neoplasms of the lung. While the 2021 WHO classification did not specifically account for the molecular characteristics of each entity, certain molecular alterations are seen consistently in a subset of lung tumors. To subclassify pulmonary neuroendocrine tumors, incorporating molecular changes into the diagnostic approach can further improve the precision and further guide the development of targeted therapies. Carcinoid tumors are clinically indolent neuroendocrine tumors with low mutation rates and usually harbor mutations in MEN1, EIF1AX, PSIP1, KMT2C, and ARID1A genes, most of which are involved in chromatin remodeling.18,19 In contrast, LCNEC usually have a higher mutation burden and smoking-related mutation signature. Genomic and transcriptomic analysis further subdivided LCNECs into two major and one minor groupings based on their mutation characteristics. One major group of LCNECs shows bi-allelic TP53, STK11/KEAP1, and KRAS alterations, frequently observed in adenocarcinomas and squamous cell carcinomas, whereas the other major group with RB1 alterations and TP53 inactivation displayed molecular features similar to SCLC.19 A minor group of LCNECs shows a MEN1 mutation, which represents a small group of tumors with carcinoid morphology but exceeds the proliferative threshold for lung carcinoids (i.e. 10 mitoses/2 mm2). Although a Ki-67 labeling index greater than 30% is considered a supportive diagnostic feature for LCNEC,20,21 its definitive diagnostic utility and the precise cutoff warrant further investigation.20
Bronchiolar adenoma/ciliated muconodular papillary tumor (BA/CMPT)
BA/CMPT was included as a new entity in the 2021 WHO classification and is usually defined as a tumor with subpleural localization less than 2.0 cm (Fig. 4) Histologic features of BA/CMPT include bland, bilayered bronchiolar-type epithelium arranged in a nodular architecture. Luminal epithelial cells can be ciliated, mucinous, or flat, with no atypia or increased mitotic activity. BA/CMPTs are further categorized into proximal and distal types.22 The proximal type usually has more ciliated and/or mucinous cells with a morphologic resemblance to proximal airway, and the distal type tends to have cuboidal, TTF-1 positive cells similar to smaller airways; such dichotomy is not mutually exclusive, and the histologic features do not always correspond to their anatomic location. While BA/CMPTs are generally considered indolent, they can mimic invasive lesions on imaging studies23 and pose a diagnostic challenge in intraoperative diagnosis.24 The continuous basal cell lining can be a clue to avoid misinterpretation in frozen sections, and the diagnosis can be confirmed by immunohistochemical staining with p63, p40, or CK 5/6. Most BA/CMPTs demonstrate characteristic molecular alterations involving BRAF, KRAS, EGFR, and HRAS genes.25
SMARCA4-deficient undifferentiated tumor (SMARCA4-UT)
SMARCA4-UT is a newly added entity in the 2021 WHO classification. These tumors have a characteristic loss of expression of BRG1 caused by an inactivating mutation in the SMARCA4 gene. BRG1 is a key member of the BAF chromatin-remodeling complex.26 Morphologically, these tumors consist of diffuse sheets of monotonous, high-grade, discohesive, round to epithelioid cells with prominent nucleoli (Fig. 5). Areas of rhabdoid differentiation can be appreciated. Originally termed “SMARCA4-deficient thoracic sarcoma”,27–29 this entity was renamed due to its smoking-associated genomic signature, clinicopathologic characteristics, and immunohistochemical profile, which suggested that these tumors are undifferentiated/de-differentiated carcinomas.30 Since a small proportion of conventional NSCLCs show SMARCA4 mutation,31 BRG1 immunohistochemical stains should be performed only in cases with appropriate clinical suspicion (i.e. high-grade, monotonous undifferentiated malignancies). SMARCA4-UTs lack the histologic (glandular formation) or immunohistochemical (keratin expression) features of carcinoma. Although SMARCA2, CD34, Sox2, SALL4, and p53 expression can be seen in SMARCA4-UTs,27 none of these markers is entirely sensitive or specific. SMARCA4-UTs tend to affect younger patients with an aggressive clinical course; median survival ranges from 4 to 7 months.27–30 Preliminary studies have shown improved clinical outcomes in patients treated with immune checkpoint inhibitors.32
Primary pulmonary hyalinizing clear cell carcinoma (HCCC)
HCCC is listed under the category of salivary gland-type tumors in the 2021 WHO classification. Originally described as a low-grade malignant neoplasm commonly involving the head and neck, roughly a dozen cases of primary pulmonary HCCC have been reported.33 These tumors are usually found in the submucosa of a large airway. Small to medium sized bland epithelioid cells with ample clear to eosinophilic cytoplasm are typically arranged in nests, cords, sheets, and thin trabeculae, in a background of extensively hyalinized stroma.33–36 Most cases lack high-grade features, such as necrosis, increased mitosis, and nuclear pleomorphism. The neoplastic cells express cytokeratin 7 and keratin AE1/AE3, the vast majority of which are positive for p63 and p40, and some cells are positive for cytoplastic mucin. Similar to their counterpart in the head and neck regions, primary pulmonary HCCC typically show EWSR1 gene rearrangement.