**6. Conclusions**

This chapter introduces an environmental impact assessment framework for denim fabrics. This framework provides an opportunity to calculate impacts of product developers' and/or designers' choices in defining the denim fabric they would like to develop using scientific LCA data. One can even compare the impact results of different types of denim fabrics without even producing the fabric itself. Calculating and sharing this detailed science-based data also represents a new level of transparency. On the contrary to the common belief of raw materials being the main impact generators in denim fabrics, the framework also proves that impacts occurred during denim fabric manufacturing, during the production of the raw and auxiliary materials, and impacts of the background processes should all be taken into consideration.

There are challenges in LCA calculations since the primary data is highly company-specific. Therefore, the chapter focused on normalizing the data and creating a scaling that can be used in decision-making.

Greenwashing is not only denims but today's one of the growing problems in every sector, in every product. Baseless claims and marketing statement caused this problem and now it is really hard to clean it up. Certifications and labels were seen as one of the solutions to this problem however, industry experiences misapplications during *Life-Cycle Assessment as a Next Level of Transparency in Denim Manufacturing DOI: http://dx.doi.org/10.5772/intechopen.110763*

the certification process in the last couple of years. There is an information pollution in the market on what is sustainable denim. This framework will help designers/ companies compare their choices about their sustainable denim fabric definitions via science-based data.


**4. Antioxidant effects of maca as a feed supplement in animal nutrition**

Most scientific research has worked with laboratory animals to know the antioxidant effects of maca. Rarely, studies on farm animals have been published in recent years. In these studies, daily doses (mg /kg BW/d) of maca or its bioactive content were calculated on their body weights (**Table 5**). In order to demonstrate antioxidant effects, laboratory animals, they are exposed to exercise-induce stress and antioxidant enzyme levels of their serum and various organs (brain, liver, muscle etc.) and exercise performance is measured [31, 42]. In farm animal nutrition, the criteria such as feed efficiency, nutrition performance, viability were recorded besides antioxidant enzyme activities. Dried, milled powder form of maca is mostly used in animal experiments.

When used in rats and mice at various doses of maca or its antioxidant compounds, some effects are occurred against the stress factors. In particular, GPx, SOD and glutathione (GSH) levels in serum, liver and brain increase, MDA and ROS (reactive oxygen species) decrease [45, 46]. Choi et al. [32] determined that the lipid soluble extract of maca contained 7.8 mg/g DM of macamide and macaene while maca powder contained at the level of 0.3–0.4 mg/g DM. When this lipid soluble extract is given at 100 mg/kg BW per day, it reduces lipid peroxidation in muscles of rats, increases GSH and exercise duration (**Table 5**) [32]. Similarly macamides (N-benzyllinoleamide, N-benzyloleamide, N-benzyloleamide), isolated from maca, reduce the oxidative stress induced by exercise and eliminate the waste products in serum [31]. But Qui et al. [29] argue that maca improves the antioxidant enzyme (CAT, SOD, GPx) activity both in blood and liver independently of macamide and macaene, and there is no correlation between antioxidant effect and these bioactive compounds. When rats are daily fed with maca polysaccharides at between 20 and 100 mg/kg BW doses, serum LA, BUN and MDA are decreased, GPx and creatine kinase are increased, especially in high dose (100 mg/ kg BW) [34, 43].

laying hens fed dry maca powder at the rate of 0.5 and 1% (w/w) no effect on nutritional performance, serum parameters and reproductive hormones was determined. But serum GPx level increased depend on the ratio of maca supplementation in diet of hens [51]. Day by day, the antioxidant effect of maca as feed additive in laboratory studies has been clarifying, but

**Species Ecotype Form Dose Unit Effect Reference**

and survival, decreasing magnesium (not significantly)

Antioxidants in Maca (*Lepidium meyenii*) as a Supplement in Nutrition

http://dx.doi.org/10.5772/intechopen.75582

decreasing magnesium

decreasing magnesium

mg/kg/d Antioxidant activity [42]

30–100 mg/kg/d Antioxidant and antifatigue activities

(significantly)

mg/kg/d Antioxidant activity [45]

mg/kg/d Antioxidant activity [35]

100 mg/kg/d Antioxidant activity

[48]

147

[51]

[49]

[32]

[29]

Fish NA Fresh Hypocotyl 5–10–15 % of feed Improving growth rate

Poultry NA Powder 0.5–1 % of feed Antioxidant activity,

Rat NA Aqueous 50–100–

extract

extract

extract

**Table 5.** Antioxidant effects of maca and its compounds in animal nutirion.

Mice NA Polysaccharide 25–50–

Rat Yellow Lipid soluble

Rat Black Petroleum ether

Mice NA Petroleum ether

Horse NA Powder 50–75 gr/day Increasing AST and GGT,

Horse Yellow Powder 20 mg/d Effects on sperm quality [47]

Rat NA Powder 1 % of feed Antioxidant activity [52]

Mice NA Powder 500–1000 mg/kg/d Antioxidant activity [46] Mice NA Macamide 40–12 mg/kg/d Antioxidant activity [31] Mice Yellow Polysaccharide 20–100 mg/kg/d Antifatigue activity [34]

> 125– 250–500

100

Yellow Antioxidant activity (slightly) Purple Antioxidant activity (slightly)

200

Maca is being both at the meals of the indigenous people and exported to the whole world. Consumers around the world are taking it as a food supplement for improving their sexual and sportive activities and energy. So researchers have similarly given priority to these issues.

not yet on the other species and more nutrition experiment is needed.

However, studies on antioxidant status of human are limited [54].

**nutrition**

**5. Antioxidant effects of maca as a food supplement in human**

In other animal species, there are effects on the criteria such as sperm quality, survival, feed conversion, nutritional performance as well as antioxidant effects of maca as a feed additive [47, 48]. When there is no effect on blood parameters in horses, Aspartate transaminase (AST) and gamma-glutamyl transpeptidase (GGT) increase [49]. In fish fed the fresh hypocotyl of maca, nutritional performance and feed conversion and viability enhanced [48, 50]. While

**Table 5.** Antioxidant effects of maca and its compounds in animal nutirion.

Li et al. [41] have reported that MP has occurred a dose depend antioxidant activity by increasing SOD, GPx and CAT enzymes and decreasing MDA in liver of mice. Also, He et al. [42] have reported that antioxidant activity with a correlation between the doses and enzymes levels in muscle against the exercise-induced oxidative stress. Similar to animal experiments, MP plays the crucial roles of antioxidant and free radical scavenger in cell cultures (**Table 4**) [39, 43].

