Con: “Is Spread Through Air Spaces an In Vivo Phenomenon or an Inducible Artifact?”
Authors: Hans Blaauwgeers, Chris Dickhoff, Giuseppe Pelosi, Wim Timens, Federica Filipello, Yuko Minami, Giulio Rossi, Erik Thunnissen
Journal: Journal of Thoracic Oncology (2024)

Since the introduction of spread through air spaces (STAS) as a new invasive pattern in the 2015 WHO classification on NSCLC,1Travis W.D. Brambilla E. Burke A.P. Marx A. Nicholson A.G. Travis W.D. Brambilla E. Burke A.P. Marx A. Nicholson A.G. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. 4th ed. IARC, Lyon, France2015Google Scholar there has been a debate on whether STAS is a true biological phenomenon or mainly an artifact induced by surgical and pathology handling of the resected specimen.2Blaauwgeers H. Russell P.A. Jones K.D. Radonic T. Thunnissen E. Pulmonary loose tumor tissue fragments and spread through air spaces (STAS): invasive pattern or artifact? A critical review.Lung Cancer. 2018; 123: 107-111Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar The definition of STAS is blurred by semantics and imaginary interpretations and lack thereof. In morphologic terms, pathologists may recognize "loose tumor cells" as single cells or clusters in the air spaces. The definition of STAS has several peculiarities and implications.3Borczuk A.C. Cooper W.A. Dacic S. et al.WHO Classification of Tumours Editorial BoardThoracic tumours.in: WHO Classification of Tumours. 5th ed. IARC, Lyon, France2021: 19-189Google Scholar(1)The actual definition of STAS has the following two components: a morphologic and a topological component. In the identification of STAS, the following three so-called patterns are recognized: single cells, micropapillary, and solid tumor cell clusters.4Kadota K. Nitadori J.I. Sima C.S. et al.Tumor spread through air spaces is an important pattern of invasion and impacts the frequency and location of recurrences after limited resection for small Stage I lung adenocarcinomas.J Thorac Oncol. 2015; 10: 806-814Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar For the topological component, one of these "patterns" of intraalveolar tumor cells needs to be found in a continuum of air spaces containing intraalveolar tumor cells back to the tumor edge (or viewed from the other direction "beyond the main tumor edge"). Nevertheless, if the tumor cells fitting in this "pattern" description are localized at a distance from the main tumor (without the continuum), then these tumor cell "patterns" are regarded as an artifact. In other words, although there is no morphologic or cytologic difference between these "patterns," the topological distribution denotes whether they are interpreted as artifact or STAS. Of note, the term "pattern" is in the lung not synonymous for "morphology." In addition to the above-mentioned definition, other aspects are relevant of the distinction of STAS:(2)Tumor floaters found by the presence of clusters of cells often randomly scattered over the tissue and at the edges of the tissue section are favored to be interpreted as an artifact.4Kadota K. Nitadori J.I. Sima C.S. et al.Tumor spread through air spaces is an important pattern of invasion and impacts the frequency and location of recurrences after limited resection for small Stage I lung adenocarcinomas.J Thorac Oncol. 2015; 10: 806-814Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar Nevertheless, no diagnostic clues are provided on how to make the distinction between when (a few) tumor cells are close to the tumor, when they are randomly scattered, or "real" STAS. In a more recent publication, the refinement of "more than one tumor cell nest involvement of at least one air space beyond the tumor edge, and continuous alveolar spread from the tumor edge to the furthest STAS"5Aly R.G. Rekhtman N. Li X. et al.Spread through air spaces (STAS) is prognostic in atypical carcinoid, large cell neuroendocrine carcinoma, and small cell carcinoma of the lung.J Thorac Oncol. 2019; 14: 1583-1593Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar was made. The minimum number should be two "cells or clusters."