Distinct Profile of Driver Mutations and Clinical Features in Immunomarker-defined Subsets of Pulmonary Large-cell Carcinoma

Natasha Rekhtman; Laura J Tafe; Jamie E Chaft; Lu Wang; Maria E Arcila; Agnes Colanta; Andre L Moreira; Maureen F Zakowski; William D Travis; Camelia S Sima; Mark G Kris; Marc Ladanyi


Mod Pathol. 2013;26(4):511-522. 

In This Article

Abstract and Introduction


Pulmonary large-cell carcinoma—a diagnostically and clinically controversial entity—is defined as a non-small-cell carcinoma lacking morphologic differentiation of either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large-cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large-cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with nine therapeutically relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large-cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%) or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large-cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large-cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.


Large-cell carcinoma is the third most common subtype of non-small-cell lung carcinoma after adenocarcinoma and squamous cell carcinoma, representing 3−9% of non-small-cell lung carcinomas.[1,2] It is defined in the 2004 World Health Organization classification of lung tumors as an 'undifferentiated non-small-cell carcinoma that lacks the cytologic and architectural features of small-cell carcinoma, glandular or squamous differentiation'.[1] In essence, large-cell carcinoma is a tumor in which the line of differentiation cannot be identified by light microscopy, as reflected by its alternative designation as 'large-cell undifferentiated carcinoma'. This designation is reserved for surgically resected tumors because the lack of morphologic differentiation in small biopsy or cytology samples is usually a reflection of incomplete sampling or poor cell preservation rather than a true lack of differentiating morphology in the entire tumor,[1,3] although this terminology has been applied inconsistently.

Large-cell carcinoma has inspired significant controversy over the years, with the main question centered on whether this tumor represents a truly distinct biological entity or an extreme in the poorly differentiated spectrum of the other major types of non-small-cell lung carcinoma, namely adeno- and/or squamous carcinomas. The latter concept is supported by the long-known observation that by electron microscopy, large-cell carcinomas commonly reveal ultrastructural features of either adeno- or squamous carcinoma.[4–6] Similarly, in more recent microarray-based expression profiling studies, these tumors were found to frequently display gene expression patterns resembling either adeno- or squamous carcinoma.[7–9] Likewise, a long-known observation from pre-immunohistochemistry era is that a subset of large-cell carcinomas elaborates cytoplasmic mucin as revealed by histochemical stains, leading to the recommendation to reclassify such tumors as variants of adenocarcinoma.[1,10] The limitation of mucin stains, however, is that their sensitivity for glandular differentiation is low (~30%),[11] and they are therefore variably utilized in routine practice. More recently, it has been noted that by immunohistochemistry, large-cell carcinomas commonly express markers typical of adeno- or squamous carcinoma,[12–18] raising the prospect that with increasing routine use of immunostains in current pathology practice, large-cell carcinoma could become an 'endangered species'.[13] However, some immunomarkers, previously utilized as 'markers of differentiation' in large-cell carcinomas, are now known to lack specificity (such as conventional p63 antibody (4A4) and 34βE12[12,16]—the squamous markers, which were recently shown to have a substantial cross-reactivity in lung adenocarcinomas[19]). Furthermore, no measure of biological accuracy or clinical value of marker-based stratification of large-cell carcinoma has been previously demonstrated.

Two recent advances—one in diagnostic pathology and the other in individualized therapy for non-small-cell lung carcinomas—make it timely to reassess the feasibility and utility of marker-based reclassification of large-cell carcinoma. First, there has been a significant progress in immunomarkers to determine the line of differentiation in non-small-cell lung carcinomas. In particular, a notable advance has been recent characterization of ΔN isoform of p63 (p40) as a highly specific squamous marker, unlike the conventional p63 antibody, which in combination with the glandular marker TTF-1 has been shown to reliably distinguish adeno- and squamous carcinomas.[20–23] Second, the treatment of patients with non-small-cell lung carcinomas has recently undergone a major paradigm shift to a highly individualized approach based on tumor histology and targetable molecular alterations.[24] In particular, the recent breakthroughs in targeted therapies have revealed fundamental molecular differences in therapeutically relevant genetic alterations between adenocarcinoma (eg EGFR,[25]KRAS,[25]ALK[26] and BRAF[27] mutations) and squamous cell carcinoma (eg PIK3CA mutations and several other recently described genetic alterations),[28] which forms the basis for a recommendation to employ predictive molecular tests differentially in patients with these tumors.[29] Given the uncertainty with the diagnostic approach and paucity of studies focused on large-cell carcinomas, the use of individualized therapies in patients with these tumors is not well established. In particular, there is little molecular data to inform a strategy for predictive molecular testing in patients with these tumors. While several studies did include a small number of large-cell carcinomas, and reported on the presence of EGFR (4%)[30] and KRAS (8−30%)[31–34] mutations in these tumors, a comprehensive screen for driver mutations in a large series of large-cell carcinomas has not been performed. Furthermore, it has not been explored whether the recent improvement in immunomarkers could translate into a more biologically precise classification of large-cell carcinomas, which could inform the selection of predictive molecular tests in patients with these tumors.

Given the above considerations, the goals of this study were to (1) establish the overall rate of targetable mutations in large-cell carcinoma, (2) determine whether the distribution of these mutations can be predicted by immunophenotyping and (3) explore whether immunomarker-defined subsets of large-cell carcinoma have distinct clinicopathologic characteristics. We therefore evaluated 102 large-cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63 as classifiers for adeno- and squamous carcinoma, respectively, and correlated the resulting subtypes with nine therapeutically relevant genetic alterations (EGFR, KRAS, BRAF, MAP2K1, PIK3CA, NRAS, AKT1, ERBB2 and ALK) as well as various clinicopathologic parameters.