Histology and molecular testing

Introduction

Lung cancer is the most aggressive human cancer and remains a common cause of death in the world, with 1.8 million new cases (13% of total cancer diagnoses) and ∼1.59 million deaths worldwide in 2012, respectively. ^, Lung cancer is broadly classified into non−small cell carcinoma (NSCC) and small cell carcinoma (SCC) according to cell type and morphology. NSCC is further classified according to the histological cell type into adenocarcinoma and squamous cell carcinoma and additional less frequent subtypes. Pulmonary adenocarcinoma is by far the most common subtype, representing approximately 50% of all lung cancers and 60% of non−small cell carcinomas. In recent times, a more accurate histological classification of NSCC is necessary, as it is critical in selecting patients for further testing of molecular alterations that are linked to effective targeted therapies. This classification of NSCC is recognized and emphasized in the 2015 World Health Organization (WHO) classification of lung tumors, where lineage-specific immunomarkers have been formally integrated into the diagnostic criteria of non−small cell lung cancer histological types, especially for poorly differentiated tumors in small biopsy setting. ^,

Histomorphological classification of pulmonary carcinomas was first proposed by Marchesani in 1924 and was subsequently incorporated in the WHO histological classification of lung tumors of 1967. Classification of pulmonary tumors has undergone multiple iterations since then, especially for adenocarcinoma. The major histological types in the 2015 WHO classification include adenocarcinoma; squamous cell carcinoma; neuroendocrine tumors including small cell carcinoma and large cell neuroendocrine carcinoma, large cell carcinoma, and sarcomatoid carcinoma; and rarer salivary gland type carcinoma ( Table 3.1 ).

Pulmonary adenocarcinoma

Adenocarcinomas are historically located in the lung periphery. However, recent publications suggest that many central lung tumors are histologically adenocarcinoma as well; specifically, up to 30% of solid- or micropapillary-predominant and even ∼20% of acinar-predominant adenocarcinomas are central tumors. ^, On gross examination, most adenocarcinomas appear as irregular tan-gray to gray-white nodules. They often show scarring, anthracotic pigment deposition, and, if close to pleura, pleural puckering. The so-called noninvasive tumors with lepidic growth of tumor cells on preexisting alveolar architecture can be difficult to detect on fresh specimens.

A recent major change in lung adenocarcinoma classification involves recognition of pure lepidic growth pattern as “noninvasive” ( Fig. 3.1 A). Tumors with <3 cm diameter are now designated as lung adenocarcinoma in situ (AIS) and tumors with <0.5 cm area of invasion are called minimally invasive adenocarcinomas (MIAs). AIS and MIA are uncommon tumors and account for ∼5% of resected adenocarcinomas.

Pulmonary adenocarcinoma is also subclassified according to cell type into nonmucinous and mucinous adenocarcinoma. Nonmucinous adenocarcinomas are more common and are further subtyped on the basis of five histological growth patterns: lepidic (noninvasive), acinar, papillary, micropapillary, or solid growth pattern (see Fig. 3.1 A−E); the latter four patterns are considered invasive patterns. As most adenocarcinomas demonstrate a mix of various growth patterns, the amount of these histological patterns should be estimated in 5% increments, and invasive adenocarcinoma is classified according to the most common or predominant pattern. This subtype classification requires the examination of the entire tumor and is therefore mainly applicable to resection specimens. For most diagnostic specimens (small biopsy or cytology specimen), a subtype classification is not necessary, although it is recommended that the primary pattern observed on biopsies should be noted in the diagnosis.

There are recommended guidelines to use standardized nomenclature for diagnosis in small biopsies, taking into account both morphology and immunohistochemistry (IHC) marker expression. The recommended terminologies include non−small cell carcinoma (NSCC); favor adenocarcinoma (when morphology is not definitive, but IHC confirms pneumocyte origin); or squamous cell carcinoma (morphology not definitive, but IHC confirms squamous lineage); or not otherwise specified (when both morphology and IHC profile are not definitive). The commonly used IHC markers for pneumocyte lineage include thyroid transcription factor 1 (TTF-1; recommended) and napsin A, while p40 (recommended) and p63 are considered most specific markers for squamous lineage. The combination of TTF-1 and p40/p63 IHCs can classify more than 90% of poorly differentiated non–small cell lung cancer (NSCLC) as favoring adenocarcinoma or squamous cell carcinoma in small biopsy specimens. In addition to the above subtypes, other less common adenocarcinoma variants include invasive mucinous adenocarcinoma (IMA) (see Fig. 3.1 F), colloid adenocarcinoma ( Fig. 3.2 A), enteric adenocarcinoma (see Fig. 3.2 B), and fetal adenocarcinoma.

