Among serological biomarkers, LDH remains to date the strongest prognostic markers in melanoma [26Solassol J, Du-Thanh A, Maudelonde T, Guillot B. Serum proteomic profiling reveals potential biomarkers for cutaneous malignant melanoma. Int J Biol Markers 2011; 26(2): 82-7.
[http://dx.doi.org/10.5301/JBM.2011.8344] [PMID: 21607923] ]. LDH is less sensitive in the early stage. However, it is indicative of the tumor growth and malignancy progression [27Hofmann MA, Gussmann F, Fritsche A, et al. Diagnostic value of melanoma inhibitory activity serum marker in the follow-up of patients with stage I or II cutaneous melanoma. Melanoma Res 2009; 19(1): 17-23.
[http://dx.doi.org/10.1097/CMR.0b013e32831bc78c] [PMID: 19104451] , 28Hofmann MA, Schicke B, Fritsch A, et al. Impact of lymph node metastases on serum level of melanoma inhibitory activity in stage III melanoma patients. J Dermatol 2011; 38(9): 880-6.
[PMID: 21658116] ]. It is still noteworthy that LDH, which occurs also in infection, inflammation and hemolysis, might lead to false positive readouts. S100 serves as diagnostic and prognostic marker for melanoma, as its expression is much higher in the malignant melanoma than in the normal tissues [29Davey GE, Murmann P, Hoechli M, Tanaka T, Heizmann CW. Calcium-dependent translocation of S100A11 requires tubulin filaments. Biochim Biophys Acta 2000; 1498(2-3): 220-32.
[http://dx.doi.org/10.1016/S0167-4889(00)00098-7] [PMID: 11108965] , 30Hsieh HL, Schäfer BW, Cox JA, Heizmann CW. S100A13 and S100A6 exhibit distinct translocation pathways in endothelial cells. J Cell Sci 2002; 115(Pt 15): 3149-58.
[PMID: 12118070] ]. It has been found that elevated level of S100, in particular S100B, is related to the poor prognosis and low survival rate [31Oberholzer PA, Urosevic M, Steinert HC, Dummer R. Baseline staging of melanoma with unknown primary site: the value of serum s100 protein and positron emission tomography. Dermatology (Basel) 2008; 217(4): 351-5.
[http://dx.doi.org/10.1159/000155878] [PMID: 18799883] , 32Beyeler M, Waldispuhl S, Strobel K, Joller-Jemelka HI, Burg G, Dummer R. Detection of melanoma relapse: first comparative analysis on imaging techniques versus S100 protein. Dermatology (Basel) 2006; 213(3): 187-91.
[http://dx.doi.org/10.1159/000095034] [PMID: 17033166] ]. Vascular endothelial growth factor (VEGF) and melanoma-inhibitory activity (MIA) protein are involved in tumor-associated angiogenesis and melanoma cell interaction with the ECM respectively. Studies have shown that elevated levels of VEGF and MIA are associated with more advanced disease and poor survival rates [33Ugurel S, Rappl G, Tilgen W, Reinhold U. Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival. J Clin Oncol 2001; 19(2): 577-83.
[PMID: 11208853] , 34Díaz-Lagares A, Alegre E, Arroyo A, et al. Evaluation of multiple serum markers in advanced melanoma. Tumour Biol 2011; 32(6): 1155-61.
[http://dx.doi.org/10.1007/s13277-011-0218-x] [PMID: 21858537] ]. Other serum biomarkers for melanoma include tyrosinase [35Visús C, Andres R, Mayordomo JI, et al. Prognostic role of circulating melanoma cells detected by reverse transcriptase-polymerase chain reaction for tyrosinase mRNA in patients with melanoma. Melanoma Res 2007; 17(2): 83-9.
[http://dx.doi.org/10.1097/CMR.0b013e3280a60878] [PMID: 17496783] ], osteopontin [36McAllister SS, Gifford AM, Greiner AL, et al. Systemic endocrine instigation of indolent tumor growth requires osteopontin. Cell 2008; 133(6): 994-1005.
[http://dx.doi.org/10.1016/j.cell.2008.04.045] [PMID: 18555776] ], IL-8 [37Sanmamed MF, Carranza-Rua O, Alfaro C, et al. Serum interleukin-8 reflects tumor burden and treatment response across malignancies of multiple tissue origins. Clin Cancer Res 2014; 20(22): 5697-707.
[http://dx.doi.org/10.1158/1078-0432.CCR-13-3203] [PMID: 25224278] ] etc.

In addition to the serum biomarkers, molecules over-expressed in the tumor tissues also function as an effective indicator of the melanoma progression and metastasis. For example, matrix metalloproteinases (MMP), which proteolytically degrade ECM proteins, have been shown to promote melanoma cell migration and tumor progression. The MMP-1/3 positive melanoma was shown to correlate with higher malignancy and decreased overall survival [38Nikkola J, Vihinen P, Vlaykova T, Hahka-Kemppinen M, Kähäri VM, Pyrhönen S. High expression levels of collagenase-1 and stromelysin-1 correlate with shorter disease-free survival in human metastatic melanoma. Int J Cancer 2002; 97(4): 432-8.
[http://dx.doi.org/10.1002/ijc.1636] [PMID: 11802203] ]. Cell adhesion molecules (CAMs) such as the carcinoembryonic antigen-related cell adhesion molecule-1 and melanoma-associated antigen (MAA), which play an essential role in cell adhesion, show abnormal expression in melanoma and participate in melanoma migration and metastasis [39Sapoznik S, Ortenberg R, Schachter J, Markel G. CEACAM1 in malignant melanoma: a diagnostic and therapeutic target. Curr Top Med Chem 2012; 12(1): 3-10.
[http://dx.doi.org/10.2174/156802612798919259] [PMID: 22196267] , 40Vergilis IJ, Szarek M, Ferrone S, Reynolds SR. Presence and prognostic significance of melanoma-associated antigens CYT-MAA and HMW-MAA in serum of patients with melanoma. J Invest Dermatol 2005; 125(3): 526-31.
[http://dx.doi.org/10.1111/j.0022-202X.2005.23798.x] [PMID: 16117794] ]. Other tissue-specific biomarkers such as cyclooxygenase-2 (Cox-2) and galectin-3 have also been shown to associate with melanoma tumor size and malignancy and overall patient survival.

