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A molecular biomarker is defined as a biological molecule in blood, other fluids, or tissues that is a sign of a normal or abnormal process, or a condition.1 The history of tumor markers has its beginnings on the demonstration of tumor-specific transplantation antigens, particularly, in chemically or virally induced cancers. Of particular relevance was the discovery of alpha-fetoprotein in 1956, currently used as marker for germ cell tumor and hepatocellular carcinoma. 2 A next landmark was the discovery of the carcinoembryonic antigen in 1965, 2 mostly utilized as a maker for colon carcinoma. Those advances encouraged the researchers to further develop approaches for biomarker discovery, thus the development and adaptation of methods and procedures for the screening and identification of biomarkers for different tumor types gained more attention. Accordingly, the field of tumor markers increased continuously and is strongly established for tumor detection, prognosis and pharmacodynamics. 3 Current molecular techniques allowed elucidation of the chemical structure and genetic origin of most established biomarkers. Although there is no general discovery and/or utilization protocol for the application of the established biomarkers in tumor pathology, their validity as pre and post operative indicators for tumor burden, therapeutic success, and postoperative recurrence has been confirmed. 3 The utilization of biomarkers in the screening of a high risk population as well as in diagnosis of cancer gained more attention in the recent decades. In this perspective, we focus on the reliability of currently utilized biomarkers in tumor pathology.

Based on their biological origin, biomarkers can be cells, molecules, genes, gene products, enzymes, etc. 1 Alterations of genes at their expression, structure or function are considered as detectable phenotypes that can be utilized as biomarkers for tumor prediction and therapy response. When those aberrations can be traced back to distinct stages of tumor development, markers will have use for detecting the existence, stage, progression, regression, resistance and recurrence. Biological products with value as tumor biomarkers include DNA, such as single nucleotide polymorphisms (SNPs), chromosomal aberrations, alteration in DNA copy number, microsatellite instability, and epigenetic alterations at the level of promoter-region methylation. Of particular interest, mutations in genes that encode for tumor-suppressor, oncogene proteins, or those encoding for proteins of the mismatch-repair machinery have been established as DNA bio-markers. For example, the detection of mutations in the oncogene Kirsten rat sarcoma viral oncogene homolog (KRAS) can be used as marker to predict metastatic spread in various tumor types. 4 A mutation in the gene that encodes the tumor suppressor p53 has been widely reported in most of sporadic colon cancers.5 Also, an inherited tumor protein p53 (TP53) mutation is associated with high risk of osteosarcoma development. 6 Moreover, mutations in cancer-related genes, such as those encoding for the tumor-suppressor protein cyclin-dependent kinase inhibitor (CDKN2A), RAS, the adenomatous polyposis coli (APC) and the retinoblastoma (RB1) have been recently recognized as a molecular biomarker for colorectal cancer prediction,7 in addition to their potential function as biomarkers for treatment prognosis or therapy selection.8 RNA-based biomarkers include over or down regulation of transcripts, as well as regulatory RNAs such as the microRNAs.9 RNA-based molecular biomarkers have been established for prediction and prognosis of different cancer types including melanoma, leukemia, lymphoma, lung, prostate and colon cancers. Also, transcript levels of enzymes, which are important for drug metabolism, have been used as predictive markers for tumor response to available therapies. 10 Although most of the DNA markers are evaluated individually, many technical procedures have been recently developed to assess the transcription levels of mRNA expression as molecular biomarker for tumor detection and prognosis. These techniques include gene expression profiling arrays, differential display, serial analysis of gene expression (SAGE), 11 and most recently next generation sequencing (NGS). Particularly, gene expression arrays allow for detection of global expression of genes and have allowed correlating gene expression profiles and biological tumor characteristics. 12 In comparison with traditional sequencing and other sequence analysis methods, NGS has dramatically increased sequencing rates and driven down sequencing costs.13 As result of the technological breakthroughs, scientists and clinicians have gained knowledge about cancer genomes (whole-genome sequencing, WGS), exomes (whole-exome sequencing, WES), RNA (coding and non-coding) transcriptomes (RNA sequencing, RNA-Seq), and the epigenome (DNA methylome and Small RNAs). 14 These approaches allow detection of somatic cancer genome alterations such as nucleotide substitutions, insertions, deletions, copy number variations, and chromosomal rearrangements. Along with increasing our understanding of the pathogenesis of cancers NGS provides biomarkers and novel targets for drug development and guided cancer therapy using existing drugs against actionable molecular targets. Protein-based markers include peptides released from tumors into serum, urine, sputum, or other body fluids, as well as phosphorylation status of some proteins, and carbohydrate determinants of some proteins.15 Examples include, serum-derived proteins such as alpha-fetoprotein (AFP), 16 β-human chorionic gonadotropin (β-HCG) 17 and lactate dehydrogenase (LDH), 18 which are approved by the American Joint Committee on Cancer (AJCC) system to stage testicular cancer. Others examples include cellular receptors such as estrogen (ER) and progesterone receptors (PR) and v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2/ Her-2/neu) status, ER-alpha (ERα) and the classic nuclear ER, and ER-beta (ERβ) in the case of breast cancer, 19 and tumor specific antigen such as prostate-specific antigen (PSA) in the case of prostate cancer. 20 Also, some of those markers have been clinically approved for the prediction of tumor responses to treatment, including chemotherapeutic drugs such as trastuzumab, cetuximab or imatinib.2123

