Recent Advances in Therapeutic Modalities Against Breast Cancer-Related Lymphedema: Future Epigenetic Landscape

Background: Lymphedema is a significant postsurgical complication observed in the majority of breast cancer patients. These multifactorial etiopathogenesis have a significant role in the development of novel diagnostic/prognostic biomarkers and the development of novel therapies. This review aims to ascertain the epigenetic alterations that lead to breast cancer-related lymphedema (BCRL), multiple pathobiological events, and the underlying genetic predisposing factors, signaling cascades pertinent to the lapses in effective prognosis/diagnosis, and finally to develop a suitable therapeutic regimen. Methods and Results: We have performed a literature search in public databases such as PubMed, Medline, Google Scholar, National Library of Medicine and screened several published reports. Search words such as epigenetics to induce BCRL, prognosis/diagnosis, primary lymphedema, secondary lymphedema, genetic predisposing factors for BRCL, conventional therapies, and surgery were used in these databases. This review described several epigenetic-based predisposing factors and the pathophysiological consequences of BCRL, which affect the overall quality of life, and the interplay of these events could foster the progression of lymphedema in breast cancer survivors. Prognosis/diagnostic and therapy lapses for treating BCRL are highly challenging due to genetic and anatomical variations, alteration in the lymphatic vessel contractions, and variable expression of several factors such as vascular endothelial growth factor (VEGF)-E and vascular endothelial growth factor receptor (VEGFR) in breast cancer survivors. Conclusion: We compared the efficacy of various conventional therapies for treating BCRL as a multidisciplinary approach. Further substantial research is required to decipher underlying signaling epigenetic pathways to develop chromatin-modifying therapies pertinent to the multiple etiopathogenesis to explore the correlation between the disease pathophysiology and novel therapeutic modalities to treat BCRL.


Introduction
2][3][4][5][6] Meanwhile, breast cancerrelated lymphedema (BCRL) is one of the significant complications that occur in breast cancer patients after the treatment for breast cancer; it has been reported that more than 20% of patients who undergo treatment for breast cancer may develop BCRL. 7,8][11] Mainly, breast cancer patients after radical mastectomy [12][13][14] are more prone to the development of lymphedema.The affected region could cause distressful life in the patients, resulting in functional problems, decreased Quality of Life (QoL), and recurrent infections.The significant pathogenesis of this disease involves dysfunctional lymph transport, and as a result, accumulation of immune cells, lipids, and interstitial fluid in the affected region occurs. 15,16Lymphedema is categorized into primary lymphedema and secondary lymphedema; primary lymphedema is induced by the malfunction of the lymphatic system, whereas secondary lymphedema is due to the iatrogenic process.Secondary lymphedema is most significantly observed in cancer therapies. 8urthermore, the lymph system is responsible for draining lymph fluid into the circulation; during lymphedema, the lymph fluid is accumulated in the interstitial space when the lymph drainage is impaired, which consequently causes edema and fosters subcutaneous tissue swelling (Fig. 1). 17ymphedema can induce dysfunction of upper extremities, discomfort, pain, heaviness, psychological changes, unsatisfactory cosmesis, and recurrent infections, [18][19][20][21] eventually exacerbating the poor QoL. 22The risk of BCRL is multifactorial, which is due to intake of chemotherapy, radiation therapy, axillary lymph node dissection, advanced disease stage, a substantial number of positive lymph nodes (>8), and a higher BMI ( ‡25 kg/m 2 ). 23,246][27] The incidence of this disease is significantly correlated to the survival time after chemotherapy or radiotherapy. 28Therefore, at present, there is a lack of perfect scientific evidence for the pathophysiology and treatment plan for lymphedema.However, BCRL is an incurable condition and current therapies, physical therapies, namely, manual lymph node drainage, shock wave therapy, laser therapy, Qigong-based exercise, and surgical interventions such as derivative microsurgery, vascularized lymph node transfer, microsurgical reconstruction are reported.][31][32][33] In this review, we critically delineated the need to explore the epigenetic landscape of lymphedema pathophysiology to develop gene-based therapies, discussed the challenges in developing effective prognosis/diagnosis strategies and multiple etiopathogenesis of lymphedema in breast cancer survivors, and also discussed the comparative efficacy of novel therapies (Table 1) for lymphedema treatment.