About half of the melanomas harbor the mutation of BRAF gene (BRAF^V600E, and possibly BRAF^V600K), which results in constant activation of BRAF kinase [6Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364(26): 2507-16.
[http://dx.doi.org/10.1056/NEJMoa1103782] [PMID: 21639808] ]. This likely induces the promotion of Ras-Raf-MEK-ERK signaling pathway and therefore the proliferation of melanoma. Like BRAF mutation, N-Ras gene mutation is also considered as the “driver mutation” and found in approximately 15-20% metastatic melanoma patients [4Johnson DB, Lovly CM, Flavin M, et al. Impact of NRAS mutations for patients with advanced melanoma treated with immune therapies. Cancer Immunol Res 2015; 3(3): 288-95.
[http://dx.doi.org/10.1158/2326-6066.CIR-14-0207] [PMID: 25736262] ]. BRAF and N-Ras mutations have been shown to link to the metastasis and poor outcome [41Mann GJ, Pupo GM, Campain AE, et al. BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in patients with stage III melanoma. J Invest Dermatol 2013; 133(2): 509-17.
[http://dx.doi.org/10.1038/jid.2012.283] [PMID: 22931913] ]. With lower frequencies, KIT mutations have been observed in mucosal and acral melanomas and melanomas with sun-damaged skin [42Kong Y, Si L, Zhu Y, et al. Large-scale analysis of KIT aberrations in Chinese patients with melanoma. Clin Cancer Res 2011; 17(7): 1684-91.
[http://dx.doi.org/10.1158/1078-0432.CCR-10-2346] [PMID: 21325067] ]. Cutaneous melanomas are characterized with mutations of BRAF, N-Ras, MEK, NF1 and KIT, while most ocular melanomas harbor the mutations of GNAQ and GNA11 [43Hodis E, Watson IR, Kryukov GV, et al. A landscape of driver mutations in melanoma. Cell 2012; 150(2): 251-63.
[http://dx.doi.org/10.1016/j.cell.2012.06.024] [PMID: 22817889] ]. In order to discriminate Spitz nevi and Spitzoid melanomas, biomarkers such as CDKN2A, RREB1, MYC and CCND1 have been utilized [44Gerami P, Li G, Pouryazdanparast P, et al. A highly specific and discriminatory FISH assay for distinguishing between benign and malignant melanocytic neoplasms. Am J Surg Pathol 2012; 36(6): 808-17.
[http://dx.doi.org/10.1097/PAS.0b013e31824b1efd] [PMID: 22588064] ].

Cytotoxic T-lymphocyte antigen-4 (CTLA-4), a protein receptor found on the surface of some T cells, functions as an immune checkpoint. By binding to the B7 on the APCs, it transmits an inhibitory signal to T cell activation, which confers vulnerability to the tumor invasion [45Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol 2009; 27: 591-619.
[http://dx.doi.org/10.1146/annurev.immunol.021908.132706] [PMID: 19132916] ]. Ipilimumab (FDA approved for melanoma therapy in 2011), the human monoclonal immunoglobulin G1 against CTLA-4, has been shown to expand anti-tumor T cell activity and inhibit immune tolerance, therefore significantly enhancing the melanoma patients overall survival rate [46Lee B, Mukhi N, Liu D. Current management and novel agents for malignant melanoma. J Hematol Oncol 2012; 5: 3.
[http://dx.doi.org/10.1186/1756-8722-5-3] [PMID: 22333219] , 47Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 2011; 364(26): 2517-26.
[http://dx.doi.org/10.1056/NEJMoa1104621] [PMID: 21639810] ]. Another anti CTLA-4 monoclonal antibody is tremelimumab, which is still undergoing human trials [48Poust J. Targeting metastatic melanoma. Am J Health Syst Pharm 2008; 65(24)(Suppl. 9): S9-S15.
[http://dx.doi.org/10.2146/ajhp080461] [PMID: 19052265] ].

Programmed death receptor-1 (PD-1) is a protein expressed on the cell surface of T cells. By binding to the ligands PD-L1 and PD-L2 expressed on tumor and stromal cells, it transmits the signal in down-regulating T cell activation and promoting self-tolerance [49Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 2012; 24(2): 207-12.
[http://dx.doi.org/10.1016/j.coi.2011.12.009] [PMID: 22236695] ]. As PD-1 is also expressed on B cells and macrophages, it is likely more potent compared to CTLA-4 in negatively inhibiting immune responses [50Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 1996; 8(5): 765-72.
[http://dx.doi.org/10.1093/intimm/8.5.765] [PMID: 8671665] ]. PD-1 inhibitors, which block PD-1 and activate immune responses, have achieved varying success in different cancers including melanoma. Nivolumab, lambrolizumab and pembrolizumab, which are anti-PD-1 antibodies, have been approved by FDA for melanoma therapy [51Hamid O, Robert C, Daud A, et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 2013; 369(2): 134-44.
[http://dx.doi.org/10.1056/NEJMoa1305133] [PMID: 23724846] , 52Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med 2013; 369(2): 122-33.
[http://dx.doi.org/10.1056/NEJMoa1302369] [PMID: 23724867] ].

One of the major challenges of engineering OVs is that the natural tropism of viruses does not match the therapeutic need. For example, the administration of adenovirus systemically, among which the majority is sequestered by hepatic macrophages and hepatocyte transduction, results in adenovirus-mediated liver toxicity [68Glasgow JN, Everts M, Curiel DT. Transductional targeting of adenovirus vectors for gene therapy. Cancer Gene Ther 2006; 13(9): 830-44.
[http://dx.doi.org/10.1038/sj.cgt.7700928] [PMID: 16439993] ]. Therefore, approaches that retarget the viruses are needed to avoid the toxic side effects and facilitate the efficient infection to the targeted tumor cells. By modifying the viral coat proteins, which is referred to as transductional targeting, the viruses are engineered to infect the tumor cells that they do not infect naturally [69Barker SD, Dmitriev IP, Nettelbeck DM, et al. Combined transcriptional and transductional targeting improves the specificity and efficacy of adenoviral gene delivery to ovarian carcinoma. Gene Ther 2003; 10(14): 1198-204.
[http://dx.doi.org/10.1038/sj.gt.3301974] [PMID: 12833129] ]. For example, the modification of hypervariable loop 5 in the capsid protein hexon in adenovirus prevents infection of hepatocytes and expand the anticancer therapeutic window [70Shashkova EV, May SM, Doronin K, Barry MA. Expanded anticancer therapeutic window of hexon-modified oncolytic adenovirus. Mol Ther 2009; 17(12): 2121-30.
[http://dx.doi.org/10.1038/mt.2009.217] [PMID: 19755961] ].

Pseudotyping, which is the first approach of transductional targeting, is to replace viral attachment proteins of one virus with the tropism determinant ligands of other viruses whose tropism is against the targeted tumor cells. Exciting results have been observed by using oncoretroviral vector pseudotyped with a modified chimeric Sindbis virus envelop glycoproteins and conjugated with antibody specific for P-glycoprotein, for targeting metastatic melanoma [71Morizono K, Xie Y, Ringpis GE, et al. Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection. Nat Med 2005; 11(3): 346-52.
[http://dx.doi.org/10.1038/nm1192] [PMID: 15711560] ].