(3)The presence of "jagged edges" of the tumor cell clusters suggested tumor fragmentation or edges of a knife cut during specimen processing. In practice, the knife blade has a width of 250 μ and the blade holder a width of 2000 μ (2 mm). The blade itself has a width of at least 10 times the size of a tumor cell, whereas the blade holder is broader than 80 tumor cells in a row (Fig. 1A and B). During cutting, a prominent force is applied on the section. An unsolved question will be whether the jagged or ragged edges are caused by the knife itself or by the force during cutting. The "jagged" or "ragged" description is at a macroscopic level appealing, like an imaginary cut in a finger, but is at microscopic level taking the broad blade into account only understandable if sheared cutting forces tear the clusters apart.(4)This cutting force may cause benign and malignant cells to be displaced. In the first manuscript, macrophages were mentioned.4Kadota K. Nitadori J.I. Sima C.S. et al.Tumor spread through air spaces is an important pattern of invasion and impacts the frequency and location of recurrences after limited resection for small Stage I lung adenocarcinomas.J Thorac Oncol. 2015; 10: 806-814Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar In the more recent manuscript of the same institute, pneumocytes and bronchial epithelial cells were specifically mentioned as an artifact when displaced from their normal environment.3Borczuk A.C. Cooper W.A. Dacic S. et al.WHO Classification of Tumours Editorial BoardThoracic tumours.in: WHO Classification of Tumours. 5th ed. IARC, Lyon, France2021: 19-189Google Scholar,5Aly R.G. Rekhtman N. Li X. et al.Spread through air spaces (STAS) is prognostic in atypical carcinoid, large cell neuroendocrine carcinoma, and small cell carcinoma of the lung.J Thorac Oncol. 2019; 14: 1583-1593Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar(5)Linear strips of cells that were lifted off of alveolar walls are also interpreted as an artifact.4Kadota K. Nitadori J.I. Sima C.S. et al.Tumor spread through air spaces is an important pattern of invasion and impacts the frequency and location of recurrences after limited resection for small Stage I lung adenocarcinomas.J Thorac Oncol. 2015; 10: 806-814Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar,5Aly R.G. Rekhtman N. Li X. et al.Spread through air spaces (STAS) is prognostic in atypical carcinoid, large cell neuroendocrine carcinoma, and small cell carcinoma of the lung.J Thorac Oncol. 2019; 14: 1583-1593Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar In a specimen with a fixation delay, the epithelial lining may be detached.6Radonic T. Dickhoff C. Mino-Kenudson M. Lely R. Paul R. Thunnissen E. Gross handling of pulmonary resection specimen: maintaining the 3-dimensional orientation.J Thorac Dis. 2019; 11: S37-S44Crossref PubMed Scopus (20) Google Scholar This is a frequent finding in resection and autopsy specimen. Nevertheless, the detachment of the cells is not restricted to the benign cells, as this general effect will also affect tumor cells creating individual tumor cells. This may lead to an overinterpretation of STAS.(6)The elephant in the room is in the context of the erythrocyte, a benign cell that is present in practically all publications that contain microscopic examples of STAS, in the neighborhood of the STAS-interpreted cells (Fig. 2). Most pathologists will see these erythrocytes and immediately skip these for diagnostic purpose and mentally regard this as an artifact. Recently, spreading of neuroendocrine epithelial cells and erythrocytes was regarded as an artifact.7Pelosi G. Nesa F. Taietti D. et al.Spread of hyperplastic pulmonary neuroendocrine cells into air spaces (S.H.I.P.M.E.N.T.S): a proof for artifact.Lung Cancer. 2019; 137: 43-47Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar The question is here: Why are the erythrocytes interpreted as an artifact and the adjacent loose tumor cells as STAS? In short, these considerations around the definition of STAS is actually on the basis of topology of at least two "loose tumor cells," which are on itself on morphologic or cytologic grounds indistinguishable from what the leading authors call "artifacts." In addition, inconsistency compared to interpretation of the loose erythrocytes from the adjacent tumor cells is arbitrary. To be useful for pathology diagnosis in daily practice, the criteria for that diagnosis have to be clear allowing a