They also describe various normal cellular signalling pathways that are altered in cancer cells and can be portrayed as overlapping and interacting integrated circuits or pathways, supporting various functions including, but not limited to, motility, viability, cell stasis, differentiation and proliferation [4]. These include the Ras-Raf-MEK-ERK signalling pathway, hedgehog signalling pathway, canonical and noncanonical Wnt signalling and the P13K/AKT MTOR pathway [5–8]. Further PTEN and AKT can act as a powerful switch between oncogenes and tumour suppressor genes [9]. FGF also plays a role in influencing signalling pathways [10].

Additional factors influence the hallmarks of cancer, namely, genetic and epigenetic instability and tumour-induced or tumour-associated inflammation. Cancer cells may have increased susceptibility to local mutagens, and there may be reduced efficiency of tumour suppressors such as TP53. Genetic mechanisms include alterations to DNA such as mutation, deletion, translocation, insertion, and aneuploidy [11]. Cell cycle can be influenced by a number of genes including Rb, p16INK4a, p15INK4b, 14–3-3, cyclin D2, cyclin E and p14ARF. Signal transduction is affected by ErbB2, RASSF1, LKB1/STK11 and APC. Apoptosis has many modifiers, but two genes involved include death-associated protein kinase gene (DAPK) and caspase-8lk. MGMT, MLH1, BRCA1, and FNACF all impact upon DNA repair. Telomerase activity via TERT and TERC impinges upon senescence [12]. The hedgehog pathway also influences these cancer signalling pathways [13].

Epigenetic mechanisms influence every stage of tumorigenesis and include those changes in gene function that are mitotically or meiotically heritable but are not associated with a change in DNA sequence [11]. Common epigenetic mechanisms include chromatin changes, histone modification, and DNA methylation [12]. Histone modifications are often covalent changes that result in switching the target gene off. There can also be microRNA changes. These small non-coding RNAs have an epigenetic role and can be oncogenic, tumour suppressor or context dependant. DNA contains the genetic code, but RNA acts as the messenger via transcription to express phenotype. Altered transcription leads to many human cancers and genetic disorders. The phenotypic expression of genes can be modified profoundly by epigenetic factors such as chromatin remodelling and histone modifications. Interestingly cancer cells usually have up to 10 genetic changes but between 100 and 1000 epigenetic modifications [11]. The epigenetic changes are important in tumour initiation and subsequent progression.

The interaction between tumour cells and the tumour microenvironment is critical. Tumour cells can elaborate tumour-associated antigens (TAA) which can assist in evasion of host immune responses. Various mesenchymal stem cells (MSCs) , regulatory T cells (Tregs), macrophages and stromal fibroblasts interact and elaborate cytokines and other proteins facilitating tumour progression. Some of the normal immune cells, natural killers (NKs), dendritic cells and cytotoxic T lymphocytes are downregulated to allow tumour growth [14].

These complex genetic, epigenetic and proteomic changes in individual tumours are being assessed not just in the research laboratory but also in the clinical setting. Next generation sequencing has permitted this and will become increasingly available over the next decade. For this huge amount of data to be processed, the discipline of bioinformatics has burgeoned. This analysis allows testing signals be converted to data which is analysed for interpretation to allow therapeutic decisions [15]. Bioinformatics involve primary, secondary and tertiary analysis of data, which requires documentation and validation. Genomic analysis pipelines can allow comparison of an individual’s genome to human reference sequences. NGS has been applied to a number of pediatric tumours that can occur in the orbit, most notably, rhabdomyosarcoma (RMS) [16, 17].

The types of cancer seen in the pediatric orbit are very different from those seen in adult practice, where carcinomas and lymphomas predominate. Rarely, carcinomas of the lacrimal gland arise in children, including adenoid cystic carcinoma. The pediatric population more commonly manifests tumours which present early and are possibly the result of disordered organogenesis. Tumours of uncertain origin occur and often fall into the undifferentiated or poorly differentiated sarcoma group, i.e. small round blue cell tumours [18]. With regard to rhabdomyosarcoma (RMS) , it often occurs in areas largely devoid of skeletal muscle. RMS can be subdivided into alveolar, embryonal and pleomorphic types. Histogenically, it is presumed to derive from primitive mesenchymal cells. Mouse studies suggest that alveolar RMS may derive from myofibres, or late myoblasts. Embryonal RMS may arise from a differentiating myoblast [18].