While pseudotyping is limited by the viral attachment proteins and the receptors exclusively present on the target cells, the other approach of transductional targeting by using adaptors, the molecules with one end binding the viral attachment protein and the other binding the target cells’ receptors, possesses more flexibility without changing the structure of viral vectors [72Waehler R, Russell SJ, Curiel DT. Engineering targeted viral vectors for gene therapy. Nat Rev Genet 2007; 8(8): 573-87.
[http://dx.doi.org/10.1038/nrg2141] [PMID: 17607305] ]. For example, the fusing of ectodomain of coxsackie virus and adenovirus receptor (CAR) and a single chain antibody against carcinoembryonic antigen allowed the adenovirus to target on tumors and ablated the liver tropism [73Li HJ, Everts M, Pereboeva L, et al. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter. Cancer Res 2007; 67(11): 5354-61.
[http://dx.doi.org/10.1158/0008-5472.CAN-06-4679] [PMID: 17545616] ]. Avidin, Biotin and monoclonal antibodies are also used in adaptor systems. For example, a biotin-acceptor peptide has been incorporated into the adenovirus fiber capsid protein and biotinylated and coupled to an avidin-binding ligand [74Parrott MB, Adams KE, Mercier GT, Mok H, Campos SK, Barry MA. Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification. Mol Ther 2003; 8(4): 688-700.
[http://dx.doi.org/10.1016/S1525-0016(03)00213-2] [PMID: 14529842] ]. MMP and MAA, as the biomarkers of melanoma, play a critical role in melanoma progression and metastasis. Efficient targeting of retrovirus to melanoma cells has been done by fusing a single chain antibody recognizing the high molecular weight MAA (HMWMAA) followed by a proline-rich linker and a MMP-2 cleavage site, to the amino terminus of murine leukemia virus (MLV-A) envelop glycoprotein [75Martín F, Chowdhury S, Neil S, Phillipps N, Collins MK. Envelope-targeted retrovirus vectors transduce melanoma xenografts but not spleen or liver. Mol Ther 2002; 5(3): 269-74.
[http://dx.doi.org/10.1006/mthe.2002.0550] [PMID: 11863416] , 76Martin F, Neil S, Kupsch J, Maurice M, Cosset F, Collins M. Retrovirus targeting by tropism restriction to melanoma cells. J Virol 1999; 73(8): 6923-9.
[PMID: 10400790] ]. Following the attachment with HMWMAA, MMP cleaves the MMP-2 cleavage site and allows the infection of MLV. Another example combining the avidin/biotin and monoclonal antibody is by first biotinylating VV to which the avidin is subsequently added and then coating the avidin bridge with biotinylated antibodies. The antibodies are against the cell surface makers of target cells [77Purow B, Staveley-OCarroll K. Targeting of vaccinia virus using biotin-avidin viral coating and biotinylated antibodies. J Surg Res 2005; 123(1): 49-54.
[http://dx.doi.org/10.1016/j.jss.2004.04.022] [PMID: 15652950] ].

The transcriptional regions consisting of promoters and enhancers regulate the kinetics and levels of mRNA production, and activators recruit RNA polymerase II for initiating the transcription of mRNA. Cancer-specific promoters can be activated in the presence of a subset of activators existing in cancerous cells but not in normal cells [78Wu L, Johnson M, Sato M. Transcriptionally targeted gene therapy to detect and treat cancer. Trends Mol Med 2003; 9(10): 421-9.
[http://dx.doi.org/10.1016/j.molmed.2003.08.005] [PMID: 14557054] ]. Therefore, transcriptional targeting in which part of the viral genome is placed under the control of cancer-specific promoter serves as a feasible way for generating tumor selectivity.

Tissue targeted virotherapy has been investigated in a variety of cancers including melanoma. Promoters of genes over-expressed in melanoma have been chosen and utilized, among which most are the above mentioned melanoma biomarkers’ promoters such as Cox-2, C-X-C chemokine receptor type 4 (CXCR-4), tyrosinase and survivin promoters [10Banerjee NS, Rivera AA, Wang M, et al. Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures. Mol Cancer Ther 2004; 3(4): 437-49.
[PMID: 15078987] , 78Wu L, Johnson M, Sato M. Transcriptionally targeted gene therapy to detect and treat cancer. Trends Mol Med 2003; 9(10): 421-9.
[http://dx.doi.org/10.1016/j.molmed.2003.08.005] [PMID: 14557054] -80Zhu ZB, Makhija SK, Lu B, et al. Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter. Gene Ther 2004; 11(7): 645-8.
[http://dx.doi.org/10.1038/sj.gt.3302089] [PMID: 15029227] ]. Cox-2 is closely associated with the progression and metastasis of melanoma, whereas benign nevi and tissues are Cox-2 negative [81Denkert C, Köbel M, Berger S, et al. Expression of cyclooxygenase 2 in human malignant melanoma. Cancer Res 2001; 61(1): 303-8.
[PMID: 11196178] ]. CXCR-4 over-expressed on human melanoma cells has also been shown to play a role in melanoma progression [82Robledo MM, Bartolome RA, Longo N, et al. Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells. J Biol Chem 2001; 276(48): 45098-105.
[http://dx.doi.org/10.1074/jbc.M106912200] [PMID: 11571298] ]. Incorporation of Cox-2 and CXCR-4 promoters in adenovirus genome has been shown to significantly enhance the virus-mediated gene expression in the malignant melanoma instead of non-malignant primary melanocytes [79Lu B, Makhija SK, Nettelbeck DM, et al. Evaluation of tumor-specific promoter activities in melanoma. Gene Ther 2005; 12(4): 330-8.
[http://dx.doi.org/10.1038/sj.gt.3302385] [PMID: 15696177] , 80Zhu ZB, Makhija SK, Lu B, et al. Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter. Gene Ther 2004; 11(7): 645-8.
[http://dx.doi.org/10.1038/sj.gt.3302089] [PMID: 15029227] ]. Tyrosinase, which plays an essential role in melanin synthesis and melanocyte differentiation, has been used as a melanoma biomarker for disease detection purposes [83Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991; 338(8777): 1227-9.
[http://dx.doi.org/10.1016/0140-6736(91)92100-G] [PMID: 1719320] ]. It has been demonstrated that incorporation of tyrosinase promoter to drive the expression of the viral genes of adenovirus leads to an obvious melanoma-selective viral cytotoxicity [84Nettelbeck DM, Rivera AA, Balagué C, Alemany R, Curiel DT. Novel oncolytic adenoviruses targeted to melanoma: specific viral replication and cytolysis by expression of E1A mutants from the tyrosinase enhancer/promoter. Cancer Res 2002; 62(16): 4663-70.
[PMID: 12183423] ]. Survivin is expressed on a variety of tumors but undetectable in terminally differentiated tissues. It is associated with tumor progression and resistance to chemo- and radio-therapies, which is likely due to its ability of modulating apoptosis pathways and inhibiting apoptotic enzymes such as caspase-3 and caspase-7 [11Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 1998; 58(23): 5315-20.
[PMID: 9850056] , 85Asanuma K, Kobayashi D, Furuya D, Tsuji N, Yagihashi A, Watanabe N. A role for survivin in radioresistance of pancreatic cancer cells. Jpn J Cancer Res 2002; 93(9): 1057-62.
[http://dx.doi.org/10.1111/j.1349-7006.2002.tb02483.x] [PMID: 12359060] ]. Lu et al. have reported that compared with Cox-2 and CXCR-4 promoters, the activity of survivin promoter is higher in melanoma cells and lower in normal tissues such as livers and epithelial melanocytes [79Lu B, Makhija SK, Nettelbeck DM, et al. Evaluation of tumor-specific promoter activities in melanoma. Gene Ther 2005; 12(4): 330-8.
[http://dx.doi.org/10.1038/sj.gt.3302385] [PMID: 15696177] ]. A continued search for a better cancer-specificity is warranted, as higher expression in tumor cells and lower expression in normal tissues for transgenes in viral vectors is always desirable.