In brief, the mechanism of antifatigue and antioxidant effects of especially aqueous polysaccharides in maca originates from improving hypoxia tolerance, eliminating metabolic wastes, serving energy source with high glucose contents and reducing oxidative damage by enhanc-

Most scientific research has worked with laboratory animals to know the antioxidant effects of maca. Rarely, studies on farm animals have been published in recent years. In these studies, daily doses (mg /kg BW/d) of maca or its bioactive content were calculated on their body weights (**Table 5**). In order to demonstrate antioxidant effects, laboratory animals, they are exposed to exercise-induce stress and antioxidant enzyme levels of their serum and various organs (brain, liver, muscle etc.) and exercise performance is measured [31, 42]. In farm animal nutrition, the criteria such as feed efficiency, nutrition performance, viability were recorded besides antioxidant enzyme activities. Dried, milled powder form of maca is mostly

When used in rats and mice at various doses of maca or its antioxidant compounds, some effects are occurred against the stress factors. In particular, GPx, SOD and glutathione (GSH) levels in serum, liver and brain increase, MDA and ROS (reactive oxygen species) decrease [45, 46]. Choi et al. [32] determined that the lipid soluble extract of maca contained 7.8 mg/g DM of macamide and macaene while maca powder contained at the level of 0.3–0.4 mg/g DM. When this lipid soluble extract is given at 100 mg/kg BW per day, it reduces lipid peroxidation in muscles of rats, increases GSH and exercise duration (**Table 5**) [32]. Similarly macamides (N-benzyllinoleamide, N-benzyloleamide, N-benzyloleamide), isolated from maca, reduce the oxidative stress induced by exercise and eliminate the waste products in serum [31]. But Qui et al. [29] argue that maca improves the antioxidant enzyme (CAT, SOD, GPx) activity both in blood and liver independently of macamide and macaene, and there is no correlation between antioxidant effect and these bioactive compounds. When rats are daily fed with maca polysaccharides at between 20 and 100 mg/kg BW doses, serum LA, BUN and MDA are decreased, GPx and creatine kinase are increased, especially in high dose (100 mg/

In other animal species, there are effects on the criteria such as sperm quality, survival, feed conversion, nutritional performance as well as antioxidant effects of maca as a feed additive [47, 48]. When there is no effect on blood parameters in horses, Aspartate transaminase (AST) and gamma-glutamyl transpeptidase (GGT) increase [49]. In fish fed the fresh hypocotyl of maca, nutritional performance and feed conversion and viability enhanced [48, 50]. While

**4. Antioxidant effects of maca as a feed supplement in animal**

ing antioxidant enzyme [38, 41].

146 Antioxidants in Foods and Its Applications

used in animal experiments.

kg BW) [34, 43].

**nutrition**

laying hens fed dry maca powder at the rate of 0.5 and 1% (w/w) no effect on nutritional performance, serum parameters and reproductive hormones was determined. But serum GPx level increased depend on the ratio of maca supplementation in diet of hens [51]. Day by day, the antioxidant effect of maca as feed additive in laboratory studies has been clarifying, but not yet on the other species and more nutrition experiment is needed.


**From Genes to -Omics: The Evolving Molecular Landscape of Malignant Peripheral Nerve Sheath Tumor**

#### **Kathryn M. Lemberg 1,2, Jiawan Wang 1,2 and Christine A. Pratilas 1,2,\***

Received: 15 May 2020; Accepted: 17 June 2020; Published: 24 June 2020

**Abstract:** Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive soft tissue sarcomas that occur with significantly increased incidence in people with the neuro-genetic syndrome neurofibromatosis type I (NF1). These complex karyotype sarcomas are often difficult to resect completely due to the involvement of neurovascular bundles, and are relatively chemotherapyand radiation-insensitive. The lifetime risk of developing MPNST in the NF1 population has led to great efforts to characterize the genetic changes that drive the development of these tumors and identify mutations that may be used for diagnostic or therapeutic purposes. Advancements in genetic sequencing and genomic technologies have greatly enhanced researchers' abilities to broadly and deeply investigate aberrations in human MPNST genomes. Here, we review genetic sequencing efforts in human MPNST samples over the past three decades. Particularly for NF1-associated MPNST, these overall sequencing efforts have converged on a set of four common genetic changes that occur in most MPNST, including mutations in neurofibromin 1 (*NF1*), *CDKN2A*, *TP53*, and members of the polycomb repressor complex 2 (PRC2). However, broader genomic studies have also identified recurrent but less prevalent genetic variants in human MPNST that also contribute to the molecular landscape of MPNST and may inform further research. Future studies to further define the molecular landscape of human MPNST should focus on collaborative efforts across multiple institutions in order to maximize information gathered from large numbers of well-annotated MPNST patient samples, both in the NF1 and the sporadic MPNST populations.

**Keywords:** MPNST; NF1; genomics

#### **1. Clinical Overview of MPNST**

MPNST are aggressive soft tissue sarcomas originating from Schwann cells in the peripheral nervous system [1,2]. Half of MPNST occur in patients with the cancer predisposition syndrome NF1, caused by germline loss of function (LOF) of one copy of the tumor suppressor gene *NF1*. In patients with NF1, most MPNST arise from within plexiform neurofibromas (pNF), which are pre-malignant tumors of the peripheral nerve [3–5]. pNF can, themselves, be a major source of disfigurement or dysfunction. MPNST can also occur sporadically or following radiation treatment in the general population, although the incidence of the latter is substantially lower. MPNST carry a high risk of sarcoma-specific death; in the absence of complete surgical resection with wide negative margins, the five-year event-free survival is ~30% [6,7]. Conventional chemotherapy and radiation often do not improve patient outcomes [8].

#### **2. Germline Loss of NF1: Correlations with NF1 Phenotype**

NF1 is one of the most common monogenic inherited syndromes with an incidence of approximately 1:3000 live births [9]. This neurocutaneous syndrome is characterized by several hallmark skin findings (café au lait macules, axillary freckling, cutaneous neurofibromas), may involve additional organ systems (including CNS, musculoskeletal, and vascular manifestations) [10], and predisposes patients to an increased risk of malignancy, with an estimated lifetime cancer risk ~60% [11]. One of the hallmark lesions in NF1 patients is the pNF, a complex lesion that grows along major nerve bundles. While benign, pNF can result in significant anatomic, functional, cosmetic, and psychological effects [12]. In patients with NF1, MPNST may arise within existing pNF and are often accompanied by rapid growth, increased pain, or other nervous system deficits. Studies correlating the pathologic changes and genetic alterations in the peripheral nerves of NF1 patients or model organisms, along the spectrum from healthy to pNF to atypical neurofibroma (ANF) to MPNST, have aided in understanding the roles of specific genetic mutations in MPNST tumorigenesis [13].