Literature Search
The literature search was performed extensively to extract the published reports using key words such as epigenetics, gene markers, breast cancer, primary lymphedema, secondary lymphedema, genetic predisposing factors for BRCL, biomarkers, conventional therapies, and surgery, the pathophysiology of BCRL, therapies, diagnostic reports in public databases such as Medline, PubMed, National Library of Medicine, and Good Scholar, which were peer reviewed.

Biomarkers Based on BCRL Histopathology
The lack of effective prognostic/early diagnostic strategies for BCRL in lymphedema patients could facilitate the advanced stages of lymphedema, in which classical methods of symptomatic therapy do not lead to a viable result.Moreover, to date, there is no full understanding of the cause of specific tissue changes associated with lymphedema. 34It is known that the impaired lymph outflow leads to disorganization of the lymphatic bed from the distal to the lesion site.However, the morphological changes (progressive lymphangiosclerosis) are substantially higher in BCRL conditions due to the anatomically justified consequences of impaired lymphatic outflow. 25hus, progressive changes in lymphatic vessels could occur in the collector zone with minimal trauma to the central lymphatic collectors (regional lymph nodes).This process is induced by the uncontrolled division of lymphatic endothelial cells (LECs) in the early stages of lymphedema.However, the markers 35 and histopathological changes during the early stages have not been examined yet through clinical studies.
During surgical dissection of lymph nodes, in the distal bed, the lymphatic capillaries experience a progressive disturbance to the outflow and confer to the formation of retrograde lymph movement, and infarction of regional collectors, which further reduce the amplitude of vessel contraction at a constant frequency, eventually leading to disorganization of the lymphatic bed as a whole.Ly6G + and CD4 + immunocytes are actively involved in the progression of such pathological changes. 36Therapeutic strategies to mitigate these pathological changes are limited.Personalization of treatment is a natural stage in the evolution of novel treatment methods against BCRL-induced chronic progressive pathologies.
To develop methods for the personalizing treatment for lymphedema, it is necessary to study the prognosis/diagnosis based on the genetic and proteomic expression pertaining to inflammation and the mitochondrial redox system as these are considered significant prognostic factors that provoke and maintain tissue disorganization and restructuring.Individually variable markers can directly affect the severity of a given disease. 37To develop a personalized and hightechnology approach to the treatment of lymphedematous pathology, it is necessary to systematize the moleculargenetic and morphological factors affecting the progression of lymphedema and the maintenance of specific pathological changes in the affected tissues. 37he etiopathogenesis of lymphedema is not fully understood, however, there is an assumption that individual genetic, immune, and morphological features play a direct role in the pathogenesis of lymphedema and in the abundance of variations in the severity of the disease. 37According to previous research reports, up to 80% of patients who underwent radical antitumor treatment develop lymphedema after several months.Furthermore, lymphedema may be developed from 10 days to 30 years after breast cancer treatment.Based on this, we can conclude that the development of lymphedema is based on secondary damage to the lymphatic outflow pathways, which leads to several pathological processes with the development of peripheral lymphadenopathy, and the disruption of metabolic processes in the interstitial space.
Current therapeutic modalities are very limited to treat secondary lymphedema, and therefore, it can cause extensive financial burden and psychological stress in BCRL patients.Therefore, novel treatment strategies are yet to be developed to enhance the overall QoL in these patients. 38The efficacy of human hormone components, namely, adrenomedullin, intermedin, and their cognate receptors such as CLR/ RAMP1-3 modulate lymphangiogenesis, enhance the LEC permeability and the activity of adipose-tissue derived progenitor cells (ADPCs), and consequently promote lymphatic vessel formation in lymphedema models. 38This report hypothesized that the combinatorial regimen of CLR/RAMP receptor ligands with ADPCs could be considered a significant therapeutic strategy to mitigate the devastating pathophysiology of lymphedema.Substantial studies using a combinatorial regimen should be examined for the lymph vessel regenerative capacity using in vivo mouse models of secondary lymphedema. 38