Mutations in ras gene, which lead to the constant activation of Ras and therefore cell over-proliferation and tumorigenesis, have been reported in 30% of all cancers [86Marcato P, Shmulevitz M, Lee PW. Connecting reovirus oncolysis and Ras signaling. Cell Cycle 2005; 4(4): 556-9.
[http://dx.doi.org/10.4161/cc.4.4.1600] [PMID: 15753655] ]. The incidence of ras mutations in melanomas is around 20% and the majority is N-ras mutations [87Bos JL. ras oncogenes in human cancer: a review. Cancer Res 1989; 49(17): 4682-9.
[PMID: 2547513] ]. It has been demonstrated that reovirus and VSV, in unaltered form, replicate preferentially and effectively in the Ras-mutated cancer cells and therefore serve as natural OVs [88Errington F, White CL, Twigger KR, et al. Inflammatory tumour cell killing by oncolytic reovirus for the treatment of melanoma. Gene Ther 2008; 15(18): 1257-70.
[http://dx.doi.org/10.1038/gt.2008.58] [PMID: 18401435] , 89Noser JA, Mael AA, Sakuma R, et al. The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells. Mol Ther 2007; 15(8): 1531-6.
[http://dx.doi.org/10.1038/sj.mt.6300193] [PMID: 17505473] ]. It has been shown that reovirus efficiently infects and kills melanoma cells and regresses the xenografted melanoma tumors. In addition, Ras signaling pathway has been shown to be involved in reovirus-induced apoptosis in human melanoma cells [9Steele L, Errington F, Prestwich R, et al. Pro-inflammatory cytokine/chemokine production by reovirus treated melanoma cells is PKR/NF-κB mediated and supports innate and adaptive anti-tumour immune priming. Mol Cancer 2011; 10: 20.
[http://dx.doi.org/10.1186/1476-4598-10-20] [PMID: 21338484] ]. Similarly, Noser et al. [89Noser JA, Mael AA, Sakuma R, et al. The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells. Mol Ther 2007; 15(8): 1531-6.
[http://dx.doi.org/10.1038/sj.mt.6300193] [PMID: 17505473] ] have reported that Ras-overexpressing cancer cells are effectively killed by VSV and the oncolytic properties of VSV are dependent on the activation of Ras/Raf/MEK/ERK signaling pathway. It is noteworthy that the activation of Ras pathway is usually coupled with the down-regulation of IFN-induced antiviral responses such as the induction of protein kinase PKR, which further favors the virus replication in cancer cells.

Dysfunction of IFN signaling pathway has been reported in different cancers such as melanoma and breast cancer. Impaired IFN pathway aids tumor progression and immune escape, as IFN signaling is critical for the proper activation of key immune cells such as T, B and natural killer (NK) cells [90Critchley-Thorne RJ, Simons DL, Yan N, et al. Impaired interferon signaling is a common immune defect in human cancer. Proc Natl Acad Sci USA 2009; 106(22): 9010-5.
[http://dx.doi.org/10.1073/pnas.0901329106] [PMID: 19451644] ]. It has been reported that several melanomas are deficient in the key components of IFN signaling pathway such as STAT1 and STAT2 [91Wong LH, Krauer KG, Hatzinisiriou I, et al. Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma. J Biol Chem 1997; 272(45): 28779-85.
[http://dx.doi.org/10.1074/jbc.272.45.28779] [PMID: 9353349] ]. One example of enhancing the viral oncoselectivity via targeting the impaired IFN pathway in tumors involves in the nonstructural protein NS1 of influenza virus which has been demonstrated to block the function of PKR induced by IFN [92Hatada E, Saito S, Fukuda R. Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells. J Virol 1999; 73(3): 2425-33.
[PMID: 9971827] ]. Influenza virus with NS1 deleted has been shown to selectively infect and lyse the melanoma cells that are PKR-deficient and regress the melanoma tumors in vivo, while failing to replicate in normal cells with intact PKR-mediated antiviral responses [93Bergmann M, Romirer I, Sachet M, et al. A genetically engineered influenza A virus with ras-dependent oncolytic properties. Cancer Res 2001; 61(22): 8188-93.
[PMID: 11719449] ]. It is worthwhile to further investigate the application of viruses with the IFN or PKR viral inhibitor deletion for melanoma virotherapies, as IFN/PKR inhibitors are expressed by a variety of viruses such as VV and retrovirus [93Bergmann M, Romirer I, Sachet M, et al. A genetically engineered influenza A virus with ras-dependent oncolytic properties. Cancer Res 2001; 61(22): 8188-93.
[PMID: 11719449] ].

Deficiency in apoptosis is a critical contributor to the therapeutic resistance of melanoma. Two pathways, the intrinsic and extrinsic pathways lead to the apoptosis. While Bcl-2 family proteins such as Bax and Bak and the induction of apoptosome and caspases are involved in the intrinsic pathway, the extrinsic pathway features the death ligands of tumor necrosis factor (TNF)-α protein family including TNF-α, Fas ligand (CD95L) and TNF-related apoptosis-inducing ligand (TRAIL) [94Eberle J, Fecker LF, Forschner T, Ulrich C, Röwert-Huber J, Stockfleth E. Apoptosis pathways as promising targets for skin cancer therapy. Br J Dermatol 2007; 156(Suppl. 3): 18-24.
[http://dx.doi.org/10.1111/j.1365-2133.2007.07855.x] [PMID: 17488402] ]. The application of tyrosinase promoter controlled CD95L has been shown to selectively induce apoptosis in melanoma cells while sparing the non-melanoma cells [95Fecker LF, Geilen CC, Hossini AM, et al. Selective induction of apoptosis in melanoma cells by tyrosinase promoter-controlled CD95 ligand overexpression. J Invest Dermatol 2005; 124(1): 221-8.
[http://dx.doi.org/10.1111/j.0022-202X.2004.23572.x] [PMID: 15654977] ]. Viruses such as Newcastle disease virus (NDV) have been demonstrated to possess the ability of naturally inducing apoptosis in various cancer cells [96Ravindra PV, Tiwari AK, Sharma B, Chauhan RS. Newcastle disease virus as an oncolytic agent. Indian J Med Res 2009; 130(5): 507-13.
[PMID: 20090097] ]. A wide array of viral proteins which have been shown to induce apoptosis include: NS3 protein of bovine viral diarrhea virus, viral protein R of human immunodeficiency virus, hemagglutinin-neuraminidase of NDV and EIA of adenovirus [96Ravindra PV, Tiwari AK, Sharma B, Chauhan RS. Newcastle disease virus as an oncolytic agent. Indian J Med Res 2009; 130(5): 507-13.
[PMID: 20090097] -99Uttenthal A, Stadejek T, Nylin B. Genetic diversity of bovine viral diarrhoea viruses (BVDV) in Denmark during a 10-year eradication period. APMIS 2005; 113(7-8): 536-41.
[http://dx.doi.org/10.1111/j.1600-0463.2005.apm_227.x] [PMID: 16086824] ]. It is appropriate to incorporate these apoptosis-inducing proteins into the OVs for expression. In addition, the over-expression of anti-apoptotic proteins is a critical factor for melanoma’s resistance to the chemotherapy. Therefore, it is feasible to increase the viral oncolysis by co-administrating with inhibitors of the anti-apoptotic proteins, such as BCL-2 inhibitors. It has been reported that the co-treatment of BCL-2 inhibitor improves the viral oncolysis of VSV in the tumors which are resistant to the treatment of virus alone [100Tumilasci VF, Olière S, Nguyên TL, Shamy A, Bell J, Hiscott J. Targeting the apoptotic pathway with BCL-2 inhibitors sensitizes primary chronic lymphocytic leukemia cells to vesicular stomatitis virus-induced oncolysis. J Virol 2008; 82(17): 8487-99.
[http://dx.doi.org/10.1128/JVI.00851-08] [PMID: 18579592] ].

