Genetic Prognostic Factors
The scant effectiveness of all conventional antitumor therapies in MPM has supported efforts to increase our knowledge of unidentified mechanisms involved in the genesis and development of the neoplasm. Tumor growth is controlled by several pathways, which are regulated by the activity of both intrinsic and extrinsic factors. Accordingly, a number of chromosomal, DNA, RNA, and gene abnormalities have recently been identified.[1,34] Their role as predictors of prognosis is currently under investigation and should be validated in larger and controlled studies.
Many chromosomal abnormalities found in MPM are correlated with patient survival.[1,34] Poor prognosis was found to be correlated with chromosome copy number deletions and alterations on the short arm of chromosome 7. Homozygous CDKN2A deletion, detected by fluorescent in situ hybridization (FISH) analysis, is another significant independent adverse prognostic factor. This locus encodes both p16INK4a and p14ARF and may be altered in MPM.[35,36] An association has been hypothesized between 9p21.3 deletion encompassing the CDKN2A locus and short-term recurrence.
DNA methylation implies a downregulation of individual genes and is associated with a poor prognosis. Patients with a low frequency of DNA methylation had significantly longer survival. Classification based on the methylation profile of patients identified subgroups characterized by different clinical outcomes.
MicroRNA expression has been shown to exhibit different and specific patterns in different histologic subtypes. Downregulation of microRNA-17 and microRNA-30c in sarcomatoid MPM and upregulation of microRNA-29c in epithelioid-type MPM are significantly associated with better survival. Increased expression of microRNA downregulates DNA methyltransferase and increases the demethylating genes, resulting in a significant decrease in proliferation, migration, invasion, and colony formation.
Gene mutations can interact with specific pathways that can be altered in MPM cells, paving the way for future targeted therapies. The TP53 gene, a tumor suppressor gene located at 17p13.1 that controls cell cycle and apoptosis, is mutated in many human cancers. TP53 alterations are considered a negative prognostic factor.
Homozygous deletions of the locus INK4a/ARF located on chromosome 9p21 may result in a deficiency of p14ARF, an inhibitor of murine double minute 2 (MDM2); MDM2 inactivates the p53 pathway, thus limiting its tumor-suppressing effect. The same locus encodes the p16INK4a protein, which inhibits the retinoblastoma protein (pRb), thereby inducing cell-cycle arrest in G1 phase. These mutations impede the functioning of the tumor suppressor pathway and are associated with poor survival.
Another frequent gene mutation is that of the neurofibromatosis 2 (NF2) gene. Mutations of this gene could be related to shorter survival.
Four-gene signatures, which include KIAA097, guanosine diphosphate–dissociation inhibitor 1, cytosolic thyroid hormone-binding protein, and an expressed sequence tag similar to the tumor antigen L6, correlated with good and poor prognostic groups. In addition, an 11-gene, oncogene-driven pathway signature is associated with a poor prognosis.
Gene expression profiling
Data from array-based studies indicate deregulation of gene expression in MPM. Gene expression analyses (transcriptome and/or quantitative reverse transcriptase-polymerase chain reaction) lead to an improvement of prognostic power. Three ratios of gene expression (TM4SF1/PKM2, TM4SF1/ARHDDIA, COBLL1/ARHDDIA) have proved capable of discriminating between high- and low-risk patients.
Aurora kinases are serine/threonine kinases that play a crucial role in cell division by controlling the segregation of chromatids. Defects in the separation process can cause genetic instability, a condition that is highly associated with tumorigenesis and poor prognosis.
Maternal embryonic leucine zipper kinase (MELK); BIRC5, an inhibitor of apoptosis; KIF4A, an adenosine(Drug information on adenosine) triphosphate–dependent microtubule-based motor protein; and SEPT9, a member of the septin family involved in cytokinesis and cell-cycle control, are upregulated in some patients with MPM, and all are associated with poor prognosis.
Molecular Pathway Factors
Tumor onset and growth is also related to a large number of deregulated molecular pathways. These involve various cell mechanisms, including growth factors, cell-cycle regulators, apoptosis, and angiogenesis. The major molecular pathways involved in MPM and their prognostic implications are summarized in Table 4.
The carcinogenic effects of asbestos fibers are the result both of direct action and of the creation of an induced inflammatory environment containing reactive oxygen and nitrogen species. These reactive species are able to affect the equilibrium between mitosis and apoptosis, moving it toward excessive cell proliferation and the onset of cancer. Genes overexpressed in this cascade are mitogen-activated protein kinase (MAPK), also known as extracellular signal–regulated kinase (ERK), transcription factor activator protein 1, nuclear transcription factor κ light chain–enhancer of activated B cells (NF-κB), and protein kinase C. Activations of the phosphatidylinositol 3 kinase plus protein kinase B (PI3K-AKT) and ERK pathways result in neoplastic cell survival and proliferation. The hyperactivation of the above-named genes may be associated with a poorer prognosis.
