Residency · Residency · Pathology

Lung Carcinoma Classification and Molecular Testing

Overview

Lung carcinoma is the leading cause of cancer death worldwide. The WHO classification emphasizes histologic subtyping and molecular profiling to guide targeted therapy and immunotherapy. Accurate classification on small biopsies and cytology specimens, with preservation of tissue for molecular testing, is a core competency for pathologists.

Major Histologic Types

Adenocarcinoma (~40% of lung cancers)

Adenocarcinoma is the most common type overall and the most common type in non-smokers. It shows a peripheral lung predominance and is positive for TTF-1, Napsin A, and CK7 while being negative for CK20.

Adenocarcinoma Subtypes (Resection Specimens)

The lepidic pattern shows growth along intact alveolar walls (formerly bronchioloalveolar carcinoma) without invasion and carries the best prognosis. The acinar pattern forms glands with central lumina. The papillary pattern produces structures with fibrovascular cores. The micropapillary pattern consists of small papillary tufts without fibrovascular cores floating in airspaces; it is aggressive with high metastatic potential. The solid pattern shows sheets of tumor cells without glands and requires mucin staining (5 or more cells with intracellular mucin per 2 HPFs) or immunohistochemistry to confirm adenocarcinoma lineage. Invasive mucinous adenocarcinoma (IMA) features columnar cells with abundant apical mucin, often in a multifocal or pneumonic pattern. It is CK7-positive, variably CK20-positive, and often TTF-1 negative, with common KRAS mutations.

Predominant Pattern Determines Classification

All patterns present should be reported with percentages, as the predominant pattern correlates with prognosis: lepidic carries the best prognosis, followed by acinar and papillary (intermediate), then solid and micropapillary (worst). Notably, a micropapillary or solid component of 5 percent or more confers worse prognosis even when not the predominant pattern.

Preinvasive Lesions

Atypical adenomatous hyperplasia (AAH) is 5 mm or smaller with mild atypia and lepidic growth. Adenocarcinoma in situ (AIS) is 3 cm or smaller with pure lepidic growth, no invasion, and 100 percent disease-free survival after resection. Minimally invasive adenocarcinoma (MIA) is 3 cm or smaller, predominantly lepidic, with invasion of 5 mm or less, and also achieves near 100 percent survival.

Squamous Cell Carcinoma (~25-30%)

Squamous cell carcinoma shows a central or hilar predominance and is strongly associated with smoking. It demonstrates keratinization and intercellular bridges. The immunophenotype is p40-positive, p63-positive, and CK5/6-positive while being TTF-1 and Napsin A negative. Subtypes include keratinizing, non-keratinizing, and basaloid.

Small Cell Lung Carcinoma (SCLC) (~15%)

SCLC is a neuroendocrine carcinoma occurring almost exclusively in smokers. The cells are small with scant cytoplasm, nuclear molding, salt-and-pepper chromatin, and a high mitotic rate. Crush artifact and the Azzopardi effect (DNA encrusting vessel walls) are characteristic. The immunophenotype includes synaptophysin, chromogranin, and CD56 positivity, with TTF-1 positive in approximately 85 percent and Ki-67 exceeding 80 percent. Combined SCLC contains a component of NSCLC (adenocarcinoma, SCC, or large cell).

Large Cell Carcinoma

Large cell carcinoma is a diagnosis of exclusion made only on resection specimens when no squamous or glandular differentiation is identified by light microscopy and neuroendocrine markers are negative. It cannot be diagnosed on small biopsies or cytology (where NSCLC-NOS should be used instead). Large cell neuroendocrine carcinoma (LCNEC) shows neuroendocrine morphology (rosettes, trabeculae, palisading) with large cell cytology, high mitotic rate, and poor prognosis similar to SCLC.

Carcinoid Tumors

Carcinoid tumors are low-grade neuroendocrine neoplasms. Typical carcinoid has fewer than 2 mitoses per 2 mm2 with no necrosis. Atypical carcinoid has 2 to 10 mitoses per 2 mm2 and/or focal necrosis. Both show organoid patterns (nests, trabeculae, rosettes) of uniform cells with moderate cytoplasm and are positive for synaptophysin and chromogranin with Ki-67 typically below 20 percent.

Small Biopsy and Cytology Approach

IHC Panel for NSCLC Subtyping

The minimum panel uses one adenocarcinoma marker (TTF-1 or Napsin A) and one squamous marker (p40 preferred over p63 for its superior specificity, or CK5/6). The minimum immunohistochemistry necessary for classification should be used to preserve tissue for molecular testing. If markers are non-contributory, the diagnosis should be rendered as "NSCLC, favor adenocarcinoma" or "NSCLC-NOS." The term "large cell carcinoma" should never be used on small biopsies.

Tissue Prioritization

The priority order is morphology and IHC for subtyping (minimum slides), then molecular testing (reflex to NGS panel for all non-squamous NSCLC and never-smokers with SCC), then PD-L1 IHC. Cytology cell blocks are equivalent to biopsies for molecular testing when adequate material is present.

Molecular Testing in Lung Adenocarcinoma

Required Biomarkers (NCCN/CAP/IASLC/AMP Guidelines)

EGFR mutations (exon 19 deletion and L858R in exon 21 are sensitizing mutations for EGFR TKIs such as osimertinib; T790M is a resistance mutation). ALK rearrangements (most commonly EML4-ALK fusion, detected by FISH, IHC with D5F3 clone, or NGS; treated with ALK inhibitors such as alectinib and lorlatinib). ROS1 rearrangements (detected by FISH or NGS; treated with crizotinib or entrectinib). BRAF V600E (treated with dabrafenib plus trametinib). KRAS G12C (treated with sotorasib or adagrasib). MET exon 14 skipping (treated with capmatinib or tepotinib). RET rearrangements (treated with selpercatinib or pralsetinib). NTRK fusions (treated with larotrectinib or entrectinib). ERBB2 (HER2) mutations (emerging therapies including trastuzumab deruxtecan).