The components in the environment where tumors exist, including the blood vessels, fibroblasts, immune cells and elements of ECM, are collectively termed as “tumor microenvironment”. The interaction between tumor cells and tumor microenvironment is critical in tumor initiation, progression and invasiveness. Also, tumor microenvironment affects the delivery and spread of the therapies such as OVs in tumors. Therefore, in this section, we will review the strategies used for increasing the efficacy of OVs and combating the melanoma progression via targeting the melanoma tumor microenvironment.

Oncolytic virotherapy may represent a unique combination of viral tumor lysis and the activation of innate and adaptive immune responses. Targeted infection of tumor cells causes the release of TAAs and localized inflammation, which subsequently induces the tumor recognition of host immune system [113Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol 2012; 30(7): 658-70.
[http://dx.doi.org/10.1038/nbt.2287] [PMID: 22781695] ]. Therefore, in this section, we will review the strategies for enhancing immune recognition of cancer specific neo-antigens and antitumor immune responses.

2.5.2. Arming OVs with immuno-stimulatory or immune-modulatory genes

Tumors have been evolving to escape the antitumor immune response and develop the therapy-resistance. The infection and lysis of tumor cells by OVs triggers the localized release of inflammatory cytokines which subsequently attract the immune cells for tumor elimination [115Pulido J, Kottke T, Thompson J, et al. Using virally expressed melanoma cDNA libraries to identify tumor-associated antigens that cure melanoma. Nat Biotechnol 2012; 30(4): 337-43.
[http://dx.doi.org/10.1038/nbt.2157] [PMID: 22426030] ]. However, it is questionable if the cytokines are sufficient for eliciting tumor-reduction immune responses. Therefore, it would be desirable to have OVs expressing cytokines effective in inducing antitumor immune responses.

The two most advanced OVs, T-vec and JX-594 are respectively the modified HSV and VV, both expressing human GM-CSF which recruits dendritic cells and NK cells and induces tumor-specific cytotoxic T lymphocytes responses [117Deng L, Fan J, Guo M, Huang B. Oncolytic and immunologic cancer therapy with GM-CSF-armed vaccinia virus of Tian Tan strain Guang9. Cancer Lett 2016; 372(2): 251-7.
[http://dx.doi.org/10.1016/j.canlet.2016.01.025] [PMID: 26803055] ]. T-vec shows significant improvement of durable response rate and overall survival rate of patients with advance melanoma [15Andtbacka RH, Kaufman HL, Collichio F, et al. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol 2015; 33(25): 2780-8.
[http://dx.doi.org/10.1200/JCO.2014.58.3377] [PMID: 26014293] ].

The induction of anti-tumor T cell responses requires two signals: (1) the interaction between the major histocompatibility complex protein presenting tumor antigens and T cell receptors, and (2) the costimulatory molecules on APCs or tumor cells [118Todo T, Martuza RL, Dallman MJ, Rabkin SD. In situ expression of soluble B71 in the context of oncolytic herpes simplex virus induces potent antitumor immunity. Cancer Res 2001; 61(1): 153-61.
[PMID: 11196154] ]. The costimulatory signals come from the binding of B7 on activated APCs to CD28 or CTLA-4 on T cells and the binding of CD40 on APCs to CD40L on T cells. While interaction of B7 and CD28 promotes T cell activation and survival, the binding of B7 and CTLA-4 transmits inhibitory signal to the activated T cells. The binding of CD40L and CD40 triggers T lymphocyte expansion and increases IL-12 release which is critical for the engagement of CTL for the antitumor immune response [119Loskog AS, Fransson ME, Totterman TT. AdCD40L gene therapy counteracts T regulatory cells and cures aggressive tumors in an orthotopic bladder cancer model. Clin Cancer Res 2005; 11(24 Pt 1): 8816-21.
[http://dx.doi.org/10.1158/1078-0432.CCR-05-1817] [PMID: 16361570] , 120Mackey MF, Gunn JR, Maliszewsky C, Kikutani H, Noelle RJ, Barth RJ Jr. Dendritic cells require maturation via CD40 to generate protective antitumor immunity. J Immunol 1998; 161(5): 2094-8.
[PMID: 9725199] ]. Adenovirus, VSV and HSV engineered to express soluble B7 or CD40L exhibited more effective melanoma regression and significantly enhanced CD4⁺ and CD8⁺ T-cell infiltration [121Galivo F, Diaz RM, Thanarajasingam U, et al. Interference of CD40L-mediated tumor immunotherapy by oncolytic vesicular stomatitis virus. Hum Gene Ther 2010; 21(4): 439-50.
[http://dx.doi.org/10.1089/hum.2009.143] [PMID: 19922169] -123Lee YS, Kim JH, Choi KJ, et al. Enhanced antitumor effect of oncolytic adenovirus expressing interleukin-12 and B71 in an immunocompetent murine model. Clin Cancer Res 2006; 12(19): 5859-68.
[http://dx.doi.org/10.1158/1078-0432.CCR-06-0935] [PMID: 17020994] ]. Similarly, potent antitumor activity along with increased T cell activity have been observed with the administration of adenovirus and VSV coding a fully monoclonal antibody specific for CTLA-4 [124Gao Y, Whitaker-Dowling P, Griffin JA, Barmada MA, Bergman I. Recombinant vesicular stomatitis virus targeted to Her2/neu combined with anti-CTLA4 antibody eliminates implanted mammary tumors. Cancer Gene Ther 2009; 16(1): 44-52.
[http://dx.doi.org/10.1038/cgt.2008.55] [PMID: 18654610] , 125Dias JD, Hemminki O, Diaconu I, et al. Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4. Gene Ther 2012; 19(10): 988-98.
[http://dx.doi.org/10.1038/gt.2011.176] [PMID: 22071969] ]. Binding of PD-L1 or PD-L2 on tumor cells to PD-1 on T cells inhibits antitumor T cell response. Combination of Anti-PD-1 blockade with virotherapy has been shown to improve the virus’ antitumor activity and result in improved therapeutic outcome [126Engeland CE, Grossardt C, Veinalde R, et al. CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy. Mol Ther 2014; 22(11): 1949-59.
[http://dx.doi.org/10.1038/mt.2014.160] [PMID: 25156126] ].