ARMS usually carries specific chromosomal translocations that result in PAX3– or PAX7–FOXO1 fusion genes, with fusion positive lesions having a poorer prognosis. ERMS commonly harbours loss of heterozygosity at 11p15.5 and gains of chromosomes 2, 8, and 12 in varying combinations3,4 [17]. Because of the relatively small number of genetic mutations in RMS , epigenetic factors are presumed to play a significant role. Recently, molecular cytogenetic alterations in RMS have been identified by array comparative genomic hybridization (aCGH). All RMS showed specific gains and losses. 12q13.12, 12q13.3 and 12q13.3-q14.1 were amplified. 1p21.1, 2q14.1, 5q13.2, 9p12, and 9q12 showed deletions. The mRNA levels of Gli1 and GEFT were elevated. Bioinformatic analysis showed that genes were enriched in functions such as immunoglobulin domain, induction of apoptosis, and defensin. This may allow the development of biomarkers and drug targets for RMS [17, 19]. Further whole-exome/whole-transcriptome sequencing, copy number (CN) and DNA methylome analyses have shed light on the genetic/epigenetic basis of RMS [16].

Looking at DNA methylation patterns, four distinct groups of RMS could be identified, which are in concordance with histological phenotype, mutation and copy number profiles and clinical course. A1and A2 subtypes correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. E1 and E2 subtypes, or largely embryonal histology, are characterized by a high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation. E2 was also characterized by p53 inactivation, which again correlated with poor outcome. The strong association with methylation status emphasizes the importance of epigenetic phenomena in RMS [16]. Another study identified differential transcriptional regulation of PAX3:FOXO1, suggesting that a histone deacetylase inhibitor might be able to convert fusion-positive lesions into a state similar to fusion-negative RMS, with a corresponding improved prognosis [20].

Ewing’s sarcoma and PNET are similar primitive neuroectodermal tumours which show CD99 positivity on immunohistochemistry, with a characteristic genetic translocation t(11:22)(q24q12). These tumours can arise in bones with intramembranous ossification, possibly from cells with neuroepithelial or mesenchymal lineage, or possibly both. Beyond the genetic changes, Ewing’s demonstrates epigenetic dysregulation. EWSR1-Fli1 is an oncogenic transcription factor with a myriad of downstream target genes that drive Ewing’s sarcoma. Histones may also be altered and chromatin remodelling conformationally allows access to DNA for tumour progression [18]. Targeting EWS/FLI with a range of different agents is currently under investigation in clinical trials [21]. Localized disease has a reasonable prognosis with 65–75% 5-year survival rates, but up to a quarter of patients have metastatic disease at presentation, with a much poorer prognosis [21].

Histiocytic lesions are relatively common in pediatric orbital practice and include Langerhans’ cell histiocytosis (LCH), predominately and less commonly juvenile xanthogranuloma (JXG) , Rosai-Dorfman disease (RDD) and Erdheim-Chester disease (ECD) [22, 23]. LCH is known to be a clonal proliferation of myeloid derived cells. The non-LCH is thought to derive from monocytic or macrophage origin. These histiocytic lesions demonstrate BRAF V600E mutations resulting in activation of MAPK and or PI3K-AKT pathways. ARAF mutations are also seen in LCH and some JXG cases. RAF inhibitors have shown significant response in BRAF V600E histiocytoses, and MEK inhibitors have also been used with fewer results available [24, 25].

Neurofibromatosis 1 results from mutations or deletions of the neurofibromin gene, located at chromosome 17p11.2 [26]. Normally the gene product downregulates RAS protein. When neurofibromin is reduced, RAS GTP causes general cell growth and predispose to malignant change. While many of the ocular manifestations of NF1 are in the realm of the pediatric ophthalmologist, the orbital surgeon will encounter both plexiform neurofibromas and optic nerve or anterior visual pathway gliomas. Plexiform neurofibromas have been associated with progression to malignant peripheral nerve sheath tumours, accompanied by additional genetic and epigenetic events, including changes to TP53, Rb gene, PTEN and CDKN2A [27]. Plexiform neurofibromas can be enormously disfiguring and troublesome for affected individuals, with many lesions inoperable because of impingement on vital structures in the skull base. The RAF/MEK/ERK pathway is the key driver for sustained growth in these lesions [28, 29]. MEK inhibitors trametinib and selumetinib have shown promise in reducing lesion size [30] (Fig. 17.1). It is likely that long-term treatment will be required to prevent rebound enlargement.