For detection purposes the analysis of tumor biomarkers will relay on techniques such as those based on molecular biology or immunodetection [e.g. polymerase chain reaction (PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA)]. 24 The application of these tests depends on protocols of extraction of biological materials such as DNA, or protein extracts from clinical samples including blood, tissues or urine. Currently, the most two common modalities for collecting patient’s tissue samples include formalin-fixed, paraffin-embedded (FFPE) samples and fine-needle aspiration cytology (FNAC) procedures. These protocolsrepresent excellent technical procedures to prepare of patient materials for clinical molecular profiling, including retrospective genomic analyses and prospective sample collection for individualized therapy or eligibility review for clinical trial enrollment. Despite of the advantages of those protocols, and particularly relevant for FNAC, one should consider the associated practical limitations specially when dealing with evaluation of large numbers of biomarkers. Most recently, identification of surrogate tumor biomarkers such as circulating tumor cells (CTCs), circulating DNA, and microRNA, represent non-invasive modalities that can support the validity of the traditional ones. 25,26 Independently of the detection protocol and the associated assessment considerations, the differential expression of the corresponding biomarkers, which are expected mostly to be tumor specific and tumor stage associated, will be key for distinguishing neoplastic from normal specimens. Similarly, since the ratio of neoplastic cells to normal cells can vary significantly from one clinical sample to the other, 27 inter-individual variation in the expression of tumor biomarkers is a critical factor to be taken into consideration. Of particular relevance for this issue, is the concept of tumor heterogeneity, which is relevant for tumorigenic and metastatic potential and can impact diagnosis, treatment selection and patients’ outcomes. The answer of critical issues is of great interest in order to improve our knowledge to in guiding treatment decisions. Critical points include gaining knowledge of the 1) correlation between the molecular characteristics of a single metastatic site with those from other metastases; 2) molecular characteristics of a primary tumor reflecting actionable characteristics of advanced metastases, and 3) molecular markers accurately reflecting unique features that shape the spectrum of multiple metastases. Response to those issues when considering the development of novel tumor biomarkers will impact prognosis, treatment response and resistance, and will contribute to the progress of precision medicine.

In this short communication, we would like to briefly discuss few of the numerous applications of tumor biomarkers. Cancer staging is the process by which the extent of the the level of tumor progression can be determined. Accordingly, tumor spread is assigned on the basis of the tumor size, and metastasis to the distant organ and lymph nodes as well as its appearance in more distant locations.The utility of tumor staging depends on its value to define prognosis, evaluate the results of treatments and clinical trials, to facilitate the exchange and comparison of information among treatment centers, and to serve as a basis for clinical and translational cancer research. 28 Establishment of molecular biomarkers to assess the tumor burden and spread is considered highly relevant for prediction of survival in addition to the determination of the type and the period of treatment. The first molecular biomarkers that were evaluated for cancer staging were circulating DNA and tumor cells. 29 Other tumor stage markers including including proliferation based markers such as proliferating cell nuclear antigen (PCNA), Ki67, and mindbomb E3 ubiquitin protein ligase 1 (MIB1) have been reported. 30 Prognostic biomarkers are used as a measurement parameter to assess the reliability of the standard therapy in patients and the subsequent clinical outcome. Therefore they will orient the clinician about of whom, and how to treat. 31 One of the salient properties of prognostic markers resides on their value in predicting recurrence after curative resection. Local recurrence and distant metastasis of most tumor types often occurs following resection of early staged tumor. 32 Thus, the ability to predict tumor patients with high-risk of early recurrence may help to control tumor recurrence and progression. Early tumor prediction is also useful for the selection of the time of surgical intervention and the appropriate neoadjuvant and adjuvant systemic therapies. 33,34 Another relevant topic refers to the utilization of molecular biomarkers as tools to identify patients who would benefit from a particular treatment, as basis for personalized medicine. Examples include B-lymphocyte antigen (CD)20, used as marker for the treatment of lymphomas with rituximab; 35 Her-2/neu used as marker for the treatment of breast cancer with trastuzumab; 36 breakpoint cluster region (BCR)- Abelson murine leukemia viral oncogene homolog 1 (ABL) translocation which serves as marker for the treatment of chronic myelogenous leukaemia (CML) with imatinib;37 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) or platelet-derived growth factor receptor-α (PDGFRA) that are established as markers for thetreatment of gastrointestinal stromal tumors (GIST) with imatinib; 38 and ER and PR with reported value for monitoring treatment with tamoxifen or aromatase based inhibitors.39 New biomarkers are also needed as the companion tests of newly developed targeted therapies. Tyrosine-protein kinases c-ros oncogene 1 (ROS1) and anaplastic lymphoma kinase (ALK) are related receptor tyrosine kinases (RTKs) of the insulin receptor family. 40 These kinases exhibit genetic aberrations such as mutations and rearrangements in glioblastomas, 41 non-small cell lung cancer (NSCLC) 42 as well as cholangiocarcinomas. 43 Because of the prevalence of these genetic aberrations in tumors, ROS and ALK have been studied as therapeutic targets for the small-molecule RTK inhibitor, crizotinib. 44 Approved for locally advanced or metastatic NSCLC having ALK gene rearrangement (ALK-positive patients), a FISH assay has become the standard diagnostic companion test for crizotinib therapy. 45 The referred examples illustrate the validity of molecular markers as evaluation parameter for therapy, since they allow for treating patients individually by targeting signaling pathways that are present or activated in their tumors. Personalized medicine is increasingly becoming established in clinical practice, supported in part by the availability of novel biomarkers. Next-generation biomarkers will help to stratify patients and treat cancer at an individualized level.

We have provided a brief insight into the reliability of biomarkers in tumor pathology. This information may be useful to inform health professionals about the biomarkers that are thought to be significant for tumor staging, prognosis and treatment. Despite of those advances more studies are essential for the detection of reliable biomarkers with the potential to improve tumor management.