Lymphedema Pathophysiology and Recent Diagnostic Reports
Dermal thickening is more significantly apparent in the later stages of this disease and confers the formation of hyperkeratosis, acanthosis, lichenification, and verrucae. 39urthermore, cellulitis, lymphangitis, and erysipelas are typically associated with chronic lymphedema. 39As per the International Society of Lymphology, stage 0 delineates subclinical lymphedema accompanied by swelling and heaviness in the limb whereas stage I indicates limb swelling and pitting without dermal fibrosis and stage II is characterized by dermal fibrosis.Stage III lymphedema to the most severe stage is accompanied by limb swelling, fat deposits, acanthosis, and verrucae. 39A plethora of studies described FIG. 1.The pathophysiology of lymphedema across arterioles and venules in the lymphatic system of BCRL patients.Damage to the lymphatic vessels through inflammation and adipose tissue deposition in the capillary beds of the lymphatic system in postsurgical BCRL patients could promote the increase in tissue fluid and fosters the formation of fibrosis and swelling.BCRL, breast cancer-related lymphedema.
the involvement of proinflammatory and inflammatory pathways in triggering the pathogenesis of lymphedema.For instance, CD4 + T cells such as Th1 and Th2 immune paradigms can trigger the release of IL-4, IL-13, and TGF-b1 across tissues and they can control collagen deposition and fibrosis in lymphedema. 40n vivo mouse models and BCRL clinical models have already depicted this paradigm pertaining to the pathogenesis of lymphedema.When these mouse models were treated with neutralizing antibodies to neutralize IL-4 or IL-13, lymphedema was reported to be prevented. 41Mainly, TGF-b1 has significant implications as the diagnostic marker in lymphedema as it could foster fibrosis, and the administration of TGF-b1 neutralizing antibodies has mitigated fibrosis in in vivo models and also impaired the migration of Th2 cells and expression of Th2 cytokines. 42However, these mechanisms still require additional research to illustrate the Th2-Th1 immune paradigm in the progression of lymphedema in human models.Furthermore, the accumulation of lymph during lymphedema is associated with the progression of adipogenesis and miscellaneous inflammatory cascades, which could result in complete failure of lymphatic impairment due to the blockade of collateral lymphatic formation.

Genetics and Lymphangiogenesis in BCRL
Gene-related alterations can promote defects in lymphangiogenesis, and lymphatic function and these mechanisms 29 yet require substantial research studies in BCRL patients.For instance, the LCP2 is reported to be involved in immune responses by modulating the T cell-induced molecular signaling, 43 and this gene has a prominent role in the development of lymphatic structures and promotes plateletdependent mechanism pertinent to the embryogenesis accompanying the separation of blood and lymphatic vessels. 44,45Copy number alterations in LCP2 and the tendency of these alterations can significantly induce their influence on other genes known to be implicated in BCRL such as interleukins, IL-10, IL-4, IL-13, and neuropilin 2. 46,47 NRP2 expression can be observed in LECs and it is upregulated in BCRL patients and acts as a significant mediator to promote angiogenesis and lymphangiogenesis through VEGFC (Fig. 2).
[55] Traditional Therapies for Lymphedema-Complex Decongestive Therapy Complex decongestive therapy (CDT) is also known as combined physical therapy; this therapy is a current standard protocol for treating BCRL, which consists of manual lymph drainage, compression therapy (consisting of compression bandages spiral-bandaging method, and a figure-of-eight method, 56 compression sleeves, or other types of compression garments), lymph-reducing exercises, skin care, and education on self-management of lymphedema. 7,23,57,58The experience of CDT is long-standing. 59For instance, there are two phases of CDT (Tables 1 and 2) during treatment; among them, phase 1 of CDT is for reducing swelling and phase 2 is for optimizing the effectiveness of CDT.CDT can

ADVANCES IN BCRL THERAPIES AND FUTURE EPIGENETIC LANDSCAPE
][62] However, the clinical outcome with CDT is always lower-than-expected in clinical practice; in addition, patient compliance is poor in CDT for BCRL 63 due to laborious, time-consuming, and expensive processes. 64Furthermore, being overweight can mitigate the efficiency of CDT 65 because the excess adipose tissue may promote the development of chronic massive lymphedema, which cannot be treated by CDT; this therapy cannot eliminate adipose tissueinduced pathophysiology in BCRL conditions by compression alone. 66In addition, regular lymphedema treatment can cause an immense financial burden on patients, and the costs are more than $14,000 a year per patient. 67There are several surgeons around the world recommending complementary and alternative therapies for treating BCRL.