Clinical and prognostic relevance of adenocarcinoma subtypes

As they are noninvasive or minimally invasive, AIS and MIA have been associated with close to 100% 5-year disease-free survival. ^, Lepidic predominant adenocarcinoma accounts for tumors with >0.5 cm invasive area or patterns. A large number of publications have demonstrated that lepidic predominant adenocarcinomas carry a good prognosis for patients.

Acinar pattern morphologically refers to round- to oval-shaped glands with central lumina (see Fig. 3.1 B). Papillary pattern shows growth of neoplastic cells along a central fibrovascular core (see Fig. 3.1 C). Papillary pattern corresponds to invasive tumor component, even in the absence of myofibroblastic stroma. Cribriform architecture corresponds to when tumor glands are fused together and is currently classified under acinar histological pattern. It is important to note that cribriform architecture reportedly has been associated with poor prognosis and is also associated with the gene-rearranged tumors such as ALK and ROS1 . Across multiple studies, acinar and papillary predominant tumors have been regarded as intermediate risk with a 5-year disease-free survival of ∼80%−85% in early stage tumors. ^,

Both micropapillary and solid tumors are considered high-grade adenocarcinoma with poor prognoses. The association with poor prognosis has been consistent across multiple large sample size series. Micropapillary tumors appear detached, grow in papillary tufts, but are devoid of the central fibrovascular cores (diagnostic for papillary pattern) (see Fig. 3.1 E). Micropapillary predominant adenocarcinoma has high rates of lymphovascular invasion, spreads through alveolar spaces, and is associated with higher rates of recurrence. ^, Solid pattern tumor is composed of sheets of polygonal cells and lacks definite morphological evidence of glandular architecture (see Fig. 3.1 D). Many of these tumors were often previously misclassified as squamous cell carcinoma when IHC markers were not an essential component of diagnostic criteria. The solid pattern adenocarcinoma, similar to micropapillary-predominant tumors, shows aggressive behavior. ^{, , ,}

IMA is defined by columnar or goblet cells with basally oriented nuclei and abundant apical intracytoplasmic mucin. IMA commonly presents as multicentric, often bilateral tumor. ^, All the above architectural patterns (excluding solid pattern) can be seen in IMA. These tumors show consistent expression for CK7, while pneumocyte marker TTF-1 can be negative in a substantial proportion of cases (60%−80%). Occasional cases can even show CDX2 (a marker of intestinal origin) or CK20 expression (a common marker for colonic origin). Establishing site of origin in IMA can often be challenging due to the lack of organ-specific cell of origin marker(s) and confounding clinical presentation that may suggest metastatic disease (multifocal bilateral disease). Metastatic mucinous tumors by and large lack organ-specific IHC markers and are often identical by morphology. Distinguishing metastatic mucinous adenocarcinomas from pancreas and hepatobiliary ducts, the lower gastrointestinal tract or ovary therefore can be quite difficult. Pulmonary IMA frequently shows mutations in KRAS (up to 60%) and NRG1 fusions. ^,

Squamous cell carcinoma

Squamous cell carcinoma (SqCC), barring rare exception, is strongly associated with smoking. SqCC is an aggressive tumor and behaves similarly to other NSCC. SqCC follows the biological spectrum of low- or high-grade dysplasia to carcinoma in situ and eventually manifests as invasive SqCC. They are historically central tumors, often requiring more extensive surgical resection (pneumonectomy). On macroscopy, these tumors can be exophytic and often become centrally necrotic and cavitating mass. These tumors are locally invasive and have a higher propensity of infiltrating adjacent structures by direct invasion. SqCC is subclassified into keratinizing, nonkeratinizing, and basaloid type. Keratinizing type SqCC is recognized by the presence of “keratin pearl” formation, keratinization of tumor cells, and distinct intercellular bridges ( Fig. 3.3 A), while nonkeratinizing SqCC lacks “keratinization” on morphology (see Fig. 3.3 B) and typically requires p40/p63 IHC positivity (see Fig. 3.3 C) to distinguish from adenocarcinoma (TTF1 or mucin positive), especially solid predominant subtype in small biopsies or large cell carcinoma.