pathologist to be consistent in time (intraobserver reproducibility) and lead to the same diagnosis between different pathologists (interobserver variability). Studies on interobserver reproducibility can be divided in local and global approach: studies reporting on reproducibility between a few pathologists within an institute or within a region and studies involving pathologists from around the globe. The latter will be most informative. A possible application of STAS could be the use in frozen sections. The sensitivity for STAS in four studies ranged from 55% to 86%.8Villalba J.A. Shih A.R. Sayo T.M.S. et al.Accuracy and reproducibility of intraoperative assessment on tumor spread through air spaces in stage 1 lung adenocarcinomas.J Thorac Oncol. 2021; 16: 619-629Abstract Full Text Full Text PDF PubMed Google Scholar, 9Zhou F. Villalba J.A. Sayo T.M.S. et al.Assessment of the feasibility of frozen sections for the detection of spread through air spaces (STAS) in pulmonary adenocarcinoma.Mod Pathol. 2022; 35: 210-217Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 10Walts A.E. Marchevsky A.M. Current evidence does not warrant frozen section evaluation for the presence of tumor spread through alveolar spaces.Arch Pathol Lab Med. 2018; 142: 59-63Crossref PubMed Scopus (48) Google Scholar One study mentions a worrying lack of consistency in sampling: STAS was only consistently present in (1) frozen section, (2) paraffin section of frozen section, and (3) other tumor sections in 39% of the cases.10Walts A.E. Marchevsky A.M. Current evidence does not warrant frozen section evaluation for the presence of tumor spread through alveolar spaces.Arch Pathol Lab Med. 2018; 142: 59-63Crossref PubMed Scopus (48) Google Scholar The reproducibility studies of STAS assessment in frozen sections revealed kappa scores of 0.51 and low 0.34, respectively.8Villalba J.A. Shih A.R. Sayo T.M.S. et al.Accuracy and reproducibility of intraoperative assessment on tumor spread through air spaces in stage 1 lung adenocarcinomas.J Thorac Oncol. 2021; 16: 619-629Abstract Full Text Full Text PDF PubMed Google Scholar,9Zhou F. Villalba J.A. Sayo T.M.S. et al.Assessment of the feasibility of frozen sections for the detection of spread through air spaces (STAS) in pulmonary adenocarcinoma.Mod Pathol. 2022; 35: 210-217Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar The concerns of STAS analysis in the frozen sections are clearly expressed in the summary of the article by Villalba et al.8Villalba J.A. Shih A.R. Sayo T.M.S. et al.Accuracy and reproducibility of intraoperative assessment on tumor spread through air spaces in stage 1 lung adenocarcinomas.J Thorac Oncol. 2021; 16: 619-629Abstract Full Text Full Text PDF PubMed Google Scholar which are regional pathologists with specific interest in pulmonary pathology: "As current accepted definitions for STAS and artifactual clusters are variably interpreted by pathologists, more precise criteria should be established and standardized, before the assessment of STAS can be implemented globally in the intraoperative setting to aid surgical decision making." Given the tendency of discohesion in malignant cells and the notion that the consistency of the cells is similar to a raw egg without a shell, other explanations for spreading of the tumor cells may be the surgeon's (1) hand, (2) instrument (Fig. 3A–C and Supplementary Fig. 1), (3) (eventual) localization procedure, (4) tearing tissue through a pinhole in video-assisted thoracic surgery, (5) iatrogenic collapse11Thunnissen E. Motoi N. Minami Y. et al.Elastin in pulmonary pathology: relevance in tumours with a lepidic or papillary appearance. A comprehensive understanding from a morphological viewpoint.Histopathology. 2022; 80: 457-467Crossref PubMed Scopus (13) Google Scholar (Supplementary File Surgery X1), the pathologist's (6) hand (Fig. 4) and (7) knife12Thunnissen E. Blaauwgeers H.J. de Cuba EM.v. Yick C.Y. Flieder D.B. Ex vivo artifacts and histopathologic pitfalls in the lung.Arch Pathol Lab Med. 2016; 140: 212-220Crossref PubMed Scopus (76) Google Scholar,13Blaauwgeers H. Flieder D. Warth A. et al.A prospective study of loose tissue fragments in non–small cell lung cancer resection specimens: an alternative view to "spread through air spaces.".Am J Surg Pathol. 