Low-level laser therapy
Low-level laser therapy (LLLT) or photobiomodulation has been applied for treating BCRL over the past 20 years.Compared with CDT or surgical management, LLLT is not invasive. 68This strategy can be used as a phototherapy using wavelengths of light in the range of 650 to 1000 nm.[71][72][73][74] FIG. 2. Pathophysiology of lymphedema: Lymphatic load into the lymphatic tissue is enhanced during the postsurgical period in breast cancer patients; and this flow is determined by both intrinsic and extrinsic forces, which fosters the lymph propulsion in the lymphatic conduct; a sequential enhancement in VEGFC and VEGFR activity, 175 which could confer microvascular permeability across VB and capillary angiogenesis, and promote the overload of the remaining lymphatic drainage capacity.Chylomicrons are the lipid-rich triglyceride-loaded content that enters into the blood circulation via the lymphatic system and the chylomicrons enter into lymphatics, which confers higher lymphedema during BRCL conditions.These events could enhance the proliferation of fibrous tissue in the lymphedema tissue. 23E-RVs, existing resistance vessels; HP imbalance, the imbalance of hydrostatic pressure difference; IFV, interstitial fluid volume; IP, interstitial pressure; VB, vascular bed; VE, vascular endothelial; VEGFC, vascular growth endothelial factor C; VEGFR, vascular endothelial growth factor receptor.

Detailed information
However, a recent study demonstrated that the combinatorial regimen of LLLT combined with conventional CDT has failed to show additional therapeutic benefits. 75tracorporeal shock wave therapy.Extracorporeal shock wave therapy (ESWT) could be used in articular and ligamental diseases, such as plantar fasciitis, lateral epicondylitis, and tendinitis. 76,77The exact mechanism of ESWT remains unknown.Based on the results of previous animal experiments, ESWT can induce upregulation of angiogenesis-related growth factors including endothelial nitric oxide synthase, nitric oxide, vascular growth endothelial factor (VEGF), bone morphogenetic protein, basic fibroblast growth factor (b-FGF), and proliferating cell nuclear antigen, sequentially promoting neovascularization.These events can enhance blood supply and cell proliferation, and eventually foster tissue regeneration to repair the tendon and articular or bone tissues.][80] The expressions of VEGFC (rat-tail and rabbit-ear models), b-FGF (rat-tail model), and VEGF receptor 3 (VEGFR3, rabbit-ear model) could be induced by low-energy ESWT and stimulate lymphangiogenesis to improve lymphedema in experimental models of rat tails and rabbit ears. 76,80A recent pilot study reported that ESWT could reduce the lymph volume in lymphedema conditions and improve the overall QoL in most of the patients with BCRL. 77Another study delineated the reduction of lymphedema on long-term ESWT for 6 months. 64These studies demonstrated the potential role of ESWT in the treatment of BCRL.

Hyperbaric oxygen therapy
The hyperbaric oxygen treatment can contribute to the enhanced barometric pressure in the patient's entire body and promote the patient with 100% oxygen for a certain period. 81he exact mechanism of hyperbaric oxygen therapy (HBOT) is still unknown, but a pilot study pointed out that the VEGF activity may be stimulated by HBOT. 82In another study, fibrosis formation could be inhibited by HBOT and reported to benefit overall improvement in lymphedema conditions. 83urthermore, HBOT can be applied in a lot of clinical scenarios such as carbon monoxide intoxication, decompression sickness, gas gangrene, necrosis, ulcers, hemorrhagic cystitis, proctitis, multiple sclerosis, and vascular dementia.][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102] Patients' overall QoL is significantly higher with HBOT for mitigating radiationinduced lesions in other organs; meanwhile, this therapy can also reduce lymphedema formation after radiotherapy for breast cancer. 83,103owever, randomized trials have failed to confirm the positive effect of HBOT in patients with lymphedema. 103The effectiveness of HBOT alone for BCRL still needs to be confirmed; the combination therapy of CDT and HBOT, unlike LLLT, is more beneficial than CDT only; therefore, HBOT may be considered an adjunct therapy with conventional treatments such as CDT for patients with BCRL. 104

Stellate ganglion block
A stellate ganglion block (SGB) is another therapeutic modality in which the injection of local anesthetic drugs could be used to block the sympathetic nerves located around the cervical sympathetic trunk. 105][108][109][110][111][112][113][114][115][116][117][118][119][120] Therefore, SGB therapy can be used as an alternative treatment for BCRL. 105Furthermore, corticosteroid administration may have an additional benefit in SGB therapy for treating BCRL. 121However, substantial research is yet to be performed to explore the underlying pharmacological mechanisms of SGB therapy for treating BCRL.