NF1 syndrome is characterized by a wide variation in phenotypic expression which partially reflects the large number of mutations in the *NF1* gene that have been identified in people with the condition [14–16]. The *NF1* gene was originally cloned nearly three decades ago [17]. It is a large gene, approximately 350 kb in length, located on human chromosome 17q11.2. There may be multiple splice variants [18] but the primary gene product codes for the NF1 protein of 2818 amino acids, which acts as a GTPase-activating protein (GAP) for RAS oncogenes [19–22]. Loss of *NF1*, therefore, leads to constitutive activation of RAS signaling [23,24] (Figure 1), likely accounting for the pro-tumor phenotype observed in patients with NF1 [25].

Historically, the diagnosis of neurofibromatosis was based on clinical symptoms and physical findings, without a requirement for clinical genetic testing [26]. With the advances in detailed sequencing e fforts, however, disruption of a copy of *NF1* in the germline may be identified in the majority of patients with NF1 [27]. Mutational analysis demonstrates a very high rate of mutations occurring in *NF1*, as evidenced by the fact that approximately 50% of cases of NF1 appear to be *de novo*. To date, hundreds of mutations associated with the syndrome have been characterized [9,14]. Identification of the specific LOF mutation in patients can be helpful for testing family members, particularly o ffspring of those a ffected, and for counseling patients about syndrome-specific risks.

Genotype–phenotype correlations associated with specific germline *NF1* alterations have been observed in a limited number of cases. Two examples are associated with limited risk forMPNST. A small in-frame deletion (c.2970\_2972del(p.Met992del)) leading to loss of a methionine in the cysteine-serine rich domain (CSRD) of *NF1* is associated with suppression of cutaneous neurofibroma (cNF) and clinically apparent pNF formation, though these individuals have an increased risk for learning disabilities (48%) and brain tumors (~5%) [28,29]. Several missense mutations a ffecting arginine 1809 (e.g., p.Arg1809Cys) have also been characterized in multiple unrelated families. These patients have a high prevalence of developmental delay and learning disabilities as well as short stature and pulmonic stenosis, but few cutaneous or plexiform neurofibromas, and low risk of malignancy [30].

By contrast, two other *NF1* genotypes have been strongly associated with a higher risk of MPNST. Microdeletion of a 1.4 Mbp segment of chromosome 17 due to homologous recombination within duplication regions of the chromosome leads to deletion of 14 functional genes [31,32]. Individuals with the microdeletion syndrome (approximately 5% of NF1 cases) tend to present with a more severe NF1 phenotype [33], including dysmorphic features, developmental delay, intellectual disability, increased number of neurofibromas, and a two-fold higher lifetime risk for MPNST (16–26%, compared to approximately 8–13% risk in the general NF1 population) [34,35]. Missense mutations in NF1 protein codons 844–848 (including Leu844, Cys845, Ala846, Leu847, and Gly848; located in the CSRD) occur in ~0.8% of studied NF1 cases and are also reported as a risk factor for severe phenotypic presentation. These patients have higher numbers of clinically apparent major pNF, symptomatic spinal neurofibromas, optic pathway gliomas, and skeletal abnormalities, and up to 10% develop malignancy, including MPNST [36].

**Figure 1.** Signaling pathways altered due to genetic changes observed in malignant peripheral nerve sheath tumors (MPNST). The most common alterations in MPNST are loss of function of multiple tumor suppressors including NF1, p16/CDKN2A, TP53, and SUZ12/EED. Loss of *NF1*, as well as epigenetic changes due to loss of PRC2 components, leads to increased signaling through the RAS/RAF/MEK and PI3K/AKT pathways. Additional molecular events observed in subsets of MPNST include mutations in *BRAF*, amplification of *EGFR* or *MET* receptor tyrosine kinases (RTKs), and changes to chromatin structure through mutations in alpha thalassemia/mental retardation syndrome X (ATRX) and other epigenetic modifiers. EGF/EGFR = epidermal growth factor/receptor; PDGF/PDGFR = platelet derived growth factor/receptor; HGF = hepatocyte growth factor; ERK = extracellular signal regulated kinase; CDK = cyclin dependent kinase; RB = retinoblastoma; TF = transcription factor; ER = endoplasmic reticulum.

#### **3. Sequencing E**ff**orts in Human MPNST Samples: Improvements in Technology with Variability in Study Design**

A collated summary of human MPNST sequencing efforts over the past two decades is shown in Table 1. MPNST have complex karyotypes with multiple chromosomal losses and gains and structural anomalies; a single recurrent translocation for diagnostic purposes has not been defined for MPNST as it has for some other mesenchymal tumors [37]. Expanded knowledge of MPNST gene alterations originated in the era of targeted gene evaluation using sequencing specific to the *NF1* locus or a small number of related genes. More recent studies have employed whole exome, whole genome, or targeted next-generation sequencing (NGS) on discovery cohorts for MPNST, with follow up studies performed by targeted gene sequencing in validation cohorts. Whole exome sequencing (WES) efforts have also been performed on patient tumors with paired neurofibroma or blood samples in a minority of cases. Individual studies vary with respect to how much additional clinical information is available (e.g., clinical background, treatment effect, comparison to neurofibroma or blood leukocytes). Some studies include sporadic and radiation-associated cases, while others focus purely on NF1-associated MPNST. In addition, in several studies multiple MPNST samples are derived from the same patient or fragments of the same tumor. These differences in study design, sample collection and annotation, and data analysis likely account for some of the differences and depth of discovery in genomic alterations across the literature. Taken together, however, a clear picture emerges of several characteristic alterations (i.e., *CDKN2A*, genes encoding PRC2 components) involved in evolution of benign nerve sheath tumor to MPNST. Less frequent alterations (i.e., *BRAF*, *MET*) identified in smaller subsets also merit additional attention in follow up evaluations, particularly as new diagnostic and treatment strategies for these tumors are being developed.

**Table 1.** Genomic sequencing studies for most common genetic alterations in human MPNST. All MPNST or neurofibromas in study reported under *n*. Reported

sequencing; CNV = copy number variation; NR = not reported; ND = not determined; cNF = cutaneous neurofibroma; pNF = plexiform neurofibroma; ANF = atypical neurofibroma.

