Genetic Alterations of Malignant Pleural Mesothelima

*Benjamin Wadowski, David T. Severson, Raphael Bueno and Assunta De Rienzo*

#### **Abstract**

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that arises from the mesothelial cells lining the pleural cavity. Asbestos is considered the major factor in the pathogenesis of this malignancy, with more than 80% of patients with a history of asbestos exposure. MPM is characterized by a long latency period, typically 20–40 years from the time of asbestos exposure to diagnosis, suggesting that multiple somatic genetic alterations are required for the tumorigenic conversion of a mesothelial cell. In the last few years, advancements in next-generation sequencing and "–omics" technologies have revolutionized the field of genomics and medical diagnosis. The focus of this chapter is to summarize recent studies which explore the molecular mechanisms underlying this disease and identify potential therapeutic targets in MPM.

**Keywords:** pleural mesothelioma, next-generation sequencing, transcriptome, exome sequencing, tumor suppressor gene

#### **1. Introduction**

Malignant pleural mesothelioma (MPM) is a lethal cancer of the mesothelial cells lining the pleural cavity and, less frequently, the pericardium, peritoneum, and tunica vaginalis [1]. Many years after the peak of asbestos use in United States, 3200 cases of MPM continue to be diagnosed annually, indicating that the U.S. population remains at risk of exposure to asbestos and development of mesothelioma [2]. There are two major histological variants: epithelioid, which accounts for about 60% of cases and has the more favorable prognosis, and sarcomatoid, whose incidence is 10%. The remaining cases demonstrate histologic characteristics of both types and are classified as biphasic [3]. The prognosis for patients with MPM is poor, with a median survival of 5–15 months [3]. However, some patients with early MPM who undergo multimodality therapy including surgical resection and chemotherapy demonstrate longer-term survival of up to 25% at 5 years [4].

Many studies have shown a causal relationship between exposure to asbestos and mesothelioma (reviewed by Bianche et al. [5]). Although it has been suggested that brief asbestos exposure is sufficient to induce disease, MPM is the consequence of prolonged exposure in most cases. However, only a small percentage of individuals exposed to asbestos develop MPM, suggesting that genetic predisposition may modulate the effect of exposure to asbestos. In addition, 20% of MPM cases with unknown asbestos exposure have been related to other risk factors such as radiation therapy and thorotrast [6].

Studies conducted on large numbers of patients indicate that the time between asbestos exposure and diagnosis of MPM is generally more than 20 years. The molecular mechanisms for the transformation of mesothelial cells are unknown; it has been suggested that asbestos induces multiple chromosomal aberrations, particularly deletions, facilitating oncogenesis [7].

Investigations prior to the advent of next-generation sequencing (NGS) revealed the complexity of the genetic alterations observed in MPM tumors by using karyotypic and comparative genomic hybridization (CGH) analyses [8, 9]. Chromosomal losses were found to be more frequent than gains and particular chromosomal regions (1p22, 3p21, 4q, 6q, 9p21, 13q13–14, 15q11–15, and 22q12) were deleted at higher frequency in MPM tissues and cell lines [10–12]. Two tumor suppressor genes (TSGs) were identified by positional cloning approaches: *CDKN2A* at 9p21 and *NF2* at 22q12. In the last few years, the genetic landscape of MPM has been characterized using high-throughput technologies [13–15]. The focus of this chapter is to summarize the major genetic changes occurring in MPM as identified by high-throughput sequencing and to describe the novel insights obtained through transcriptomic studies.

#### **2. Exome sequencing studies**

NGS technologies have allowed the sequencing of DNA and RNA at unprecedented speed, uncovering potential driver genes and creating novel biological applications [16]. In the last decade, NGS has been used to detect driver genetic mutations in cancer and provide new insights into tumorigenesis.

Shotgun pyrosequencing was used to characterize RNA expression levels and mutations of four patients in the first effort to investigate MPM by NGS. Several different mutations were found in the four transcriptomes. In addition, RNA editing gene deletions and gene silencing were identified [17].

In 2010, the first whole genome sequence of one MPM tumor and matching normal tissue was conducted using a combination of sequencing-by-synthesis and pyrosequencing methodologies [18]. This study showed that aneuploidy and chromosomal rearrangements were more numerous than point mutations in this tumor. One large deletion in the dipeptidyl peptidase like 10 (*DPP10*) gene, altering the expression of the corresponding transcript, was further investigated in 53 additional MPM tumors. Patients expressing *DPP10* had statistically longer survival compared to patients lacking *DPP10* expression [18].

In 2016, Bueno et al. conducted an extensive analysis of the mutational landscape of MPM. Ninety-nine MPM tumors were examined by whole exome sequencing, whereas additional 103 samples were characterized by targeted exome sequencing [13]. *BAP1*, *NF2*, *TP53*, *SETD2*, *DDX3X*, *ULK2*, *RYR2*, *CFAP45*, *SETDB1* and *DDX51* were found to be significantly mutated (q-score ≥ 0.8), and recurrent mutations were found in *SF3B1* (2%) and *TRAF7* (2%).

