MiR-205 and MiR-375 microRNA assays to distinguish squamous cell carcinoma from adenocarcinoma in lung cancer biopsies

Abstract

Introduction: Identification of adenocarcinoma (AC) and squamous cell carcinoma (SCC) histology of non-small-cell lung cancer (NSCLC) in biopsies is clinically important but can be inaccurate by routine histopathologic examination. We quantify this inaccuracy at a cancer center, and evaluate the utility of a microRNA-based method to histotype AC/SCC in biopsies.

Methods: RNA was extracted from tissue sections with greater than 90% tumor content that were macro- or micro-dissected from formalin-fixed, paraffin-embedded biopsy specimens. MicroRNAs in RNA from the biopsies and from resected tumors were quantified by TaqMan reverse transcription-polymerase chain reaction assays and normalized against the RNU6B housekeeping RNA. Publicly available microRNA expression datasets were examined.

Results: NSCLC subtyping of small biopsy specimens by routine histopathologic examination either failed or mistyped the histology of 21% of 190 cases. Using 77 resectates, an reverse transcription-polymerase chain reaction-based assay of microRNAs miR-21, miR-205, and miR-375 was developed to identify AC and SCC subtypes of NSCLC. This method identified the AC/SCC histotypes of 25 biopsies with an accuracy of 96%, and correctly histotyped all 12 cases for which the histology had been mistyped by routine histopathologic examination of the biopsy. Examination of publicly available datasets identified miR-205 and miR-375 as microRNAs with the best ability to histotype AC and SCC, and that levels of the two microRNAs in AC or SCC are unaffected by the pathologic stage of the tumor or the age or race of the patient.

Conclusions: Histotypic microRNA assays can aid the subtyping of NSCLC biopsies as AC or SCC by standard histopathologic methods.

Figure 1. Histotypic microRNAs to distinguish lung squamous cell carcinoma (SCC) and adenocarcinoma (AC) identified in analyses of data from the Cancer Genome Atlas

Count per million microRNAs in primary AC (n = 334, black) and SCC (n = 300, gray) tumor or tumor-adjacent normal lung tissues (n = 79, white) for 10 of the 12 microRNAs identified in the analyses are depicted as mean and 95% confidence interval values.

Figure 2. Correlation between small RNA quantifications obtained by reverse transcription (RT)-PCR using two amounts of tissue RNA

Measurements obtained using 0.2 ng RNA of 14 resected lung cancers of U6B and miR-21, -205 and -375 as quantification cycle (C_q) values, and of miR-205 relative to U6B and miR-21 (Lebanony score), or only U6B (miR-205 ΔC_q^U6B) are plotted against those obtained with 5 ng RNA. The Pearson correlation coefficient (r) with 95% confidence interval, and the linear regression line generated with the least squares fitting technique are also shown.

Figure 3. Expression of miR-205 in lung squamous cell carcinoma (SCC) and adenocarcinoma (AC) resectates

Reverse transcription (RT)-PCR was used to quantify miR-205 relative to U6B and miR-21 (Lebanony score), or only U6B (miR-205 ΔC_q^U6B) in 5 ng RNA for 49 AC and 28 SCC cases. For each type of measurement, panel A shows dot-plots of individual values along with histology-specific group means and standard deviations; panel B shows receiver operating characteristic curves, along the area under curve (AUC) with 95% confidence interval, for the miR-205 measurements to discriminate SCC from AC; and, panel C shows the measurement values in terms of molarity along with linear regression lines and their slopes (m) and Y intercepts (b) determined by the least squares fitting technique. Molarity equivalent of the Lebanony score could be determined for only 56 of the total 77 cases.

Figure 4. Expression of miR-205 and miR-375 in lung squamous cell carcinoma (SCC) and adenocarcinoma (AC) resectates

Reverse transcription (RT)-PCR was used to quantify miR-205 relative to U6B and miR-21 (Lebanony score), and of miR-375 relative to U6B (miR-375 ΔC_q^U6B) in 5 ng RNA for 49 AC (gray) and 28 SCC (black) cases. Dotted lines indicate the median and standard deviation (SD) values of miR-375 ΔC_q^U6B and Lebanony scores of the AC cases with scores >2.5.