2017; 41: 1226-1230Crossref PubMed Scopus (0) Google Scholar (Fig. 1 and Supplementary File 2 Pathology).Figure 4Lobectomy specimen with elevated pleura above the tumor and different moments of gross cutting. Photos made in Italy received with the courtesy of M. Papotti, MD, and A. Pittaro, MD. Note that with the intention to cut in the vertical direction only, the left hand is holding (compressing) the tumor. Supplementary File 2 Pathology.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Wild-type epithelial cells are attached to each other with adhesion molecules. Detachment will lead to apoptosis. Adhesion molecules play a role in the apoptotic process. In pulmonary adenocarcinoma, down-regulation of E-cadherin is a frequent finding with increased risk for metastases, which is supported in model systems and leads to a reduction in cohesion, a characteristic of malignant cells, which is also recognized in other organs. For example, discohesion of the tumor cells in cytologic samples of breast cancer is used as a criterion for malignancy, not as a proof for invasion, as ductal carcinoma in situ may have the same dissociation. Remarkably, in the lung, the discohesion of the loose tumor cells is, if interpreted as STAS, in the WHO 2021 classification arbitrarily interpreted as invasive.3Borczuk A.C. Cooper W.A. Dacic S. et al.WHO Classification of Tumours Editorial BoardThoracic tumours.in: WHO Classification of Tumours. 5th ed. IARC, Lyon, France2021: 19-189Google Scholar The discohesion is a clear morphologic characteristic of the micropapillary pattern. Peculiar in this context is that in the study of Yagi et al.14Yagi Y. Aly R.G. Tabata K. et al.Three-dimensional histologic, immunohistochemical, and multiplex immunofluorescence analyses of dynamic vessel co-option of spread through air spaces in lung adenocarcinoma.J Thorac Oncol. 2020; 15: 589-600Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar E-cadherin remained present, a feature fitting more with radial noninvasive growth as found in lepidic-featuring adenocarcinoma of either precursor or outgrowth type. Gross et al.15Gross D.J. Hsieh M.S. Li Y. et al.Spread through air spaces (STAS) in non−small cell lung carcinoma: evidence supportive of an in vivo phenomenon.Am J Surg Pathol. 2021; 45: 1509-1515Crossref PubMed Scopus (0) Google Scholar evaluated additional resection specimen (lobectomy or local resection) from patients who underwent a previous local resection and had STAS. In the second specimen, samples were taken close to the staple margin and found to have occult STAS tumor cells. They report this finding as evidence supportive of an in vivo phenomenon. Nevertheless, a plausible alternative explanation is that this finding is a consequence of clamping edema in a series of patients with a selection bias for lymphangioinvasion (Fig. 3C). The detailed arguments are provided in Supplementary File 2 Pathology. Since STAS was included in the WHO classification of 2015, close to 200 manuscripts have been published on this subject. Not surprisingly, STAS is significantly associated with many of the known adverse prognostic factors, which are in detail provided in Supplementary File 2 Pathology. In summary, there is no incontrovertible evidence that STAS, as currently presented, is indeed a biological phenomenon and not rather an inducible artifact with inconsistency in sampling. On top of this, the reproducibility of its presence is at best moderate. Inclusion of STAS in the WHO classification as an established and widely agreed-on phenomenon is premature.2Blaauwgeers H. Russell P.A. Jones K.D. Radonic T. Thunnissen E. Pulmonary loose tumor tissue fragments and spread through air spaces (STAS): invasive pattern or artifact? A critical review.Lung Cancer. 2018; 123: 107-111Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar STAS, including the underlying biology of dissociation, needs further investigation. Hans Blaauwgeers: Writing—original draft preparation. C. Dickhoff, G. Pelosi, Wim Timens, Federica Filipello, Yuko Minami: Writing—review and editing. Erik Thunnissen: Conceptualization, Writing original draft—review and editing, Supervision. Download .docx (.19 MB) Help with docx files Supplemental File 1 Download .docx (.74 MB) Help with docx files Supplemental File 2