In 2018, The Cancer Genome Atlas (TCGA) program performed a comprehensive molecular profiling of 74 primary MPM samples including exome sequencing, copy-number arrays, mRNA sequencing, noncoding RNA profiling, DNA methylation, and reverse-phase protein arrays [15]. The significantly mutated genes in this study were *BAP1*, *NF2*, *TP53*, *LATS2*, and *SETD2*. Furthermore, this study identified a new near-haploid molecular MPM subtype.

The TCGA study performed a comparison of the significantly mutated genes between the Bueno and TCGA cohorts [15]. This analysis identified five genes that were frequently mutated in both studies: BRCA1-associated protein-1 (*BAP1*), neurofibromin 2 (*NF2*), tumor protein P53 (*TP53*), SET domain containing 2, histone

**93**

**Table 1.**

*Number of mutations in each gene in the two studies.*

*Genetic Alterations of Malignant Pleural Mesothelima DOI: http://dx.doi.org/10.5772/intechopen.93756*

**2.1 BAP1**

**Gene symbol**

lysine methyltransferase (*SETD2)*, and SET domain bifurcated histone lysine methyltransferase 1 (*SETDB1*). The large tumor suppressor kinase 2 (*LATS2*) gene was found frequently altered in the TCGA cohort alone, whereas four additional genes, DEAD-box helicase 3 X-linked (*DDX3X*), Unc-51-like autophagy-activating kinase 2 (*ULK2*), ryanodine receptor 2 (*RYR2*), and DEAD-box helicase 51 (*DDX51*) were identified as commonly mutated in the series from Bueno et al. (**Table 1**).

*BAP1* is located on the short (p) arm of chromosome 3, at position 21.1., a region frequently deleted in MPM [9]. This gene encodes for a deubiquitinase involved in cell cycle regulation, modulation of gene transcription, cellular differentiation, and DNA repair [19]. *BAP1* is one of the most commonly mutated genes in MPM [13, 15, 20, 21]. Germline *BAP1* mutations have been linked to the development of *BAP1* tumor predisposition syndrome, which includes uveal and cutaneous melanoma, atypical Spitz tumors, renal cell carcinoma, and MPM. In all these malignancies but MPM, *BAP1* mutations are associated with poor prognosis [22, 23]. In contrast, some studies have shown that patients with MPM carrying *BAP1* mutations have longer overall survival compared to patients with wild-type *BAP1* [24, 25]. In one study, BAP1 immunohistochemistry (IHC) was performed using tissue microarray including 229 MPM tumors. The results showed that loss of BAP1 nuclear staining was associated with longer median survival of 16.11 months (95% CI: 12.16–20.06) versus 6.34 months for patients with nuclear BAP1 staining (95% CI: 5.34–7.34) (P < 0.01) [24]. Baumann et al. compared the survival in 23 patients with MPM carrying germline mutations in *BAP1* with a control group of MPM patients from the Surveillance, Epidemiology, and End Results (SEER) database and found a 7-fold

increase in long-term survival in patients with BAP1 mutation [25].

**Gene ID Chromosomal** 

monly used as a diagnostic marker in MPM [26, 27].

Given its prevalence in MPM, loss of nuclear BAP1 expression by IHC is com-

Recently, *BAP1* status has been associated with drug response [28, 29]. I*n vitro* studies showed MPM cell lines carrying *BAP1* mutations were significantly less sensitive to gemcitabine compared to wild-type cells. Silencing of *BAP1* in MPM

**location**

*BAP1* ENSG00000163930 3p21.1 55 17 72 *NF2* ENSG00000186575 22q12.2 39 19 58 *TP53* ENSG00000141510 17p31.1 17 10 27 *SETD2* ENSG00000181555 3p21.31 18 8 26 *SETDB1* ENSG00000143379 1q21 7 3 10 *LATS2* ENSG00000150457 13q12.11 2 9 11 *DDX3X* ENSG00000215301 Xp11.4 8 0 8 *RYR2* ENSG00000198626 1q43 4 1 5 *ULK2* ENSG00000083290 17p11.2 4 0 4 *DDX51* ENSG00000185163 12q24.33 3 0 3 Total 157 67 224

**Number of mutations in Bueno's cohort**

**Number of mutations in Hmeljak's cohort**

**Total**

lysine methyltransferase (*SETD2)*, and SET domain bifurcated histone lysine methyltransferase 1 (*SETDB1*). The large tumor suppressor kinase 2 (*LATS2*) gene was found frequently altered in the TCGA cohort alone, whereas four additional genes, DEAD-box helicase 3 X-linked (*DDX3X*), Unc-51-like autophagy-activating kinase 2 (*ULK2*), ryanodine receptor 2 (*RYR2*), and DEAD-box helicase 51 (*DDX51*) were identified as commonly mutated in the series from Bueno et al. (**Table 1**).