**Table 1.** *Cont.*

109

#### **4. Somatic NF1 Mutations in Tumors Including MPNST**

Consistent with its role as a classical tumor suppressor gene, loss of heterozygosity (LOH) or "second-hit" somatic mutations in the inherited wild-type *NF1* allele have been detected in a variety of tumors in patients with NF1, including pheochromocytomas [50], breast cancer [51], and hematologic malignancies [52]. Somatic LOH analysis using PCR markers performed on the *NF1* locus in dermal neurofibromas identified deletions in a subset of tumors in several early studies [53,54]; those cases known to be familial were analyzed further and shown to have deletions in the non-germline allele, demonstrating that somatic inactivation of *NF1* occurs in these benign lesions. Several studies have compared germline and somatic *NF1* mutations in MPNST. In a single study which investigated 34 MPNST from 27 NF1 patients, germline mutations were identified by lymphocyte DNA in 22 cases—these included one large 1.4 Mbp genomic deletion, one two-exon deletion, and smaller mutations (missense, nonsense, frameshift, and splicing anomalies) in the remainder [55]. In the same cohort, somatic *NF1* mutations were identified in 31 out of 34 MPNST samples—of these, 28 (91% tumors) were large genomic deletions that partially or entirely deleted the *NF1* gene. The authors speculate that in some cases somatic *NF1* mutations arise upon aberrant intrachromosomal recombination of the *NF1* gene during mitosis. Similarly, another report screened 47 MPNST from patients with or without NF1 syndrome (*n* = 25 and 22 cases, respectively). Of the somatic *NF1* mutations identified (*n* = 10/25 NF1-associated and 9/22 sporadic), approximately 55–60% involved large genomic copy number changes (i.e., deletions) in both NF1 and sporadic MPNST [32]. By contrast, in MPNST analyzed from NF1 patients with the 1.4 Mbp germline *NF1* microdeletion, the *NF1* somatic hit is typically a small (e.g., missense) mutation [31]. Interestingly, in a single patient with clinical NF1 syndrome who developed asynchronous cNF, a primary breast tumor, and later gluteal MPNST, WES revealed three distinct *NF1* somatic mutations compared to the germline mutation noted in the blood [51].

#### **5. Acquired Mutations during Transformation from pNF**

#### *5.1. Loss of CDKN2A*/*B: Correlations with the pNF to ANF Transition*

*NF1* LOH is considered to be an initiating event in pNF formation as confirmed in several animal models [56]. Several additional mutations are necessary for malignant transformation. ANF (now re-classified as atypical neurofibromatous neoplasms of uncertain biological potential, ANNUBP) are precursor lesions to NF1-associated MPNST, representing an intermediate step from the malignant transformation of pNF into MPNST [57–59]. Alterations to chromosome 9q have been observed in a high proportion of ANF and MPNST [48,60]; one study noted deletion at 9p21.3, identified in 94% (15/16) of ANF and in 70% (16/23) of high-grade MPNST but not in pNF [57]. This locus encompasses several candidate tumor suppressors, including *CDKN2A*/*B*. *CDKN2A* encodes two gene products each the result of di fferential splicing: p16ink4a (a negative regulator of CDK4 and CDK6 cyclin dependent kinases) and p19Arf, a negative regulator of the *TP53* E3 ligase MDM2. Several early studies on human NF1-associated MPNST specimens identified deletions within the short arm of chromosome 9, in the region of *CDKN2A*, as well as low expression of p19, while these were not detected in neurofibroma samples [61,62]. A more recent study identified frequent somatic deletions of *CDKN2A*/*B* (69%) and *SMARCA2* (42%), apart from recurrent *NF1* somatic mutations (81%), in 16 ANF [48]. These studies indicate that *CDKN2A*/*B* deletion is the first step in the progression of pNF toward ANF and eventually MPNST.

#### *5.2. LOH and Mutation in the Tumor Suppressor TP53: Not Universal in Human MPNST*

Copy number variation and mutations in the tumor suppressor gene *TP53* have been identified in some cases of NF1-associated MPNST. Early studies on small subsets of NF1-associated neurofibrosarcomas identified deletions on chromosome 17 outside of the *NF1* locus [63,64], which included the coding region for *TP53*. Screening for *TP53* inactivation in a panel of 20 MPNST identified LOH in over half of the tumors tested [55]. The first genetically-engineered mouse (GEM) model for MPNST made use of LOH of both *NF1* and *TP53* from mouse chromosome 11 as the tumor initiating event [65]. Numerous subsequent studies have focused on identifying the true incidence of *TP53* mutation in human MPNST; from compiled data on 25 studies including 114 MPNST (both NF1- associated and sporadic), *TP53* mutations were observed in 14% of MPNST, with LOH in 39% of cases (Table 1) [39]. WES of NF1 tumor samples from a single patient with pNF, MPNST, and metastatic sites also identified loss of one copy of *TP53* in the MPNST and metastatic lesion, but not the primary pNF [66]. Genetic changes in *TP53* are thus present in some MPNST but not necessary for all cases of pNF malignant transformation.

#### *5.3. Loss of PRC2 or H3K27me3: Recurrently and Specifically Occurs in MPNST*

Components of the epigenetic regulatory PRC2are recurrently and specifically inactivated in MPNST (Table 1). Chi and colleagues identified genomic alterations in *EED* (37%, or 19/52) and *SUZ12* (48% or 25/52) in MPNST, alongside frequent somatic alterations in *CDKN2A* (81%, 42/52) and *NF1* (87%, 45/52) [43]. Bettegowda and colleagues simultaneously reported PRC2 loss via *EED* (2%, 1/50) and *SUZ12* (32%, 16/50) mutations in 50 MPNST [42]. De Raedt et al. similarly reported alterations in *EED* in 29% (15/51) and *SUZ12* in 63% (32/51) of NF1-associated MPNST [41]. PRC2-component loss in MPNST is associated with complete loss of histone H3 trimethylation at lysine 27 (H3K27me3) and increased level of H3K27 acetylation (H3K27Ac), which can serve as biomarkers to improve upon the accuracy of the diagnosis of MPNST [41,43]. *SUZ12* loss potentiates the effects of NF1 loss by amplifying RAS-driven transcription through effects on chromatin that triggers an epigenetic switch [41]. Further detail on the role and function of PRC2 elements in MPNST is found in the review article by Zhang et al. dedicated to this topic, also included in this Special Issue on *Genomics and Models of Nerve Sheath Tumors* [67]. Collectively, the highly recurrent and specific inactivation of PRC2 components, *NF1*, and *CDKN2A*/*B* posits their critical and potentially cooperative roles in MPNST pathogenesis.