A variety of cytokines have been incorporated into OVs (Table 1) to activate various lymphocytes and attain enhanced antitumor immune response. For example, IL-2, the T cell growth factor, not only promotes T cell activation and proliferation, but also activates NK cells and increases NK cells’ lysing ability. IL-2 has been engineered into viruses such as NDV and HSV, intended to stimulate the activation of T and NK cells [127Vigil A, Park MS, Martinez O, et al. Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus. Cancer Res 2007; 67(17): 8285-92.
[http://dx.doi.org/10.1158/0008-5472.CAN-07-1025] [PMID: 17804743] -129Carew JF, Kooby DA, Halterman MW, Kim SH, Federoff HJ, Fong Y. A novel approach to cancer therapy using an oncolytic herpes virus to package amplicons containing cytokine genes. Mol Ther 2001; 4(3): 250-6.
[http://dx.doi.org/10.1006/mthe.2001.0448] [PMID: 11545616] ]. Other cytokines engineered into OVs for promoting the antitumor activity through enhancing immune cell activation include IL-12, IL-15, IL-18, CCL-3 and IFN-γ etc [12Edukulla R, Woller N, Mundt B, et al. Antitumoral immune response by recruitment and expansion of dendritic cells in tumors infected with telomerase-dependent oncolytic viruses. Cancer Res 2009; 69(4): 1448-58.
[http://dx.doi.org/10.1158/0008-5472.CAN-08-1160] [PMID: 19190348] , 13Stephenson KB, Barra NG, Davies E, Ashkar AA, Lichty BD. Expressing human interleukin-15 from oncolytic vesicular stomatitis virus improves survival in a murine metastatic colon adenocarcinoma model through the enhancement of anti-tumor immunity. Cancer Gene Ther 2012; 19(4): 238-46.
[http://dx.doi.org/10.1038/cgt.2011.81] [PMID: 22158521] , 123Lee YS, Kim JH, Choi KJ, et al. Enhanced antitumor effect of oncolytic adenovirus expressing interleukin-12 and B71 in an immunocompetent murine model. Clin Cancer Res 2006; 12(19): 5859-68.
[http://dx.doi.org/10.1158/1078-0432.CCR-06-0935] [PMID: 17020994] , 130Choi IK, Lee JS, Zhang SN, et al. Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ2 or IL-18Rα. Gene Ther 2011; 18(9): 898-909.
[http://dx.doi.org/10.1038/gt.2011.37] [PMID: 21451575] -134Su C, Peng L, Sham J, et al. Immune gene-viral therapy with triplex efficacy mediated by oncolytic adenovirus carrying an interferon-gamma gene yields efficient antitumor activity in immunodeficient and immunocompetent mice. Mol Ther 2006; 13(5): 918-27.
[http://dx.doi.org/10.1016/j.ymthe.2005.12.011] [PMID: 16497559] ]. The expression of these cytokines results in overall increase of therapeutic effect, despite the effect that some cytokines such as GM-CSF and IFN-γ cause reduced virus replication and early clearance.

Table 1
Melanoma virotherapy.

In the past decade, there has been a rapid development of viruses with oncolytic activity as the clinical or preclinical anticancer therapeutics. More and more studies have shown that rather than using OVs as tumor therapy alone, it would be likely to realize OVs’ full therapeutic potential by combining with other treatment strategies such as radiotherapy, chemotherapy or immunotherapy.

A major part of the current standard therapeutic strategy for melanoma includes a range of targeted treatment entities based on specific gene mutations (BRAF inhibitors), pathways or enzymes (MEK inhibitors, tyrosine kinase inhibitors) and growth factors (VEGF inhibitors) [6Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364(26): 2507-16.
[http://dx.doi.org/10.1056/NEJMoa1103782] [PMID: 21639808] , 19Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012; 380(9839): 358-65.
[http://dx.doi.org/10.1016/S0140-6736(12)60868-X] [PMID: 22735384] , 53Gonzalez D, Fearfield L, Nathan P, et al. BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel. Br J Dermatol 2013; 168(4): 700-7.
[http://dx.doi.org/10.1111/bjd.12248] [PMID: 23360189] -60Tarhini AA, Frankel P, Margolin KA, et al. Aflibercept (VEGF Trap) in inoperable stage III or stage iv melanoma of cutaneous or uveal origin. Clin Cancer Res 2011; 17(20): 6574-81.
[http://dx.doi.org/10.1158/1078-0432.CCR-11-1463] [PMID: 21880788] ]. Melanoma associated biomarkers like LDH, MIA, VEGF and MMP have shown a notable prognostic value where the level of these biomarkers has closely correlated with the advancement of the disease [33Ugurel S, Rappl G, Tilgen W, Reinhold U. Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival. J Clin Oncol 2001; 19(2): 577-83.
[PMID: 11208853] , 34Díaz-Lagares A, Alegre E, Arroyo A, et al. Evaluation of multiple serum markers in advanced melanoma. Tumour Biol 2011; 32(6): 1155-61.
[http://dx.doi.org/10.1007/s13277-011-0218-x] [PMID: 21858537] , 38Nikkola J, Vihinen P, Vlaykova T, Hahka-Kemppinen M, Kähäri VM, Pyrhönen S. High expression levels of collagenase-1 and stromelysin-1 correlate with shorter disease-free survival in human metastatic melanoma. Int J Cancer 2002; 97(4): 432-8.
[http://dx.doi.org/10.1002/ijc.1636] [PMID: 11802203] ]. In the context of FDA approved therapies targeting melanoma biomarkers such as BRAF, MEK and VEGF, it is safe to say that biomarkers can not only serve as prognostic tools but are also useful in developing new therapies targeting melanoma. Targeted therapies fail to provide expected outcomes in many patients due to the genetic variability of melanoma, as melanoma tumors can be genetically heterogeneous and may or may not harbor mutations in target genes of therapeutic interest. Efficacy of treatment strategy for melanoma increases significantly when multiple targeted therapies are used in combination versus use of a single therapeutic agent, indicating the high level of dependence of targeted melanoma therapies on the genetic profile of the cancer. While nearly 50% of melanoma patients usually respond to BRAF inhibitors, the number of non-responding patients decreased by 40% when BRAF inhibitors were used along with MEK inhibitors as a combination therapy [6Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364(26): 2507-16.
[http://dx.doi.org/10.1056/NEJMoa1103782] [PMID: 21639808] , 19Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012; 380(9839): 358-65.
[http://dx.doi.org/10.1016/S0140-6736(12)60868-X] [PMID: 22735384] , 55Sosman JA, Kim KB, Schuchter L, et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med 2012; 366(8): 707-14.
[http://dx.doi.org/10.1056/NEJMoa1112302] [PMID: 22356324] , 160Larkin J, Del Vecchio M, Ascierto PA, et al. Vemurafenib in patients with BRAF(V600) mutated metastatic melanoma: an open-label, multicentre, safety study. Lancet Oncol 2014; 15(4): 436-44.
[http://dx.doi.org/10.1016/S1470-2045(14)70051-8] [PMID: 24582505] ]. In regard of this, immunotherapy (IFN-α and IL-2) and oncolytic virotherapy that are not heavily dependent on genetic heterogeneity of melanoma would circumvent limitations associated with targeted therapies that target specific gene mutations.

