Sequential Binary Gene Ratio Tests Define a Novel Molecular Diagnostic Strategy for Malignant Pleural Mesothelioma

Abstract

Purpose To develop a standardized approach for molecular diagnostics, we used the gene-expression ratio bioinformatic technique to design a molecular signature to diagnose MPM from among other potentially confounding diagnoses and differentiate the epithelioid from the sarcomatoid histological subtype of MPM. In addition, we searched for pathways relevant in MPM in comparison to other related cancers to identify unique molecular features in MPM. Experimental Design We performed microarray analysis on 113 specimens including MPMs and a spectrum of tumors and benign tissues comprising the differential diagnosis of MPM. We generated a sequential combination of binary gene-expression ratio tests able to discriminate MPM from other thoracic malignancies. We compared this method to other bioinformatic tools and validated this signature in an independent set of 170 samples. Functional enrichment analysis was performed to identify differentially expressed probes. Results A sequential combination of gene-expression ratio tests was the best molecular approach to distinguish MPM from all the other samples. Bioinformatic and molecular validations showed that the sequential gene ratio tests were able to identify the MPM samples with high sensitivity and specificity. In addition, the gene-ratio technique was able to differentiate the epithelioid from the sarcomatoid type of MPM. Novel genes and pathways specifically activated in MPM were identified. Conclusions New clinically relevant molecular tests have been generated using a small number of genes to accurately distinguish MPMs from other thoracic samples supporting our hypothesis that the gene-expression ratio approach could be a useful tool in the differential diagnosis of cancers.

BAP1, a tumor suppressor gene driving malignant mesothelioma

“…Also comparable, was the association of somatically inactivated BAP1 with the epithelioid MM subtype (36). Since BAP1 can influence the regulation of gene expression epigenetically or through deubiquitination of transcription factors (see below), it is plausible that these different patterns of gene expression can lead to variations in MM tumor subtypes (35,40,41).…”

Molecular markers and new diagnostic methods to differentiate malignant from benign mesothelial pleural proliferations: a literature review

“…In this context, we can mainly categorize the identification of reliable diagnostic biomarkers as the following: historical MPM biomarkers, including immunohistochemical ones (such as GLUT-1, p53, desmin, EMA, IMP-3) (5-13) and PE soluble ones analyzable by ELISA (such as mesothelin and fibulin-3) (41-47), which have been and are still extensively studied; emerging biomarkers recently introduced into clinical practice (BAP1 analyzable by IHC and p16 by FISH) (3); suggested MPM signatures based on miRNAs and mRNA expression panels (50-52); and new diagnostic tools based on molecular panels and classification algorithms (53,(55)(56)(57).…”

“…Application of a sequential method allowed the authors to overcome limits due to the binary nature of individual gene expression ratios. Moreover, the gene ratio signature showed a sensitivity and specificity comparable or even better than those achieved by the more complex algorithm k-nearest neighbor (KNN) and linear discriminant analysis (53). One limitation of this study is that for microarray analysis, 0.75 µg of total RNA and 1 µg for RT-PCR were utilized; these amounts can be obtained from fresh frozen tissues, but they can be scarcely obtained from FFPE or cytological specimens.…”

“…The gene signature was identified by an Illumina whole genome microarray analysis on 113 fresh frozen tissues from 39 MPM patients (24 epithelioid, 7 biphasic and 8 sarcomatoid), 7 normal pleural samples and other common thoracic malignancies and then validated by RT-PCR in a validation cohort of 170 samples, including 100 MPM tissues (63 epithelioid, 27 biphasic and 10 sarcomatoid), 12 normal pleural samples and 58 other tumors. In detail, they developed four gene ratio-based tests: one to distinguish MPM from normal pleura (UBE2T, AGENCOURT_14535501, MAGED1, ADCY4, PAK4, and MYH11), a second to distinguish MPM from all sarcomas (MSLN, TGFBR3, ANXA8, TCEAL7, KRT8, and PCDH18), a third to distinguish MPM from renal cell carcinoma (NFKBIZ, ARHGAP2, ARL6IP6, HPN, and LOC648293), and a fourth to distinguish MPM from thymoma (KRT18, PRSS16, RGS16, and BCL11A) (53). In addition, the authors added to their analysis their previously validated diagnostic test for discerning MPM and lung adenocarcinoma (54).…”

Surgery for Malignant Pleural Mesothelioma

“…If VATS is chosen, a single port should be used in the line of a future thoracotomy incision to minimize seeding of the track with tumor cells. The role of fine needle aspiration for the diagnosis of MPM is evolving since the creation of a molecular test developed by our group at Brigham and Women's Hospital [15,16].…”