MS4A1 dysregulation in asbestos-related lung squamous cell carcinoma is due to CD20 stromal lymphocyte expression

Our plain-English summary of MS4A1 dysregulation in asbestos-related lung squamous cell carcinoma is due to CD20 stromal lymphocyte expression, by C.M. Wright, S.M. Savarimuthu Francis, M.E. Tan, et al (PloS One, 2012).

The study

• Authors: C.M. Wright, S.M. Savarimuthu Francis, M.E. Tan, M.U. Martins, C. Winterford, M.R. Davidson, E.E. Duhig, B.E. Clarke, N.K. Hayward, I.A. Yang, R.V. Bowman, K.M. Fong
• Journal: PloS One
• Published: 2012;7(4):e34943
• Cited by: 23 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Asbestos-related lung cancer accounts for 4-12% of lung cancers worldwide. We have previously identified ADAM28 as a putative oncogene involved in asbestos-related lung adenocarcinoma (ARLC-AC). We hypothesised that similarly gene expression profiling of asbestos-related lung squamous cell carcinomas (ARLC-SCC) may identify candidate oncogenes for ARLC-SCC. We undertook a microarray gene expression study in 56 subjects; 26 ARLC-SCC (defined as lung asbestos body (AB) counts >20AB/gram wet weight (gww) and 30 non-asbestos related lung squamous cell carcinoma (NARLC-SCC; no detectable lung asbestos bodies; 0AB/gww). Microarray and bioinformatics analysis identified six candidate genes differentially expressed between ARLC-SCC and NARLC-SCC based on statistical significance (p<0.001) and fold change (FC) of >2-fold. Two genes MS4A1 and CARD18, were technically replicated by qRT-PCR and showed consistent directional changes. As we also found MS4A1 to be overexpressed in ARLC-ACs, we selected this gene for biological validation in independent test sets (one internal, and one external dataset (2 primary tumor sets)). MS4A1 RNA expression dysregulation was validated in the external dataset but not in our internal dataset, likely due to the small sample size in the test set as immunohistochemical (IHC) staining for MS4A1 (CD20) showed that protein expression localized predominantly to stromal lymphocytes rather than tumor cells in ARLC-SCC. We conclude that differential expression of MS4A1 in this comparative gene expression study of ARLC-SCC versus NARLC-SCC is a stromal signal of uncertain significance, and an example of the rationale for tumor cell enrichment in preparation for gene expression studies where the aim is to identify markers of particular tumor phenotypes. Finally, our study failed to identify any strong gene candidates whose expression serves as a marker of asbestos etiology. Future research is required to determine the role of stromal lymphocyte MS4A1 dysregulation in pulmonary SCCs caused by asbestos.

In plain English

Researchers in Queensland, Australia compared gene activity in surgically removed lung squamous cell carcinomas from 26 patients classed as asbestos-related (at least 20 asbestos bodies per gram wet weight of non-tumor lung) and 30 patients with no asbestos bodies found. The two groups did not differ significantly in age, sex, smoking or tumor stage. Microarray analysis flagged six genes. A second laboratory method confirmed only two, CARD18 and MS4A1, both higher in the asbestos-related tumors by a relatively small amount. MS4A1 was also slightly higher in the authors' earlier asbestos-related lung adenocarcinoma data (fold change 1.2) and in an outside dataset (fold changes 1.59 and 1.38). Staining of 52 tumors found the MS4A1 protein, CD20, in no tumor cells. It came mainly from immune cells (lymphocytes) in the surrounding tissue. No gene-based classifier correctly sorted more than 57% of tumors. The authors concluded that the MS4A1 difference was a surrounding-tissue signal of uncertain significance. They named not separating tumor cells from surrounding tissue as a limitation.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory gene expression (microarray) study comparing lung tumor tissue from patients with and without lung asbestos bodies, with checks by qRT-PCR, independent test sets and immunohistochemistry
• Who was studied: Training set of 56 surgically resected primary lung squamous cell carcinomas from The Prince Charles Hospital, Queensland, Australia: 26 classed as asbestos-related and 30 as non-asbestos related. There was also an internal test set of 12 cases (3 asbestos-related, 9 non-asbestos related) and an external dataset of 28 lung cancers of various histologies from Wikman and colleagues. Dates of collection are not given.
• Exposure: Asbestos exposure was defined by lung asbestos body count in non-tumor lung tissue, measured by bleach digestion. Cases were classed as asbestos exposed at 20 or more asbestos bodies per gram wet weight and as non-exposed when none were found. Fiber types are not specified.
• Main results:
  • The asbestos-related and non-asbestos related groups showed no statistically significant differences in age, sex, smoking history or tumor stage, in either the training or the test set.
  • In the 56 training tumors, 484 of 8,239 probes differed between the asbestos-related (n = 26) and non-asbestos related (n = 30) groups at p less than 0.05. The authors expected 412 to differ by chance.
  • Six genes met the thresholds of p less than 0.001 and more than 2-fold difference on microarray. Only two, CARD18 and MS4A1, were confirmed by qRT-PCR. Both were higher in asbestos-related tumors, by a relatively small amount.
  • In the internal test set of 12 squamous cell carcinomas, MS4A1 expression was 1.7-fold higher in tumor tissue than in the same patients' normal lung. CARD18 was 73-fold higher in tumor than in normal lung.
  • In the authors' earlier lung adenocarcinoma data, MS4A1 (but not CARD18) was higher in asbestos-related than in non-asbestos related adenocarcinoma, with fold change 1.2 and P = 0.021.
  • In the external Wikman dataset, both MS4A1 probes were higher in asbestos-related tumors (fold change 1.59 for probe 1 and 1.38 for probe 2).
  • In 52 tumors stained for CD20 (the MS4A1 protein), no staining was seen in tumor cells. Staining in most cases came from stromal lymphocytes.
  • CD20-positive lymphocytes inside tumor cell nests were seen in seven tumors: six asbestos-related and one non-asbestos related.
  • None of six prediction models correctly classified more than 57% of the squamous cell carcinomas.
  • The best model (K-nearest neighbor, k = 1) misclassified 15 of 26 asbestos-related tumors. It had sensitivity 42.3%, specificity 70%, positive predictive value 55% and negative predictive value 58.3% (permutation p = 0.296).
  • The authors reported that the gene expression profiles could not separate tumors by lung asbestos body count with enough sensitivity or specificity to be useful as a diagnostic test.
• The authors' conclusion: The authors concluded that the higher MS4A1 expression in asbestos-related versus non-asbestos related lung squamous cell carcinoma is a signal from the surrounding (stromal) tissue of uncertain significance, and that the study found no strong gene marker of asbestos cause.
• Limitations the authors state:
  • Tumor samples were not microdissected or enriched for tumor cells, so signals from surrounding tissue could dominate. The authors said they would use microdissected samples if redesigning the study.
  • The probes in the original 484-probe profile had very high false discovery rates.
  • The internal test set was small. The authors attributed the large CARD18 tumor-versus-normal difference to this small size.

Topics

Medical terms in this study, explained in our Lexicon: Squamous Cell Carcinoma, Lung Cancer, Lung Adenocarcinoma, Asbestos Body Formation, Asbestos Bodies, Statistical Significance, Etiology.

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