The OVs’ ability of causing tumor lysis, stimulating local inflammatory responses and serving as gene delivery vehicles, supports them as promising therapeutic agents for melanoma. While OVs are able to induce cell lysis and local inflammation that are not relying on genetic heterogeneity of melanoma, some viruses, in native forms, preferentially infect melanoma cells with aberrant genetic properties. Viruses such as reovirus, NDV and VSV have been shown to effectively kill the Ras-mutated tumor cells or lead to apoptosis, which make them as natural OVs for melanoma [88Errington F, White CL, Twigger KR, et al. Inflammatory tumour cell killing by oncolytic reovirus for the treatment of melanoma. Gene Ther 2008; 15(18): 1257-70.
[http://dx.doi.org/10.1038/gt.2008.58] [PMID: 18401435] , 89Noser JA, Mael AA, Sakuma R, et al. The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells. Mol Ther 2007; 15(8): 1531-6.
[http://dx.doi.org/10.1038/sj.mt.6300193] [PMID: 17505473] , 96Ravindra PV, Tiwari AK, Sharma B, Chauhan RS. Newcastle disease virus as an oncolytic agent. Indian J Med Res 2009; 130(5): 507-13.
[PMID: 20090097] ]. In addition, for those viruses without natural oncoselectivity, modifications targeting them to the genetic abnormalities of melanoma, often represented as biomarkers, have shown significant effectiveness in enhancing their oncolytic activity. For example, promoters of melanoma biomarkers such as Cox-2, CXCR-4, tyrosinase and survivin have been incorporated into the viral genomes, which result in significantly enhanced oncospecificity [10Banerjee NS, Rivera AA, Wang M, et al. Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures. Mol Cancer Ther 2004; 3(4): 437-49.
[PMID: 15078987] , 78Wu L, Johnson M, Sato M. Transcriptionally targeted gene therapy to detect and treat cancer. Trends Mol Med 2003; 9(10): 421-9.
[http://dx.doi.org/10.1016/j.molmed.2003.08.005] [PMID: 14557054] -80Zhu ZB, Makhija SK, Lu B, et al. Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter. Gene Ther 2004; 11(7): 645-8.
[http://dx.doi.org/10.1038/sj.gt.3302089] [PMID: 15029227] ]. A single chain antibody recognizing the HMWMAA, which is another melanoma biomarker, has been engineered into the virus for transductionally targeting melanoma cells [77Purow B, Staveley-OCarroll K. Targeting of vaccinia virus using biotin-avidin viral coating and biotinylated antibodies. J Surg Res 2005; 123(1): 49-54.
[http://dx.doi.org/10.1016/j.jss.2004.04.022] [PMID: 15652950] ]. Beyond the genetic and epigenetic alterations within the melanoma cells, the interaction between the melanoma cells and the microenvironment critically determines the transformation process of melanoma. Microenvironmental factors such as growth factors (VEGF, FGF, PDGF and TGF) and proteolytic enzymes (MMPs) involve in angiogenesis and tissue remodeling, highly promoting melanoma progression and metastasis. Modifications of OVs to target these microenvironmental factors have resulted in enhanced viral penetration and spread and melanoma regression [103Frentzen A, Yu YA, Chen N, et al. Anti-VEGF single-chain antibody GLAF-1 encoded by oncolytic vaccinia virus significantly enhances antitumor therapy. Proc Natl Acad Sci USA 2009; 106(31): 12915-20.
[http://dx.doi.org/10.1073/pnas.0900660106] [PMID: 19617539] , 110Cheng J, Sauthoff H, Huang Y, et al. Human matrix metalloproteinase-8 gene delivery increases the oncolytic activity of a replicating adenovirus. Mol Ther 2007; 15(11): 1982-90.
[http://dx.doi.org/10.1038/sj.mt.6300264] [PMID: 17653103] , 111Choi JW, Lee YS, Yun CO, Kim SW. Polymeric oncolytic adenovirus for cancer gene therapy. J Control Release 2015; 219: 181-91.
[http://dx.doi.org/10.1016/j.jconrel.2015.10.009] [PMID: 26453806] ].

OVs represent a class of novel agents as they can be engineered to harness the benefits of targeted therapy and immunotherapy, by inducing cancer cell death along with induction of a strong innate and adaptive immune response against cancer cells. The greatest success has been achieved with HSV (T-vec) and VV (JX-594) when developing OVs for cancer treatment. Some other viruses that are being studied in pre-clinical and clinical trials include HF10 (HSV), reovirus, NDV, VSV and coxsackie virus [115Pulido J, Kottke T, Thompson J, et al. Using virally expressed melanoma cDNA libraries to identify tumor-associated antigens that cure melanoma. Nat Biotechnol 2012; 30(4): 337-43.
[http://dx.doi.org/10.1038/nbt.2157] [PMID: 22426030] , 127Vigil A, Park MS, Martinez O, et al. Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus. Cancer Res 2007; 67(17): 8285-92.
[http://dx.doi.org/10.1158/0008-5472.CAN-07-1025] [PMID: 17804743] , 152Galanis E, Markovic SN, Suman VJ, et al. Phase II trial of intravenous administration of Reolysin(®) (Reovirus Serotype-3-dearing Strain) in patients with metastatic melanoma. Mol Ther 2012; 20(10): 1998-2003.
[http://dx.doi.org/10.1038/mt.2012.146] [PMID: 22871663] , 161Watanabe D, Goshima F, Mori I, Tamada Y, Matsumoto Y, Nishiyama Y. Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10. J Dermatol Sci 2008; 50(3): 185-96.
[http://dx.doi.org/10.1016/j.jdermsci.2007.12.001] [PMID: 18226503] , 162Shafren D, Quah M, Wong Y, Andtbacka RH, Au G. Combination of a novel oncolytic immunotherapeutic agent, coxsackievirus a21 and pd-1 blockade significantly reduces tumor growth and improves survival in an immune competent mouse melanoma model. Ann Oncol 2014; •••: 25. [PMID: WOS:000346901000307].]. TAAs and immunostimulatory cytokines have been engineered into OVs, intended to attain enhanced immune recognition of tumor antigens and antitumor immune responses [115Pulido J, Kottke T, Thompson J, et al. Using virally expressed melanoma cDNA libraries to identify tumor-associated antigens that cure melanoma. Nat Biotechnol 2012; 30(4): 337-43.
[http://dx.doi.org/10.1038/nbt.2157] [PMID: 22426030] ]. Using the viruses as the platforms for expressing immunostimulatory cytokines such as GM-CSF is appealing as it is likely to increase the local concentration of the gene products and minimize the side effects due to the systemic exposure [163Simmons AD, Moskalenko M, Creson J, et al. Local secretion of anti-CTLA-4 enhances the therapeutic efficacy of a cancer immunotherapy with reduced evidence of systemic autoimmunity. Cancer Immunol Immunother 2008; 57(8): 1263-70.
[http://dx.doi.org/10.1007/s00262-008-0451-3] [PMID: 18236040] ]. While in some instances the expression of cytokines showed no inhibitive effect in virus replication, in some other cases the incorporation of cytokines such as IL-2, IFN and GM-CSF resulted in significantly decreased virus titer [126Engeland CE, Grossardt C, Veinalde R, et al. CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy. Mol Ther 2014; 22(11): 1949-59.
[http://dx.doi.org/10.1038/mt.2014.160] [PMID: 25156126] , 128Zhao H, Janke M, Fournier P, Schirrmacher V. Recombinant Newcastle disease virus expressing human interleukin-2 serves as a potential candidate for tumor therapy. Virus Res 2008; 136(1-2): 75-80.
[http://dx.doi.org/10.1016/j.virusres.2008.04.020] [PMID: 18538434] , 164Kirn DH, Wang Y, Le Boeuf F, Bell J, Thorne SH. Targeting of interferon-beta to produce a specific, multi-mechanistic oncolytic vaccinia virus. PLoS Med 2007; 4(12): e353.
[http://dx.doi.org/10.1371/journal.pmed.0040353] [PMID: 18162040] -166Lei N, Shen FB, Chang JH, et al. An oncolytic adenovirus expressing granulocyte macrophage colony-stimulating factor shows improved specificity and efficacy for treating human solid tumors. Cancer Gene Ther 2009; 16(1): 33-43.
[http://dx.doi.org/10.1038/cgt.2008.46] [PMID: 18670453] ]. To cope with this issue, strategies have been designed to fuse the cytokine transgene to an exogenous cytokine-expressing regulator, which regulates the expression of the cytokine at specific time [167Chen H, Sampath P, Hou W, Thorne SH. Regulating cytokine function enhances safety and activity of genetic cancer therapies. Mol Ther 2013; 21(1): 167-74.
[http://dx.doi.org/10.1038/mt.2012.225] [PMID: 23281444] ].

