Simultaneous Multi-Antibody Staining in Non-Small Cell Lung Cancer Strengthens Diagnostic Accuracy Especially in Small Tissue Samples

Abstract

Histological subclassification of non-small cell lung cancer (NSCLC) has growing therapeutic impact. In advanced cancer stages tissue specimens are usually bioptically collected. These small samples are of extraordinary value since molecular analyses are gaining importance for targeted therapies. We therefore studied the feasibility, diagnostic accuracy, economic and prognostic effects of a tissue sparing simultaneous multi-antibody assay for subclassification of NSCLC. Of 265 NSCLC patients tissue multi arrays (TMA) were constructed to simulate biopsy samples. TMAs were stained by a simultaneous bi-color multi-antibody assay consisting of TTF1, Vimentin, p63 and neuroendocrine markers (CD56, chromogranin A, synaptophysin). Classification was based mainly on the current proposal of the IASLC with a hierarchical decision tree for subclassification into adenocarcinoma (LAC), squamous cell carcinoma (SCC), large cell neuroendocrine carcinoma (LCNEC) and NSCLC not otherwise specified. Investigation of tumor heterogeneity showed an explicit lower variation for immunohistochemical analyses compared to conventional classification. Furthermore, survival analysis of our combined immunohistochemical classification revealed distinct separation of each entity's survival curve. This was statistically significant for therapeutically important subgroups (p = 0.045). As morphological and molecular cancer testing is emerging, our multi-antibody assay in combination with standardized classification delivers accurate and reliable separation of histomorphological diagnoses. Additionally, it permits clinically relevant subtyping of NSCLC including LCNEC. Our multi-antibody assay may therefore be of special value, especially in diagnosing small biopsies. It futher delivers substantial prognostic information with therapeutic consequences. Integration of immunohistochemical subtyping including investigation of neuroendocrine differentiation into standard histopathological classification of NSCLC must, therefore, be considered.

Napsin A/p40 antibody cocktail for subtyping non‐small cell lung carcinoma on cytology and small biopsy specimens

“…The distinction between ACA and SQCC can be challenging in small biopsy and cytology specimens, particularly in cases in which the tumor is poorly differentiated, the sample is paucicellular, and/or the morphology is difficult to evaluate due to preparation artifact. Many authors have advocated antibody panels to enhance the sensitivity and specificity of NSCLC subtyping, and several have explored the use of multiplexed antibody “cocktails” to minimize the number of slides necessary for accurate NSCLC subtyping …”

“…Many authors have advocated antibody panels to enhance the sensitivity and specificity of NSCLC subtyping, 8,14,22,26,27 and several have explored the use of multiplexed antibody "cocktails" to minimize the number of slides necessary for accurate NSCLC subtyping. [28][29][30][31][32][33][34][35] We evaluated the ability of a 2-antibody cocktail targeting napsin A and p40 to subtype NSCLC on a single histologic section. In NSCLC TMAs constructed from resected ACA and SQCC specimens, the napsin A1/ p406 and napsin A-/p401 immunophenotypes using the cocktail were 100% specific for ACA and SQCC, respectively.…”

A microRNA-based prediction algorithm for diagnosis of non-small lung cell carcinoma in minimal biopsy material

“…Of course, certain diagnostic biopsies may contain even lower fractions of tumour cells. In such cases, diagnosis in NSCLC biopsies can be also facilitated by specific immunohistochemical markers (Kayser et al , 2013). It is likely that a combination of the two methods may significantly complement each other, resulting in a highly accurate diagnostic test.…”

Into the Depths: Techniques for in Vitro Three-Dimensional Microtissue Visualization

“…This technique was also validated using rhodamine phalloidin to visualize cellular F-actin staining and cytoskeletal organization in BEAS-2B microtissue sections (Figure 2C). Similar to tissue arrays, fixing, embedding and sectioning the whole agarose hydrogel also enables uniform staining, visualization, and assessment of multiple microtissues within a single sample (22–24). Additional samples fixed in Optimal Fix (American MasterTech Scientific, Inc., Lodi, CA), an alcohol-based fixative, were similarly embedded but yielded poor results during cryosectioning due to a lack of proper OCT infiltration in the hydrogel (data not shown).…”