#### **6. Less Common Recurrent Variants Identified with Modern Sequencing Investigations of MPNST**

MPNST demonstrate complex genomic imbalances and chromosomal aberrations [58,59]. In addition to the common deletions of tumor suppressor genes *NF1*, *CDKN2A*, *TP53* and LOF in the PRC2 genes *EED* and *SUZ12*, several other recurrent genomic events have been identified in NF1-associated and sporadic MPNST. Significant findings from these studies are highlighted in Table 2 and described below.

**Table 2.** Less frequent genomic alterations identified in MPNST.

**Table 2.** *Cont.*

polymorphism; NR = not reported; ND = not determined.

*Genes* **2020**, *11*, 691

#### *6.1. BRAF Mutation: An Alternate Mechanism for Activation of RAS Signaling*

In addition to loss of *NF1* and PRC2function, *BRAF* mutations are reported as an alternate mechanism for aberrant activation of RAS signaling in MPNST, albeit at a lower frequency (ranging from 0–9.7%) [32,47,69,78,79], and occurring more commonly in sporadic than NF1-associated cases [78]. Strongly activating kinase mutations (BRAF V600E) occurred in five out of ten *BRAF*-mutant *NF1*-wild type MPNST (*n* = 84; Table 2) [47]. *BRAF* amplification has also been described, with a frequency of 31% in another study cohort consisting of 51 MPNST [40]. Brohl et al. suggest that the relative strength of RAS-activating mutations may determine whether *BRAF* and *NF1* mutations (or *NRAS*/*KRAS* and *NF1*) co-occur and thereby serve together to result in ERK signaling hyperactivation [45].

#### *6.2. EGFR, MET and Other Receptor Tyrosine Kinases: Frequent Copy Number Gains in MPNST*

A variety of oncogenic receptor tyrosine kinases (RTK) are frequently altered in MPNST. In MPNST, alterations in RTK usually take the form of amplification, rather than single nucleotide variations that result in constitutively activated kinases (Table 2). Several early aCGH studies revealed amplifications of *HGF*, *MET*, *EGFR*, *PDGFRA*, and *IGF1R* in approximately 25% to 40% of analyzed MPNST [38,40]. These studies and others [71,72,80] suggest a putative role of these genes and their respective biological pathways in the initiation and/or progression of MPNST.

Notably, *HGF* and its receptor *MET*, co-located at chromosome 7q, are highly expressed in a relatively large panel of human MPNST samples, and increased phospho-MET expression level directly correlates with shorter MPNST patient survival [81]. A single patient study revealed progressive amplifications of *HGF*, *MET* and *EGFR* in a patient with MPNST harboring early *NF1* and *TP53* loss, using longitudinal genomic analysis from pNF, to MPNST, to metastatic recurrence. These studies further justify investigation of the role of RTK signaling, in particular HGF/MET, on the progression of MPNST.

## *6.3. AURKA Amplification*

Dramatic upregulation (7.9-fold) of *AURKA* (the gene encoding aurora kinase A) was observed through RAS-driven transcriptome analysis on a GEM model and 14 human MPNST samples compared with normal nerves. Further analysis using SNP-array and qPCR confirmed copy number gains in the *AURKA* locus in eight out of 13 primary MPNST and five out of five MPNST cell lines but not neurofibromas [74]. Reducing the expression and activity of Aurora kinase using shRNA knockdown and a kinase inhibitor MLN8237, respectively, inhibits MPNST cell survival in vitro and in vivo, and supports the role of aurora kinase as a rational therapeutic target for MPNST [82].

#### *6.4. Tyrosine Kinase 2 Overexpression in MPNST*

NGS on a set of seven NF1-associated MPNST identified a predicted pathogenic mutation in tyrosine kinase 2 (*TYK2*) in two out of seven tumors [75]. TYK2 P1104A mutated tumors demonstrated strong immunoreactivity, whereas TYK2 wild type tumors were not immunoreactive. Strong TYK2 expression as assayed by immunohistochemical staining was observed in 63% of MPNST in an independent tissue set, while only 11% of pNF samples stained for TYK2. Ablation of TYK2 expression in human and murine MPNST cells resulted in increased cell death in vitro and decreased tumor growth in a murine model [83]. The example of *TYK2* suggests the role that sequencing e fforts can play in development of novel markers of MPNST biology.

#### *6.5. ATRX Mutation and Evidence for Alternative Lengthening of Telomeres*

In addition to the role of PRC2 in MPNST chromatin regulation, the chromatin regulator ATRX (Alpha Thalassemia/Mental Retardation Syndrome X) has been identified as mutated in a subset of MPNST [75]. Loss of ATRX function is involved in alternative lengthening of telomeres (ALT), a telomerase-independent means of telomere maintenance which prevents tumor cell senescence

and promotes tumorigenesis. Subsequent studies on a larger subset of MPNST identified decreased nuclear expression of ATRX and demonstrated a correlation between aberrant ATRX expression and decreased overall survival in NF1-associated MPNST [84]. In a separate study a small subset (*n* = 3) of NF1-associated MPNST that were ALT-positive were analyzed by NGS and found to have *ATRX* mutations in two out of three cases [76]. While this study did not identify inferior overall survival (OS) for ALT-positive MPNST compared to those with normal telomere length, short telomeres were significantly correlated with improved OS.

#### *6.6. Beyond SUZ12: Less Common Variant Mutations in Other Chromatin Modifying Genes*

In addition to loss of *SUZ12* and *EED*, several studies have demonstrated additional alterations in PRC2 components or associated chromatin modifying genes. Sohier and colleagues detected a novel sequence change in the histone lysine demethylase *KDM2B* by WES (c3376C > T) in one out of eight human MPNST. This change is thought to potentially impact protein function; in an additional set of 14 tumors assayed by qPCR, KDM2B expression was reduced [44]. Whole genome and whole exome sequencing on an additional subset of NF1-associated MPNST identified mutations in additional chromatin associated genes including *CHD4*, *AEBP2*, *EPC1*, and *EZH2*, particularly in tumors with intact *SUZ12* [42].