PD-L1 Testing

The Tumor Proportion Score (TPS) measures the percentage of viable tumor cells with partial or complete membranous staining. A TPS of 50 percent or greater qualifies for first-line single-agent pembrolizumab. A TPS of 1 to 49 percent supports pembrolizumab in combination with chemotherapy. A TPS below 1 percent directs toward chemotherapy with or without immunotherapy combinations. The primary companion diagnostic antibody clone is 22C3 (Dako) for pembrolizumab, with SP263, SP142, and 28-8 as alternatives. Interobserver and inter-platform variability remains a known limitation.

NGS Panel Approach

Comprehensive genomic profiling is preferred over sequential single-gene testing because it detects mutations, fusions, copy number alterations, tumor mutational burden (TMB), and microsatellite instability simultaneously. TMB of 10 or more mutations per megabase is associated with immunotherapy response. Liquid biopsy (ctDNA) is acceptable when tissue is insufficient and is FDA-approved for EGFR and ALK testing.

Staging Essentials for the Pathologist

Under the TNM 8th edition, tumor size is measured for the invasive component only (excluding the lepidic component). Spread through air spaces (STAS), defined as tumor cells beyond the edge of the main tumor in airspaces, is recognized as a form of invasion and is associated with recurrence. Pleural invasion is assessed using the PL0 to PL3 system, and elastic stain (VVG or orcein) is recommended for evaluation; PL1 or greater upstages the T classification. Lymph node assessment follows the N1 (ipsilateral peribronchial/hilar), N2 (ipsilateral mediastinal), and N3 (contralateral) system.

<image>A medical illustration of lung adenocarcinoma growth patterns. Five panels showing the histologic subtypes: Panel A (Lepidic): Tumor cells growing along intact alveolar walls without stromal or vascular invasion, preserving alveolar architecture. Panel B (Acinar): Well-formed glands with central lumina infiltrating desmoplastic stroma. Panel C (Papillary): Papillary structures with fibrovascular cores lined by malignant columnar cells. Panel D (Micropapillary): Small papillary tufts without fibrovascular cores floating freely in alveolar spaces, ring-like arrangements. Panel E (Solid): Sheets of polygonal tumor cells without glandular differentiation, with intracytoplasmic mucin highlighted by mucicarmine stain inset.</image>

<image>A medical illustration of small biopsy lung cancer classification using IHC. Panel A: Poorly differentiated carcinoma on H&E that cannot be classified by morphology alone. Panel B: TTF-1 immunostain showing diffuse nuclear positivity, confirming adenocarcinoma lineage. Panel C: p40 immunostain showing diffuse nuclear positivity in a different case, confirming squamous cell carcinoma. Panel D: Synaptophysin immunostain showing diffuse positivity in small cells with nuclear molding and high mitotic rate, confirming small cell carcinoma. Each panel includes the recommended diagnostic terminology for small biopsy reporting.</image>

<image>A medical illustration of PD-L1 scoring and molecular targets. Panel A: PD-L1 IHC (22C3 clone) showing strong membranous staining in >50% of tumor cells (TPS >=50%), with a diagram illustrating the scoring method counting only viable tumor cells. Panel B: ALK IHC (D5F3 clone) showing diffuse strong granular cytoplasmic staining in a lung adenocarcinoma, with corresponding ALK FISH inset showing split red and green signals confirming rearrangement. Panel C: An infographic showing the molecular testing algorithm for lung adenocarcinoma: tissue acquisition, morphologic classification, reflex to NGS panel and PD-L1, with the actionable targets listed (EGFR, ALK, ROS1, BRAF, KRAS G12C, MET ex14, RET, NTRK, HER2).</image>

Clinical Pearls

On small biopsies, the minimum IHC needed for classification (one adenocarcinoma marker plus one squamous marker) should be used with remaining tissue preserved for molecular testing. Over-staining small biopsies is a common and preventable error that depletes material needed for genomic profiling. Micropapillary and solid patterns, even as minor components (5 percent or more), are associated with significantly worse outcomes and should always be reported with percentages in resection specimens.

Invasive mucinous adenocarcinoma (formerly mucinous BAC) is characteristically TTF-1 negative and KRAS-mutated; negative TTF-1 should not be taken as evidence against a lung primary in this setting. STAS (spread through air spaces) is now recognized as a form of invasion, and its presence in what would otherwise qualify as AIS or MIA upgrades the diagnosis to invasive adenocarcinoma.

All non-squamous NSCLC should undergo reflexive molecular testing; even squamous cell carcinoma in never-smokers should be tested, as 5 to 10 percent harbor actionable mutations. PD-L1 scoring is subject to significant interobserver variability, particularly at the 1 percent and 50 percent thresholds; training and standardization are essential, and equivocal cases should be discussed at tumor board.

References

• WHO Classification of Tumours Editorial Board. Thoracic Tumours. 5th ed. IARC; 2021.
• Lindeman NI, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. J Mol Diagn. 2018;20(2):129-159.
• Travis WD, et al. The 2015 World Health Organization classification of lung tumors. J Thorac Oncol. 2015;10(9):1243-1260.
• Tsao MS, et al. PD-L1 immunohistochemistry comparability study in real-life clinical samples. J Thorac Oncol. 2018;13(12):1836-1843.
• Amin MB, et al. AJCC Cancer Staging Manual. 8th ed. Springer; 2017.

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