Among the subtypes of SqCC, basaloid type has been reported as a more aggressive tumor than the other two subtypes, although more recent reports have not validated this. More importantly, the tumor morphology may resemble neuroendocrine carcinoma (small cell or large cell neuroendocrine carcinoma) and a diagnosis typically requires establishing squamous lineage marker (p40/p63) expression.

Neuroendocrine carcinomas

The current WHO classification divides pulmonary neuroendocrine tumors into four histological variants: typical carcinoid tumor, atypical carcinoid tumor, small cell carcinoma (SCC), and large cell neuroendocrine carcinoma (LCNEC). Typical carcinoids are low-grade tumors with good prognosis, while atypical carcinoids are intermediate-grade tumors with varying behavior. Among neuroendocrine carcinoma, SCC and LCNEC are both high-grade tumors, with a dismal prognosis.

Small cell lung carcinoma

SCC is a high-grade neuroendocrine carcinoma that accounts for approximately 13% of newly diagnosed lung cancers. It also has the strongest association with smoking among the lung cancer histological subtypes. ^,

SCC is a malignant epithelial neoplasm characterized by relatively small cells with minimal cytoplasm, high nuclear-to-cytoplasmic ratio, finely granular chromatin, and absent or inconspicuous nucleoli. The tumor cells are usually densely packed and frequently show tumor cell nuclei conforming to adjacent tumor cell nuclei, a feature known as nuclear molding. The tumor cell shapes can vary from round, ovoid, to spindled/fusiform, but the tumor cell size is typically less than 3× the diameter of adjacent resting lymphocytes. Necrosis is common and can encompass large geographic areas. In addition, peritumoral vessels can show encrustation of basophilic nuclear DNA (the Azzopardi effect). Mitotic figures are found greater than 10 per 2 mm ² (or ≈10 high power fields) and frequently show counts up to 60 per 2 mm ² . The Ki-67 proliferation index is greater than 50% and generally averages greater than 80% ( Fig. 3.4 ).

IHC is routinely used in the diagnostic workup of SCC. Broad-spectrum cytokeratin (AE1/AE3 and/or Cam5.2) is used to confirm the epithelial nature of tumor cells and can show a variety of immunostaining patterns, which range from diffuse cytoplasmic, paranuclear, to dotlike staining patterns. Neuroendocrine markers are essential and usually consist of a panel including chromogranin A, synaptophysin, and NCAM/CD56; chromogranin A (most specific) and synaptophysin are commonly expressed in SCC, but they lack the sensitivity of NCAM/CD56. While NCAM/CD56 is the most sensitive, it is also the least specific marker (e.g., NK cells will express CD56) and requires contextual morphological interpretation with membranous staining on tumor cells. INSM1, a zinc-finger transcription factor implicated in neuroendocrine differentiation, is a recent and promising IHC marker to confirm neuroendocrine lineage in this context. SCC is commonly positive for TTF-1 (90%−95%). Since a key feature of the tumor is its high proliferation index, the proliferation activity as assessed by Ki-67 immunostaining is useful for distinguishing SCC from carcinoid/atypical carcinoid tumors, especially in biopsy samples with crushing artifact.

Comprehensive genomic profiling efforts have shed light into the molecular underpinnings of SCC. Consistent with its strong association with smoking, the genomes of SCC have extremely high rates of mutation and C:G>T:A transversions. The most common genomic alterations include TP53, RB, and MLL2, with nearly all cases of SCC harboring bi-allelic inactivation of TP53 and RB1 , sometimes by complex rearrangements. This highly suggests that loss of activity in both p53 and RB1 pathways is obligatory in SCC pathogenesis. In addition, up to 25% of SCC have inactivating mutations in NOTCH family genes and up to 13% will have alterations in the TP73 gene. From the genomic standpoint, SCC appears to be a relatively homogenous population and lacks recurrent druggable targets. However, emerging data from gene expression and epigenetic studies of SCC strongly suggest four distinct subtypes defined by their association and differential expression of transcription regulators ASCL1 , NeuroD1 , Yap1 , and Pou2F3. This area of research is being actively investigated with the hopes of finding effective therapeutic interventions within each subclass.

Large cell neuroendocrine carcinoma

LCNEC is an NSCC that displays neuroendocrine morphology and expresses neuroendocrine markers as detected by IHC. LCNEC usually presents as a large peripheral lung mass, often with invasion into adjacent structures (pleura or chest wall). By morphology, LCNEC displays a broad range of growth patterns evident in neuroendocrine tumors, including organoid nesting, trabecular growth, rosette-like structures, and peripheral palisading patterns. The tumor cells are large with moderate to abundant cytoplasm, and the tumor cell nuclei is generally bigger than 3× the diameter of adjacent resting lymphocytes. Similar to SCC, these tumors have high mitotic rates (>10 per 2 mm ² ) and they often display Ki-67 proliferation rates ranging from 40% to 80% ( Fig. 3.5 ).