#### *6.7. Evidence for Alterations in the HIPPO Pathway in a Subset of MPNST and Schwann Cell Derived Tumors*

Several studies have found evidence for alterations in the HIPPO–YAP pathway in MPNST. Analysis of aCGH from 51 MPNST samples [40] revealed an increase in the copy number of HIPPO effector gene loci, including *TAZ*, *CTGF* and *BIRC5* and a loss of HIPPO inhibitory gene loci, such as *LATS2* and *AMOTL2* [77]. In agreement with these findings, transcriptome sequencing of human MPNST samples from two additional patient cohorts revealed elevated YAP-activated gene expression in MPNST relative to normal nerves and NF1-associated neurofibromas [85,86]. Genomic alterations in the HIPPO pathway appear to occur in additional NF1 patient tissues including somatic mutations in seven of 33 cNF described in a recent study and as germline mutations (e.g., missense, frameshift and occasionally insertion) in seven of nine NF1 patients from the same dataset [87]. Together these studies validate the role of HIPPO pathway in neurofibroma biology and as a driver of MPNST tumorigenesis [77,87].

#### **7. Beyond Genomics: The State of Understanding MPNST Transcriptomes, Proteomes, Epigenomes, and Metabolomes**

In addition to the genomic alterations described above, these and other studies on human MPNST have revealed downstream e ffects on MPNST gene product expression and signaling. These investigations have confirmed or supplemented the genomic data by assaying downstream pathway e ffects in human MPNST. Several studies have broadly analyzed gene expression in human MPNST samples using microarray or RNAseq approaches [48,88]; these data can be examined in relation to known genetic changes to generate additional hypotheses for e ffects on downstream signaling pathways. Recent work compared gene expression in multiple functional pathways across pNF, ANF/ANNUBP, and MPNST and found that some ANNUBP share signaling pathway characteristics that more closely resemble pNF (e.g., ERK/MAPK) and others (e.g., AKT/mTOR) are more similar to MPNST [88]. Phospho-proteome arrays may be used to investigate kinase signaling in relation to various genomic alterations or therapeutic interventions in MPNST; to date this has primarily been used in MPNST cell lines or animal models (see article by Grit et al. in this Special Issue on Genomics and Models of Nerve Sheath Tumors) [89]. Methylation analysis on MPNST has revealed overall decreased histone and DNA methylation [90], and has also revealed how methylation changes in MPNST can a ffect expression of other tumor suppressor genes (e.g., *PTEN*) in MPNST [91]. Parallel methylation analysis and proteomic analysis on a set of nine MPNST samples characterized the relationship between PRC2 LOF on histone and DNA modification and consequent gene product expression. This work found that PRC2 loss was associated with increased pro-growth and immune evasion protein expression [92]. To date global metabolomics profiling has not been reported on human MPNST specimens; several recent e fforts have examined metabolic shifts in animal models of MPNST in response to preclinical therapeutic interventions [93–95].

#### **8. Translating Molecular Landscape of MPNST into Improved Therapies for Patients**

One overarching goal of improved molecular characterization of MPNST is to translate genomic discoveries into improved treatments for this classically chemo-refractory tumor. As a result of improved understanding of MPNST genomic variants, several targeted therapies have been trialed in preclinical MPNST models. For example, the MET-specific tyrosine kinase inhibitor capmatinib has shown promise, particularly in combination with the MEK inhibitor trametinib, in an NF1-MET driven MPNST GEM model [70]. BRAF mutant MPNST may also respond to targeted therapy; one case report described a dramatic response to the RAF inhibitor vemurafenib in a patient with sporadic metastatic MPNST harboring the BRAF V600E mutation [96]. E fforts to target histone acetylation in a preclinical MPNST model with loss of SUZ12 shrank tumors when combined with MEK inhibition [41], while other DNA methyltransferase inhibitors appear to a ffect immune surveillance of MPNST [92]. It is likely that in the near future MPNST clinical trials will incorporate therapies inhibiting components of the epigenetic machinery.


**2.2 Evolution of Flavonoid metabolism**

The enzymes, chalcone isomerase (CHI) and isoflavone reductase in Chlamydomonas, dihydrokaempferol-4-reductase and naringenin chalcone synthase (CHS) in *Phaeodactylum*, and CHI and dihydroflavonol reductase in *Ectocarpus* were created as a result of several evolutionary processes in representatives of bryophytes (mosses), liverworts, and hornworts. CHI-like enzymes were discovered in certain proteobacteria and fungi, and they may have been acquired by horizontal gene transfer. Contrarily, the recruitment and gene duplication of polyketide synthases and oxoglutarate-dependent dioxygenases from primary metabolism, respectively, led to the evolution of CHS and F3H. The first three flavonoids, chalcones, flavanols, and flavones, were created as a result of the CHS, CHI, and F3H activities. These metabolites, which have not altered in 500 million years, are essential intermediates in today's irreducibly complicated flavonoid manufacturing pathways in plants [17, 18].

The number of key events that sparked the flavonoid pathway's gradual rise, variety, and evolutionary successes are:

• the recruitment of enzymes from fundamental metabolisms, such as the polyketide, phenylpropanoid, and shikimate pathways

*Flavonoids: Recent Advances and Applications in Crop Breeding DOI: http://dx.doi.org/10.5772/intechopen.107565*

**Figure 2.**

*Distribution of flavonoids in plant kingdom and their respective structures.*

• the flexibility of flavonoid pathways and their capacity to shift intermediate molecule fluxes towards the production of complex scaffolds of quite varied chemicals depending on the needs of the local ecosystem.

More than 10,000 of these compounds have been found in over 9000 current plant species as a result of these evolutionary processes, making flavonoids one of the most extensively distributed routes in plants today [16, 19].

#### **2.3 The flavonoid biosynthetic pathways**

From a genetic standpoint, a lot of work has been done to decipher the flavonoids' biosynthesis routes. Flavonoid synthesis mutants have been discovered in a variety of plant species. The first important experimental models in this system were snapdragon (*Antirrhinum majus*), petunia (*Petunia hybrida*), and maize (*Zea mays*), which led to further discovery of several structural and regulatory flavonoid genes. Arabidopsis (*Arabidopsis thaliana*) has recently contributed to the study of flavonoid pathway regulation and subcellular localization [20].