Pneumatic compression device
A pneumatic compression device (PCD) is used as an adjuvant therapy to mitigate lymphedema to improve the overall quality of life in BCRL patients. 66Therapy involving PCD can reduce lymph volume during lymphedema.So, the frequency of visits, treatments, and complications after discharge of breast cancer survivors can be minimized with regular PCD therapy, and this method is a cost-effective intervention. 122owever, a meta-analysis reported that a combinatorial regimen of CDT and PCD failed to produce additive efficacy for mitigating lymphedema-induced pathophysiology. 123

Mesenchymal stem cell therapy
LECs can undergo differentiation induced from stem cells to promote angiogenesis and neovasculature, consequently conferring the generation of new lymphatic vessels, recovery of impaired lymphatic networks, and reconstruction of the lymphatic circulation pathway. 124Adipose-derived stromal stem cells (ASCs) also play a significant role in the treatment of lymphedema.Some reports delineated that the administration of ASCs are beneficial for enhancing the clinical outcomes in BCRL patients due to the reduction in the overall volume of the lower extremity and minimal requirement for subsequent compression therapy.There are no serious adverse effects observed in these patients. 125,126ASCs can stimulate the regeneration of lymphatic vessels and enable restoration of the disrupted lymphatic circulation in a murine tail lymphedema model 127 as ASCs exhibit the pluripotent capacity to differentiate into multiple cell lineages; therefore, the differentiation of ASCs can be conducive to the formation of LECs across deteriorated lymph tissue.
Expression of VEGF-E and VEGFR3 is significantly higher during the process of ASC differentiation to LECs, which further enhances lymphangiogenesis. 128,129Thus, stem cell therapy is a promising strategy for the treatment of BCRL, however, clinical trials are required to decipher and confirm the efficacy of this stem cell therapy in the management of BRCL. 124

Surgical management
1][132] A few studies reported that breast reconstruction by deep inferior epigastric perforator-flap or greater omentum flap with vascularized lymph node transfer from inguinal or mesenteric lymph nodes is promising. 131,133,134owever, surgical intervention is always invasive and it can cause secondary adverse effects to the patients who suffered breast cancer resection.The complications including postoperative infection also cannot be overlooked.

Acupuncture
3][154] A study at the Memorial Sloan-Kettering Cancer Center (MSKCC) has reported that acupuncture therapy as an adjunct therapy can decrease the overall volume of extremities during lymphedema and minimize the infection rate or other severe side effects. 155The efficacy of acupuncture alone still needs to be evaluated for treating BCRL.