Recent next-generation sequencing studies of pulmonary LCNEC have suggested the plausible biological relationship between this highly aggressive tumor with SCC and NSCC. ^, Genomic profiling reveals three distinct subtypes: small cell–like subtype (SCC-like), NSCLC-like subtype, and carcinoid-like subtype. SCC-like LCNEC tumors are characterized by similar genomic alterations also seen in SCC, such as comutations/loss in TP53 and RB1, MYC amplifications, and other SCC-like alterations. On the other hand, NSCLC-like LCNEC tumors lack comutations in TP53 and RB, rather harbored genetic alterations more characteristic of NSCLC (particularly adenocarcinoma), including mutations in STK11, KRAS, and KEAP1 . Lastly, the carcinoid-like LCNEC subtype comprises a minority of LCNEC tumors that show relatively lower mutation burden and are characterized by MEN1 mutations. These tumors are often difficult to distinguish from atypical carcinoids as they can share similar morphological features. In fact, recent multiomics studies of pulmonary neuroendocrine tumors, including typical and atypical carcinoids, LCNEC, and SCC, showed that there are two prognostically distinct subtypes within the atypical carcinoid group (10-year overall survival of 88% and 27%). This prognostically worse group is being termed “supracarcinoid” and may be analogous to a well-differentiated neuroendocrine tumor with high-grade features (G3 NET) found in other organs.

Other pulmonary carcinomas

NUT carcinoma is a rare, highly aggressive tumor, defined by the presence of NUT gene fusions on chromosome 15q ( Fig. 3.6 A). Diagnosis of NUT carcinoma requires the demonstration of NUT fusion protein expression by IHC (see Fig. 3.6 B).

Lymphoepithelioma-like carcinoma is characterized by sheets of poorly differentiated epithelial tumor cells with syncytial growth pattern, large nuclei, and prominent nucleoli (see Fig. 3.6 C). By immunohistochemistry, it stains positive for p40/p63, indicating squamous cell lineage. It is most seen in Asian patients and is not associated with smoking. The diagnosis of this tumor requires documenting the presence of Epstein-Barr virus genome by positive Epstein-Barr early protein (EBER)1 chromogenic in situ hybridization (see Fig. 3.6 D).

Salivary gland−type tumors as a group are rare. The common histological subtypes are similar to salivary gland tumor counterparts and comprise mucoepidermoid carcinoma, adenoid cystic carcinoma, epithelial-myoepithelial carcinoma, and pleomorphic adenoma. These tumors typically present as central-type tracheal or endobronchial lung tumors and are not associated with smoking. Diagnostic workup encompasses careful attention to histology and architecture and confirming with IHC markers, when applicable. Mucoepidermoid and adenoid cystic carcinomas have low-grade and high-grade morphologies. However, by and large, these tumors are typically less aggressive than the more common and traditional lung NSCC (adenocarcinoma or SqCC).

Sarcomatoid carcinomas encompass poorly differentiated non−small cell carcinomas displaying mesenchymal features. There are five subtypes, including pleomorphic carcinoma (see Fig. 3.2 C), spindled cell carcinoma, giant cell carcinoma, carcinosarcoma, and pulmonary blastoma. Diagnosis requires that the tumor contains at least 10% of the mesenchymal component (spindled cells or giant cells) or is a carcinoma purely composed of spindled or giant cells. Next-generation sequencing studies revealed that this unique subtype of non−small cell carcinoma is associated with a higher frequency of Met exon 14 skipping mutation.

Before the routine-use immunohistochemistry to subclassify non−small cell carcinoma was practiced, many solid adenocarcinomas or nonkeratinizing squamous cell carcinomas were classified as large cell carcinoma. Currently, this category is reserved for undifferentiated carcinoma with either null or unclear immunohistochemical lineage marker expression, or the latter is not available.