#### *2.3.1 Following are the biosynthetic pathways of some major flavonoids*

The Figure shows the eight branches of flavonoid biosynthetic pathway (showed in the eight different colored boxes) and four important intermediate metabolites (represented by the green boxes) (**Figure 3**). The abbreviated forms of enzyme names and flavonoid compounds mentioned in the figure are as follows: (i) ANR: anthocyanidin reductase; (ii) ACCase: acetyl-CoA carboxylase; (iii) AS: aureusidin synthase; (iv) 4CL: 4-coumarate: CoA ligase; (v) CHS: chalcone synthase; (vi) CHI: chalcone isomerase; (vii) CHR: chalcone reductase; (viii) C4H: cinnamic acid 4-hydroxylase; (ix) CH2′GT: chalcone 2′-glucosyltransferase; (x) CH4′GT: chalcone 4′-O-glucosyltransferase; (xi) ANS: anthocyanidin synthase; (xii) CLL-7: cinnamate–CoA ligase; (xiv) FNS: flavone synthase; (xv) F6H: flavonoid 6-hydroxylase; (xvi) IFS: isoflavone synthase; (xvii) HID: 2-hydroxyisoflavanone dehydratase; (xviii) FNR: flavanone 4-reductase; (xix) F8H: flavonoid 8-hydroxylase; (xx) F3'5'H: flavanone 3′,5′-hydroxylase; (xxi) F3H: flavanone 3-hydroxylase; (xxii) DHK: dihydrokaempferol; (xxiii) DHM: dihydromyricetin; (xxiv) DFR: dihydroflavonol-4-reductase; (xxv) DHQ: dihydroquercetin; (xxvi) FLS: flavonol synthase; (xxvii) OMT: O-methyl transferases; (xxviii) PAL: phenylalanine ammonia lyase; (xxix) UFGT: UDP-glucose flavonoid 3-Oglucosyltransferase; (xxx) LAR: leucoanthocyanidin reductase [21].

#### *<IP_ADDRESS> Phenylpropanoid pathway*

The phenylpropanoid route produces flavonoids from phenylalanine, whereas the shikimate pathway produces phenylalanine [22, 23]. The general phenylpropanoid route refers to the first three steps of the phenylpropanoid pathway [24]. The aromatic amino acid phenylalanine is transformed to p-coumaroyl-CoA in this route. The typical phenylpropanoid route begins with the deamination of phenylalanine to trans-cinnamic acid, which is catalyzed by the enzyme phenylalanine ammonia lyase (PAL) [25]. In plants, PAL also has a significant role in controlling the transfer of carbon from primary to secondary metabolism [26]. The second step in the general phenylpropanoid route is catalyzed by the activity of C4H, a cytochrome P450

**Figure 3.**

monooxygenase found in plants that hydroxylates trans-cinnamic acid to produce p-coumaric acid [27]. The quantity of lignin, a crucial phenylpropanoid metabolite, in *Populus trichocarpa* and *Arabidopsis thaliana*, is connected to the degree of C4H expression [24, 28]. In the third step, 4-coumarate (4CL) catalyzes the production of p-coumararoyl-CoA by incorporating a coenzyme A (CoA) unit into p-coumaric acid [29].


**3. Overview of methods of treatment of uveal melanoma**

These days there are a lot greater treatment choices other than enucleation, which was the main alternative for a large portion of a century ago. The more moderate treatment choices mean to save the influenced eye and hold vision. Treatment of uveal melanoma relies upon different variables including age of the patients, foundational strength of the patient, state of the contrary eye, tumor size and area.

Neverthesess, metastases cannot be prevented. Based on the theoretical models, clinically manifest metastases are likely to occour 5 or 6 years onset of the systemic dissemination. By the time we diagnosis uveal melanoma, micrometastases may have been spread as of now. Along these lines, metastatic sickness happening after therapy is not unprecedented. Roughly 50% of the patients will kick the bucket from the sickness inside 10 to 15 years of enucleation. When a metastasis is found the endurance is under 7 months. In the event that a metastasis emerges as a lone injury in the liver, expanded endurance might be acquired by nearby resection of the tumor mass.

Tumor area and size are considered to be two of the primary factors in deciding on the treatment of ocular melanoma. There is no reason to save the eye if a small melanoma in a necessary place completely destroyed vision. It is important to remember this - patients who have undergone enucleation and individuals who have undergone radiation treatment respond appropriately when they receive information about the nature of their patients after treatment. The most important for them was tumor endurance.

Treatment using radiation is a typical therapy for intraocular melanoma that utilizes high energy radiation to kill tumor cells. Radiation treatment can regularly safeguard some vision, albeit once in a while this is lost at any rate since radiation harms different pieces of the eye. The structure of the eye is saved and this is mainly the advantage of this sort of treatment.

Radiation treetment can be divided into two categories. External radiation treatment that utilizes a machine outside the body to send radiation toward the tumor, and the second type is inside radiation treatment that utilizes a radioactive substance fixed in needles, seeds, wires, or catheters that are set legitimately into or close to the tumor. The manner in which the radiation treatment is given relies upon the sort and phase of the tumor being dealt with. In ophthalmooncology field we utilize both photon pillar light and furthermore proton beam irradiation.

The metastatic free survival rate, the local control and the late toxicity were studied in patients that underwent fractionated Stereotactic Radiation Therapy (fSRT) for uveal melanoma. These patients had a median follow-up 32 months and were given five fractions of 10 Gy. The results showed that fSRT is an effective treatment for uveal melanoma with a good local control. There were performed 15 enucleations after irradiation mainly because of neurovascular glaucoma [16].

Plaque therapy is the most often utilized framework for delivering radiation The other methods are Gamma Knife or methods that include proton beam. Radiation plaque treatment which offers great tumor control, can frequently safeguard helpful vision, and has a fundamental visualization that is practically identical to that of enucleation. Enucleation remains the standard strategy for the board of the biggest melanomas of the choroid and ciliary body. The Collaborative Ocular Melanoma Study (COMS) is randomized clinical trial assessing essential enucleation versus beam radiation done externaly followed by enucleation in the management of patients with choroidal melanomas. The study demonstrated that the two options to be used in same medium sized tumors. COMS studied also treatment of large tumors and found out that combined external radiotherapy followd by enucleation shown that there is no limit in orbital recurrence of the tumor mass [10–13].