Kinesio-tape
Kinesio tape (K-tape) is an elastic strip with an acrylic adhesive that is used for relieving pain and disability during injuries.The mechanism of K-tape facilitates adhering and lifting the skin in BRCL patients and increases the space beneath the skin and between muscles; therefore, the lymph fluid, interstitial fluid, or blood can flow efficiently and mitigate edema or congestion induced by lymphedema.A ran-domized clinical trial has shown that K-tape can be a better alternative tool to replace bandages in CDT, especially for patients with poor short-stretch bandage compliance. 156netic Variations and Future Epigenetic Alterations in BCRL Previous evidence described the genetic screening of hereditary syndromes accompanied by the genetic mutations in genes (Table 3) such as FOXC2, FLT-4, and SOX18 for lymphedemadistichiasis, Milroy disease, and hypotrichosis-lymphedematelangiectasia, respectively.Furthermore, gene mutations such as CCBE1, FAT4, GJC2, VEGFC, PTPN14, GATA2, HGF, and PIEZO1 could induce generalized lymphatic dysplasia, inherited lymphedema type 1C and 1D, lymphedema-choanal atresia, Emberger, and lymphedema-lymphangiectasia, and hereditary lymphedema III, respectively.Hence, future studies require the epigenetic alterations pertinent to these genes triggering the above lymphedema types.In addition, several genetic algorithms were developed to develop genetic markers as molecular diagnostic markers pertinent to BRCL and it is crucial to explore the epigenetic landscape and other chromosomal abnormalities underlying the lymphangio dysplastic syndromes and lymphedema incidence postsurgery in breast cancer conditions.
The above clinical syndromes have been associated with lymphedema as a crucial pathophysiological component.Hence, it is highly significant to explore the de novo germinal variations pertinent to these genes.Substantial ongoing research using genome-wide association analysis, and whole-Exmore sequencing should focus on the underlying epigenetic landscape of the incidence of secondary lymphedema after breast cancer surgery or mastectomy due to the damage to the lymphatic syndrome.Therefore, it is possible to identify the single/multiple and interacting genetic or epigenetic variants pertinent to the lymphedema incidence for efficient early diagnosis in breast cancer patients. 157ead and neck cancer (HNC) survivors exhibit divergent patterns of methylation in inflammatory signaling, chemokine signaling, TLR-signaling, or natural killer-mediated signaling, and these pathways are also reported to play a significant role in the pathophysiology of postsurgical intervention in HNC patients. 158Hence, it is crucial to examine the differentially methylated probes in genes related to these signaling repertoires involved in the lymphedema pathophysiology.Genetic variation has a significant influence on the incidence of lymphedema in females after mastectomy.0][161][162][163] For instance, mutations occurring in the GJC2 could induce alterations in amino acids in connexin 47, which is associated with lymphedema. 164Several single nucleotide polymorphisms are reported to be confined to the coding regions of genes pertinent to lymphedema development and progression. 165,166pigenetic alterations are resulted by alterations in DNA methylation and posttranslational histone alterations.In addition, the changes in noncoding RNA expression could cause epigenetic changes.These alterations are reported to be highly evident in early breast cancer pathogenesis and useful to predict these as biomarkers to foster early detection of breast cancer. 167There are no specific reports exploring the specific epigenetic modifications that lead to the pathophysiology of BRCL.DNA methylation factors such as attachment of methyl groups to the CpG nucleotides subsequently form 5-methyl-cytosine in the presence of DNA methyltransferases. 168These DNA methylation-mediated epigenetic modifications are yet to be explored in the BCRL pathophysiology.Furthermore, the histone modifications are mainly evident in the H2A, H2B, H3, and H4 proteins. 169osttranslational modifications of histone proteins do not exert any influence on the DNA sequence but influence the transcription process by inducing alterations in the chromatin structure.This can occur by modification of the noncondensed transcriptionally active site to the condensed inactive site.Posttranslational modifications such as sumoylation, DNA methylation, and acetylation are evident in the tail region of these histone proteins. 170These alterations are associated with the formation of H3K4me2 and H3K4me3 across the gene region of the promoter, subsequently causing alterations in oncoprotein expression to foster cancers. 171,172These histone alterations are yet to be explored in the pathophysiology as these modifications may play a prominent role in the DNA-mediated cellular processes in BCRL. 173

Conclusion
Very limited reports are available to delineate the complete pathophysiology and multiple etiopathogenesis of BRCL in breast cancer survivors; therefore, a significant understating of the factors pertinent to the lymphatic system damage in BRCL and the chain of complex progressive events occurring in this condition may benefit the patients by prescribing a suitable combinatorial traditional therapeutic modality.For instance, the accumulated hypertrophic fat moieties can confer the dissipation of lymphatic capillaries consequently inducing damage to the fluid and liquid transport and promoting fat deposition in the periphery of BCRL patients.A better understanding of various gene expression levels is crucial to delineate the single, multiple variants based on the epigenetic landscape of lymphedema; protein-related marker expression is also ascertained to diagnose the disease at an early stage.Furthermore, lymphatic anatomy is crucial to develop novel therapeutic modalities and devices for treating lymphedema.
Yet, large-scale, randomized clinical trials are required to examine their efficacy; in the future, it is crucial to explore the comparative efficacy of the above therapies mentioned in this review to enhance the overall QoL of patients with BCRL.

Table 3 .
Gene Mutations Pertinent to the Clinical Syndromes Where Lymphedema Is a Significant