Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

Abstract

Malignant pleural mesothelioma (MPM), a rare malignancy with a poor prognosis, is mainly caused by exposure to asbestos or other organic fibers, but the underlying genetic mechanism is not fully understood. Genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. We found that the MPM had R282W, a key TP53 mutation, and genome-wide allelic loss or loss of heterozygosity, a distinct genomic alteration not previously described in MPM. We identified frequent inactivating SETDB1 mutations in this patient and in 68 additional MPM patients (mutation frequency: 10%, 7/69) by targeted deep sequencing. Our observations suggest the possibility of a new genetic mechanism in the development of either MPM or multiple primary cancers. The frequent SETDB1 inactivating mutations suggest there could be new diagnostic or therapeutic options for MPM.

Keywords: SETDB1; exome sequencing; genome-wide allelic loss; malignant pleural mesothelioma; multiple primary cancer.

Figure 1. Clinical diagnosis of lung adenocarcinoma and MPM

A. A 2 cm ground-glass opacity (arrow), later diagnosed as lung adenocarcinoma, was identified in the patient's left lower lobe on chest CT scan. No pleural thickening or implants were noted on preoperative imaging. B. Immunohistochemical staining for calretinin confirmed the diagnosis of MPM (original magnification 20X).

Figure 2. TP53 mutation and allele frequency of all variants

A. Sanger sequencing confirmed TP53 R282W mutation identified in exome and targeted deep sequencing of MPM. B. Allele frequency of variants identified in MPM with genome-wide allelic loss. Frequency of mutant alleles was higher (> 0.8) in most variants except for two located on chromosome 7.

Figure 3. Genome-wide allelic loss identified in a MPM patient with multiple primary cancers

A. Scatter plots show genome-wide allelic view of all variants including known SNPs identified in the patient's MPM. Allelic fraction represents ratio of wild-type to variant counts in an exome sequencing. B-allele fraction plot in the upper panel represents allelic ratio between wild-type and variant in the tumor sample. In the lower panel, the values (log2 ratio) of the x-axis are an allelic fraction of tumor normalized to that of matched normal pleura. The majority of variants on all chromosomes except chromosomes 7 and 20 show losses of either wild-type or variant allele in tumor. B. An example of genome-wide allelic view of tumor with frequent genetic alterations, but only with focal allelic loss. B-allele fraction plot of tumor sample in the upper panel and allelic fraction plot of tumor normalized to that of matched normal in the lower panel were newly generated from exome sequencing data previously reported [32].

Figure 4. Frequent SETDB1 mutations in MPM patients

Two types of deletion mutations from MPM patients, shown by an Integrative Genomics Viewer (IGV). A. 17 bp deletion (677_693del17) and B. In-frame deletion (3747_3749del) of SETDB1 were identified from different MPM patients.

Figure 5. Map of SETDB1 mutations identified in MPMs

Nine SETDB1 somatic mutations identified in this study and reported previously were mapped in domains of SETDB1. Four of five truncating mutations were located at the N-terminal (5′) regions before Tudor (Tud) domains. All three missense and one deletion mutation were found in the SET domain. Tud: Tudor; MBD: methyl-CpG-binding domain; pS: pre-SET; PS: post-SET [–31].