While some OVs are being studied for their efficacy as monotherapy, some are under investigation in combination with other therapies such as chemotherapy, radiotherapy, targeted therapy and more recently adoptive T cell therapy. T-vec is under investigation in several clinical studies where its efficacy is being assessed when combined with ipilimumab (CTLA4 inhibitor), and the results of a phase Ib/2 study of T-vec+ipilimumab suggest the enhanced synergistic efficacy with higher complete response and objective response rate [168Puzanov I, Milhem MM, Andtbacka RH, Minor DR, Hamid O, Li A, et al. Primary analysis of a phase 1b multicenter trial to evaluate safety and efficacy of talimogene laherparepvec (T-VEC) and ipilimumab (ipi) in previously untreated, unresected stage IIIB-IV melanoma. J Clin Oncol 2014; 32(15) [PMID: WOS:000358613204315].]. Similar synergistic effects have also been observed when OVs were combined with anti-PD-1 and IL-2 [157Rajani K, Parrish C, Kottke T, et al. Combination therapy with reovirus and anti-pd-1 blockade controls tumor growth through innate and adaptive immune responses. Mol Ther 2016; 24(1): 166-74.
[http://dx.doi.org/10.1038/mt.2015.156] [PMID: 26310630] ]. Adoptive T cell therapy, with priming of T cells against tumor antigens followed by transfusion of the primed T cells in cancer patients, has shown consistent efficacy in clinical trials for melanoma [169Weber JS. At the bedside: adoptive cell therapy for melanoma-clinical development. J Leukoc Biol 2014; 95(6): 875-82.
[http://dx.doi.org/10.1189/jlb.0513293] [PMID: 24732024] ]. Adoptive T cell therapy is also being investigated as combination therapy along with OVs. It has been shown that oncolytic adenovirus expressing IFN-γ combined with adoptive T cell therapy resulted in overall objective response of 38.5% in melanoma patients [170Khammari A, Nguyen JM, Saint-Jean M, et al. Adoptive T cell therapy combined with intralesional administrations of TG1042 (adenovirus expressing interferon-γ) in metastatic melanoma patients. Cancer Immunol Immunother 2015; 64(7): 805-15.
[http://dx.doi.org/10.1007/s00262-015-1691-7] [PMID: 25846669] ].

Although combination therapies have shown significant improvement and encouraging results, careful attention is still paid to the clinical trial designs, timing and dosing regimens, biosafety and biodistribution. For example, when combining the OVs and immunotherapy such as anti-PD-1, the timing of immunotherapy administration would be critical to avoid augmenting the anti-viral immune response or any earlier clearance of the virus. Rajani et al. [157Rajani K, Parrish C, Kottke T, et al. Combination therapy with reovirus and anti-pd-1 blockade controls tumor growth through innate and adaptive immune responses. Mol Ther 2016; 24(1): 166-74.
[http://dx.doi.org/10.1038/mt.2015.156] [PMID: 26310630] ] have demonstrated that administration of anti-PD-1 7 days after the intratumoral injection of virus is optimal to maximize the virus spread within the tumors. When combining OVs with chemotherapies such as CPA, CPA is able to eliminate the neutralizing antibodies against the virus and allow the virus to cause toxicities in systemic organs [153Qiao J, Wang H, Kottke T, et al. Cyclophosphamide facilitates antitumor efficacy against subcutaneous tumors following intravenous delivery of reovirus. Clin Cancer Res 2008; 14(1): 259-69.
[http://dx.doi.org/10.1158/1078-0432.CCR-07-1510] [PMID: 18172278] ]. Therefore, it is essential to critically supervise the toxicity and the real-time levels of neutralizing antibodies against the virus, and carefully make dose-escalation decisions. For the combinational therapy of OVs and immunotherapy, it has been proposed to additionally combine with the systemic treatment such as T-reg depletion, to exert even higher synergistic efficacy [125Dias JD, Hemminki O, Diaconu I, et al. Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4. Gene Ther 2012; 19(10): 988-98.
[http://dx.doi.org/10.1038/gt.2011.176] [PMID: 22071969] ]. However, one should be cautious to avoid autoimmune reactions when combining T cell enhancement with T-reg depletion, as immunotherapy such as anti-CTLA-4 treatment is already closely related to decreased number of myeloid derived suppressor cells [171Martens A, Wistuba-Hamprecht K, Geukes Foppen M, et al. Baseline Peripheral Blood Biomarkers Associated with Clinical Outcome of Advanced Melanoma Patients Treated with Ipilimumab. Clin Cancer Res 2016; 22(12): 2908-18.
[http://dx.doi.org/10.1158/1078-0432.CCR-15-2412] [PMID: 26787752] ].

Although it is reasonable to expect sufficient efficacy exerted by a one-shot curative agent, the immune “barriers” such as virus neutralization and sequestration make it more realistic to attain the optimal therapeutic effect with serial OVs for priming the antitumor immune responses. Strategies have been developed to prime the antitumor responses with one viral vector and boost with another, in order to minimize the anti-viral immune responses [172Irvine KR, Chamberlain RS, Shulman EP, Surman DR, Rosenberg SA, Restifo NP. Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors. J Natl Cancer Inst 1997; 89(21): 1595-601.
[http://dx.doi.org/10.1093/jnci/89.21.1595] [PMID: 9362157] , 173Marshall JL, Hoyer RJ, Toomey MA, et al. Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses. J Clin Oncol 2000; 18(23): 3964-73.
[PMID: 11099326] ].

In summary, this review describes OVs and their mechanisms in melanoma tumor regression. We also discuss different combinational therapies, which overcome the limitations associated with each therapeutic agent such as chemo-, radio- and targeted- therapies. Nonetheless, the quest for identifying new melanoma biomarkers that can channelize efforts for developing newer targeted therapies for melanoma should be continued.