Cyclooxygenase-2 (COX-2) is implicated in many events in the tumorigenic process, producing highly reactive products that can affect cell growth, immune response, apoptosis, and angioneogenesis. High COX-2 expression is a marker of poor prognosis in MPM, and may be a target for selective COX-2 inhibitors (eg, celecoxib(Drug information on celecoxib) [Celebrex], rofecoxib(Drug information on rofecoxib) [Vioxx]).
Growth factor receptors
Many growth factors appear to be highly expressed in MPM, and these may have some prognostic significance.[1,34] Epidermal growth factor (EGF) and its receptor (EGFR), a membrane receptor tyrosine kinase (RTK), are highly expressed in MPM and are statistically associated with unfavorable prognosis. In vitro inhibition of EGFR by specific RTK inhibitors resulted in control of tumor growth and inhibition of angiogenesis.
High expression of platelet-derived growth factor (PDGF) and/or its receptor (PDGFR) is strongly associated with shorter survival. PDGFR is also an RTK that can be inactivated by selective inhibitors.
Vascular endothelial growth factor (VEGF) is a growth factor linked to angiogenesis and is overexpressed in MPM. High expressions of VEGF and of its receptor (VEFGR) are correlated with the density of microvessels and high tumor necrosis.
An inverse relationship between placental growth factor (PlGF) expression and survival has recently been demonstrated in operated MPM, suggesting a central role for this factor in the recurrence and progression of disease.
Hepatocyte growth factor (HGF) is a multifunctional factor that induces cell proliferation. The receptor for HGF is synthesized from c-MET, a proto-oncogene with an RTK located on chromosome 7q31. Overexpression of HGF and c-MET is associated with increased angiogenesis.
Many attempts at using RTK inhibitors as MPM therapy have failed. These unsatisfactory outcomes show that inhibition of a single factor is probably not sufficient to obtain a tumor suppressor effect; rather, the use of multiple inhibitors should be considered.
Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, play a role in tumor angiogenesis and invasion. Increases in the inactive proprotein form of MMP-2 and total MMP-2 have been associated with poor survival on multivariate analysis. High expression of MMP-14 has been associated with lower survival, and the MMP14 gene has been proposed as a potential MPM biomarker.
Wnt signaling pathway
The Wnt signaling pathway is a network of proteins that transfers signals from cell surface receptors to DNA using β-catenin protein (Figure 2), thereby stimulating developmental processes, cell proliferation, and cell polarity. This pathway may be altered in MPM because of promoter hypermethylation of regulatory genes. Its inhibition results in tumor reduction and apoptosis, whereas overexpression may portend a less favorable prognosis.
The Hippo pathway is a novel and promising cell growth inhibitor pathway; it involves signal transduction from membrane receptors (Fat) into the nucleus (Figure 2). Alterations in the Hippo pathway predispose to cell overgrowth. Merlin, the protein encoded by NF2, regulates cell growth by signaling via the Hippo pathway to inhibit the function of the transcriptional coactivator and candidate oncogene yes-associated protein 1 (YAP1), which also interferes with the Wnt pathway. NF2 normally acts as a tumor suppressor gene and as a gatekeeper in asbestos-related MPM. Disruption of NF2 signaling plays a major role in the development of MPM as demonstrated by the high rate of mutations seen in this tumor.
This pathway is related to many other pathways, including the NF-κB pathway, and to inhibitors of apoptosis. Complex proteasome subunits are upregulated in MPM, whereas other proteins, such as FAS-associated factor 1 (FAF1), are downregulated and tend to be associated with a poorer prognosis.
The cell cycle is organized into many checkpoints regulated by the combination of proteins called cyclins with cyclin D–dependent kinases (CDKs). These multimeric complexes are inhibited by CDK inhibitors such as p21 and p27 (Figure 2). The p53 tumor suppressor pathway is also involved in cell-cycle regulation, providing transcriptional control of various cell-cycle regulatory proteins, including p21, which is induced by activation of the p21 gene. p27 and p21 have been associated with a statistically significant decrease in survival.
As mentioned above, inactivation of genes located on the INK4/ARF locus and encoding the p14ARF and p16INK4a proteins leads to cell-cycle deregulation with uncontrolled proliferation. These events are associated with a poorer prognosis.
The monoclonal antibody MIB-1 recognizes a nuclear protein, antigen Ki67, that is expressed in proliferating cells; MIB-1 can be used to assess the growth fraction in normal and neoplastic tissues. A close correlation between high expression of MIB-1 and aggressive biological behavior of MPM has been demonstrated.