**2. Terminology, history, and background**

The most common types of regenerative medicine therapies include platelet rich plasma (PRP), stem cell therapy from bone marrow aspirate concentrate (BMAC) and adipose tissue grafts, and exosomes. Regenerative therapies are growing worldwide

(**Figure 1**). They are increasing from 164% in 1 year with a total of 4.1 billion dollars in total global funding [2]. These therapies can be used in conjunction or individually, and there is no set algorithm or protocol that dictates superiority [3]. Stem cell therapy in this article will refer to adult stem cells, which are multipotent and have no ethical concerns related to their use, as opposed to embryonic stem cells [4]. Typically, candidates include patients with chronic peripheral joint pain that have not responded to steroid injections, or cannot tolerate the medication due to side effects, including but not limited to hyperglycemia, hypertension, ineffective wound healing, or adrenal gland suppression. Although there is no set protocol, some studies recommend implementing a series of three injections for PRP [5]. However, some physicians may grade progress and response to the initial injection as a rationale for a repeat injection.

#### **2.1 Platelet rich plasma**

PRP is the most common and readily available treatment option. It is the plasma fraction of blood with a high platelet concentration, as well as clotting factors, growth factors, chemokines, cytokines, and other plasma proteins [6]. This therapy helps promote stem cell migration as well as healing [7]. Commonly injected into joints and tendons for repair, PRP was first coined in the 1970's and used to describe the platelet count in peripheral blood, used to transfuse patients with thrombocytopenia [6]. PRP is obtained from blood after centrifuge, which helps separate components based on density gradients (**Figure 2**). Devices used to simplify the preparation of PRP are said to amplify the concentration of PRP 2–5 times the baseline [7]. Despite the limited clinical evidence that exists, PRP has been used to initiate healing for a variety of cases, most commonly including osteoarthritis (OA), lateral epicondylitis, rotator cuff tears, ligament and tendon injuries [7]. Recent randomized control trials actually demonstrate benefit in tendinopathy [7]. It has also been suggested that PRP can play a role in elimination neuropathic pain, thought to be secondary to a cascade of inflammation followed by repair via axon and tissue regeneration [8]. PRP has also been used as an intervertebral disc injection for low

**21**

**2.3 Bone marrow aspirate concentrate**

*Regenerative Medicine*

**2.2 Adipose tissue grafts**

*Blood components after centrifugation.*

**Figure 2.**

*DOI: http://dx.doi.org/10.5772/intechopen.93717*

back pain with promising clinical results, however more randomized controlled trials are needed [3]. Success of this therapy will ultimately depend on the preparation

Stem cell therapy uses non-embryonic adult stem cells described as multipotent stem cells, and in the clinical setting refers to therapy with mesenchymal (often from adipose tissue) and hematopoietic stem cells (often from bone marrow aspirate) [3]. These cells are present in a variety of tissues (adipocytes, chondrocytes, myocytes) and are thought to play a role in immune modulation [3]. The most common source of mesenchymal stem cells (MSC) are found in adipose tissue, first discovered in 1964 by Rodell [2]. There are approximately 500 to 2500× times more MSC's when compared to bone marrow [3]. Adipose derived stem cells (ASC's) are the most promising stem cells identified in humans, since adipose tissue is easily obtained in large quantities with small donor site discomfort [4]. Sites of harvest include the abdomen, upper arm, thigh, and trochanteric fat deposits. Common mechanisms to obtain fat include liposuction or lipectomy, followed by homogenization and enzymatic digestions. Traditional cosmetic liposuction can remove large volume (>4 kg) or small volume (<4 kg) adipose tissue, however for purposes of adipose tissue grafting only 100–200 mL may be needed [9]. The resultant material is then centrifuged. Each gram of adipose tissue yields 5 × 10^3 stem cells, significantly greater than bone marrow [4]. It is important to note that stem cell harvesting is more invasive that a simple blood draw for PRP, and could thus lead to an increase risk for infection or complication for patients undergoing MSC harvesting [10]. If performed under local or tumescent anesthesia, there is minimal to no recovery time. To date, hundreds of trials are listed on the United States National Institutes of Health website (NIH) for the use of ASC's. Examples of applications include soft tissue regeneration, skeletal tissue repair, myocardial infarction, immune disorders such as lupus, multiple sclerosis, Crohn's disease, diabetes.

Bone marrow aspiration is a procedure in which bone marrow is collected, usually from the pelvic iliac crest [11]. The procedure is very similar to PRP, in which the

and composition of the injectate, location, and type and extent of injury.

*Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.93717*

*Pain Management - Practices, Novel Therapies and Bioactives*

(**Figure 1**). They are increasing from 164% in 1 year with a total of 4.1 billion dollars in total global funding [2]. These therapies can be used in conjunction or individually, and there is no set algorithm or protocol that dictates superiority [3]. Stem cell therapy in this article will refer to adult stem cells, which are multipotent and have no ethical concerns related to their use, as opposed to embryonic stem cells [4]. Typically, candidates include patients with chronic peripheral joint pain that have not responded to steroid injections, or cannot tolerate the medication due to side effects, including but not limited to hyperglycemia, hypertension, ineffective wound healing, or adrenal gland suppression. Although there is no set protocol, some studies recommend implementing a series of three injections for PRP [5]. However, some physicians may grade progress and response to the initial injection as a rationale for a repeat injection.

*An atlas of regenerative medicine therapies worldwide. Adapted from alliance for regenerative medicine.*

PRP is the most common and readily available treatment option. It is the plasma

fraction of blood with a high platelet concentration, as well as clotting factors, growth factors, chemokines, cytokines, and other plasma proteins [6]. This therapy helps promote stem cell migration as well as healing [7]. Commonly injected into joints and tendons for repair, PRP was first coined in the 1970's and used to describe the platelet count in peripheral blood, used to transfuse patients with thrombocytopenia [6]. PRP is obtained from blood after centrifuge, which helps separate components based on density gradients (**Figure 2**). Devices used to simplify the preparation of PRP are said to amplify the concentration of PRP 2–5 times the baseline [7]. Despite the limited clinical evidence that exists, PRP has been used to initiate healing for a variety of cases, most commonly including osteoarthritis (OA), lateral epicondylitis, rotator cuff tears, ligament and tendon injuries [7]. Recent randomized control trials actually demonstrate benefit in tendinopathy [7]. It has also been suggested that PRP can play a role in elimination neuropathic pain, thought to be secondary to a cascade of inflammation followed by repair via axon and tissue regeneration [8]. PRP has also been used as an intervertebral disc injection for low

**20**

**2.1 Platelet rich plasma**

**Figure 1.**

**Figure 2.** *Blood components after centrifugation.*

back pain with promising clinical results, however more randomized controlled trials are needed [3]. Success of this therapy will ultimately depend on the preparation and composition of the injectate, location, and type and extent of injury.