Pathological staging of lung carcinoma—AJCC 8th edition

The UICC and AJCC 8th edition of the lung cancer staging classification system developed by the International Association for the Study of Lung Cancer (IASLC) staging and prognostic factor committee has been implemented internationally in 2017 and in the United States in 2018, respectively. The main updates correspond to the T-descriptor or tumor size with some minor changes in M-descriptor; the N-staging is unchanged. The in situ carcinomas (AIS or squamous in situ carcinoma/SCIS) and MIA are now included as Tis. Changes in the T-descriptor correspond to (1) tumor size, (2) staging of multiple/synchronous lung tumors, and (3) updating tumor spread/extent. The early-stage pT1 tumors (previously pT1a, pT1b in the seventh edition) are now expanded to three subcategories in the eighth edition, viz., pT1a (≤1 cm), pT1b (>1–2 cm), and pT1c (>2−3 cm). Prior pT2 and pT3 tumors from the seventh edition have been updated as well. A lung tumor that measures >3−5 cm corresponds to pT2 tumor (pT2a: >3−4 cm, pT2b: >4−5 cm), while pT3 tumors range from >5 to 7 cm. Any tumor >7 cm is now classified as pT4.

In the new eighth edition, the pathological T stage is based primarily on size of the “invasive,” non-lepidic histological component of adenocarcinoma. The semiquantitative estimation of “noninvasive” lepidic component and morphological distinction of other invasive patterns (i.e., acinar, papillary, micropapillary and solid patterns) now carries utmost importance. Intrapulmonary metastasis remains unchanged in the 8th edition. Ipsilateral same-lobe tumor nodule is staged as pT3, while the ipsilateral different-lobe tumor nodule (pT4) and/or contralateral tumor nodule (M1a) are staged, similar to the seventh edition.

The staging of patients with multifocal lung tumor nodules, which was ambiguous in the seventh edition, has been updated in a series of manuscripts. It is essential to distinguish synchronous separate primary lung tumors and metastatic tumors under the eighth edition staging scheme. Various criteria incorporating clinical and radiological features, histomorphology, and molecular profiles have been published in order to aid and distinguish synchronous versus metastatic tumors. This distinction is relatively straightforward in situations of different cell types (e.g., adenocarcinoma vs. SqCC); however, it can be challenging when dealing with same cell-type tumors. For adenocarcinomas, the initial step typically includes assessment of comprehensive histological patterns of two or more tumors. Multifocal lung tumor nodules are considered synchronous tumors when discordant but separate primary tumors when concordant histologies are encountered.

Regarding staging of lung cancers with multiple ground-glass opacities on imaging studies, which show low-grade morphology (lepidic predominant spectrum) on pathology (AIS or MIA), these tumors are now considered synchronous and staged with the letter “m” in parenthesis, and staging is performed for the higher-stage tumor. Pneumonic-type adenocarcinoma, most common with invasive mucinous adenocarcinoma histology, is staged on the basis of the size of the tumor and location (pT3 in single lobe, pT4 involving different ipsilateral lobe, or M1a involving contralateral lobes). In the current eighth edition, tumors involving the main bronchus are now staged as pT2 tumors (regardless of distance from carina), while diaphragm and recurrent laryngeal nerve involvement/s have now been upstaged to pT4 tumors.

The definition of M1a (separate tumor nodule(s) in a contralateral lobe; tumor with pleural or pericardial nodule(s) or malignant pleural or pericardial effusion) has not changed. The M1b category, however, has been expanded to distinguish between extrathoracic oligometastatic (M1b) and tumors with multiple metastatic disease (M1c).

Predictive biomarker and molecular testing

Before 2008, the treatment of lung cancer patients was largely determined by broad histological classification (non−small cell carcinoma, NSCC vs. small cell carcinoma, SCC) and the stage (early vs. advanced) of the tumor. While this remains the basis of overall treatment strategy, biomarker testing has now become the pillar of treatment decision for advanced-stage NSCLC patients. This change was initiated by the discovery of mutations on exon 18–21 of the epidermal growth factor receptor (EGFR) gene, which results in the constitutive activation of the tyrosine kinase of the receptor. ^, It was discovered that small molecule inhibitors targeting the EGFR kinase can induce tumor cell apoptosis and tumor shrinkage, giving rise to the concept of “oncogene addiction” and “driver” oncogene. ^, During the next decade, additional driver oncogenes were identified and therapies were developed and approved for clinical use. These include anaplastic lymphoma kinase (ALK), ROS1, BRAF V600E, and neurotrophic receptor tyrosine kinase (NTRK). More recently, targeted therapies have been approved targeting KRAS, MET, and RET, which are discussed in Chapter 12 . At the time of writing, clinical trials for additional new targets including HER2 and NRG1 are ongoing and show promising results ( Figure 3.7 ).