Apoptosis is a process characterized by programmed cell death; it is used to control cellular proliferation and destruction. In MPM, the expression of genes that regulates apoptosis is altered. Apoptosis is normally induced by extrinsic and intrinsic pathways (Figure 2). The extrinsic pathway is activated by ligands of the tumor necrosis factor family, the tumor-related apoptosis-inducing ligands (TRAIL). Most MPM cells are resistant to apoptosis induced by TRAIL; this may be due to overexpression of the caspase-8 inhibitor FLIP/CFLAR and by methylation of TRAIL receptors. Downregulation of TRAIL, as well as overexpression of NF-κB, which protects the cell from apoptosis, may have an impact on the prognosis.
The intrinsic pathway is triggered by internal apoptotic signals and involves release of cytochrome c from the mitochondrial intermembrane space. Mitochondrial membrane permeability is regulated by the B-cell lymphoma 2 (Bcl-2) family of proteins, which includes proapoptotic proteins (eg, Bax, Bak, Bad, Bid, Bim) and antiapoptotic proteins (eg, Bcl-2, Bcl-xL, and Mcl-1). Elevated expression of Bcl-xL was seen in all MPM cell lines, and downregulation of Bcl-xL improves sensitivity to chemotherapeutic agents, thereby influencing prognosis. Conversely, reduced levels of Bax protein, which has proapoptotic activity, have been associated with a poor outcome.
The phosphatase and tensin homolog (PTEN) enzyme is a tumor suppressor that functions as a negative regulator of the combined PI3K-AKT pathway and mammalian target of rapamycin (mTOR) pathway, which promote cell growth and impede apoptosis (Figure 2). Hence, PTEN inhibits cell division and directs cells to programmed death. High expression of PTEN has been correlated with better survival, and its inactivation may account for poorer prognosis.
Another apoptotic protein is glucose transporter-1 (GLUT1), which regulates glycolysis. Its overexpression, measured with immunohistochemistry in MPM samples, has proved to be correlated with lower survival.
The length of human telomeres is believed to be directly proportional to the number of possible cell divisions and is therefore correlated with cell life expectancy. Telomere lengthening is promoted by the enzyme telomerase, which can prolong the cell lifespan. Deregulated telomerase activity could be at the basis of unlimited growth of the tumor cell. Telomerase activity was found overexpressed in MPM, and a significant correlation with tumor relapse and short disease-free survival has been documented.
Aquaporin 1 (AQP1)
AQP1 is a protein in the cell membrane that is involved in the transport of water, in cell motility, and in proliferation. Its expression in ≥ 50% of tumor cells proved an independent prognostic factor in MPM regardless of treatment. Labeling immunohistochemistry for AQP1 should be included in the routine work-up of patients with MPM. An agonist or blocker of AQP1 may be found to be useful for treatment.
Calretinin is a vitamin D–dependent calcium-binding protein involved in calcium signaling. This protein plays a role in message targeting and intracellular calcium buffering. Low expression of calretinin was independently associated with a poor prognosis in MPM patients undergoing extrapleural pneumonectomy.
A body of literature has developed covering the numerous investigations that have been conducted on MPM. Despite relevant advances in many areas, including improvements in diagnosis, staging, and the clinical course of treated patients, MPM remains a rare but highly lethal disease characterized by a markedly aggressive local evolution with progressive loss of pulmonary function. Thus, MPM is a continuing challenge for thoracic surgeons and for medical and radiation oncologists. More recently, geneticists and biologists interested in better understanding the behavior of the disease have joined the ranks of those attempting to address the challenges of MPM.
The therapeutic role of surgery is still vigorously debated, despite a remarkable decrease in operative morbidity and mortality. The decision to operate is dependent on many factors, which necessarily include specific features and effects of the disease, along with the experience of the surgeon and the results of the preoperative and intraoperative evaluations. Now, in selected patients, surgery combined with chemotherapy and radiation therapy seems to provide the greatest benefit in terms of survival and quality of life.
Attention should also be paid to the study of prognostic factors, novel biomarkers, and genetic abnormalities. These all might be of help in formulating an early diagnosis as well as in selecting a more accurately targeted treatment. Until the suggested novel gene and immunologic therapies have demonstrated their effectiveness, the best approach that can be offered to patients remains as extensive a surgical cytoreduction as possible, followed by adjuvant chemo- and radiotherapy. Still, an adequate knowledge and evaluation of prognostic factors can help in defining the multidisciplinary approach to therapy in order to reduce the mortality from this lethal disease.
Finally, it is important that the rarity of this neoplasm not be an obstacle to research into the optimal general approach to treatment and management.
Financial Disclosure: The authors have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.