Breast fibromatosis: Making the case for primary vs secondary subtypes.

Fibromatosis of the breast is a rare condition that can be locally aggressive. The mainstay of treatment remains wide local excision, with varied adjuvant therapy as needed. The authors describe their experience in the treatment of a series of patients and propose the classification of primary and secondary breast fibromatosis. A single-institution retrospective analysis of patients treated for breast fibromatosis from 2003 to 2017 was completed. Demographic data, pertinent past medical history, and treatment modalities were reviewed. Primary breast fibromatosis was defined as arising in the absence of previous surgery or radiation therapy to the ipsilateral breast. Secondary breast fibromatosis was defined as arising in the setting of previous surgery or radiation therapy to the ipsilateral breast. A total of 16 patients were included with the median age 40 (28-64) years. The average size of the lesion was 6.37 cm (range of 1.5-15 cm). Mean follow-up time was 65 months. Surgical excision was completed in 14 patients, with two recurrences. There were no recurrences in patients with surgical margins >1 cm. Two patients were treated nonsurgically. There were seven patients with primary fibromatosis of the breast and nine patients with secondary fibromatosis of the breast. Fibromatosis of the breast is difficult to diagnose prior to surgical excision. We advocate for the multi-disciplinary treatment of this disease process with an aggressive surgical approach to achieve margins >1 cm.

Regulatory T Cells Mediate Local Immunosuppression in Lymphedema.

Patients who suffer from lymphedema have impaired immunity and, as a result, are at an increased risk for infections. Furthermore, previous studies have shown that lymphadenectomy impairs acquisition of adaptive immune responses and antibody production in response to foreign antigens. Although it is clear that antigen presentation in lymph nodes plays a key role in adaptive immunity, the cellular mechanisms that regulate impaired immune responses in patients with lymphedema or following lymphatic injury remain unknown. We have previously found that axillary lymph node dissection, both clinically and in a mouse model, results in a marked increase in the number of regulatory T cells in the ipsilateral limb. In this study, we focus on the role of regulatory T cells in immunosuppression and show that regulatory T-cell proliferation in tissues distal to site of lymphatic injury contributes to impaired innate and adaptive immune responses. More importantly, using Foxp3-DTR transgenic mice, we show that depletion of regulatory T cells in the setting of lymphatic injury restores these critical immune-mediated responses. These findings provide additional evidence that immune responses following lymphatic injury play a key role in mediating the pathology of lymphedema.

Th2 cytokines inhibit lymphangiogenesis.

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.

Regulation of inflammation and fibrosis by macrophages in lymphedema.

Lymphedema, a common complication of cancer treatment, is characterized by inflammation, fibrosis, and adipose deposition. We have previously shown that macrophage infiltration is increased in mouse models of lymphedema. Because macrophages are regulators of lymphangiogenesis and fibrosis, this study aimed to determine the role of these cells in lymphedema using depletion experiments. Matched biopsy specimens of normal and lymphedema tissues were obtained from patients with unilateral upper extremity breast cancer-related lymphedema, and macrophage accumulation was assessed using immunohistochemistry. In addition, we used a mouse tail model of lymphedema to quantify macrophage accumulation and analyze outcomes of conditional macrophage depletion. Histological analysis of clinical lymphedema biopsies revealed significantly increased macrophage infiltration. Similarly, in the mouse tail model, lymphatic injury increased the number of macrophages and favored M2 differentiation. Chronic macrophage depletion using lethally irradiated wild-type mice reconstituted with CD11b-diphtheria toxin receptor mouse bone marrow did not decrease swelling, adipose deposition, or overall inflammation. Macrophage depletion after lymphedema had become established significantly increased fibrosis and accumulation of CD4(+) cells and promoted Th2 differentiation while decreasing lymphatic transport capacity and VEGF-C expression. Our findings suggest that macrophages home to lymphedematous tissues and differentiate into the M2 phenotype. In addition, our findings suggest that macrophages have an antifibrotic role in lymphedema and either directly or indirectly regulate CD4(+) cell accumulation and Th2 differentiation. Finally, our findings suggest that lymphedema-associated macrophages are a major source of VEGF-C and that impaired macrophage responses after lymphatic injury result in decreased lymphatic function.

Decellularized Lymph Nodes as Scaffolds for Tissue Engineered Lymph Nodes.

BACKGROUND: The lymphatic system is commonly injured during cancer treatment. However, despite the morbidity of these injuries, there are currently no options for replacing damaged lymphatics. The purpose of this study was to optimize methods for decellularization of murine lymph nodes (LN) and to determine if these scaffolds can be used to tissue engineer lymph node-like structures. METHODS AND RESULTS: LNs were harvested from adult mice and subjected to various decellularization protocols. The degree of decellularization and removal of nuclear material was analyzed histologically and quantitatively using DNA isolation. In addition, we analyzed histological architecture by staining for matrix proteins. After the optimal method of decellularization was identified, decellularized constructs were implanted in the renal capsule of syngeneic or allogeneic recipient mice and analyzed for antigenicity. Finally, to determine if decellularized constructs could deliver lymphocytes to recipient animals, the matrices were repopulated with splenocytes, implanted in submuscular pockets, and harvested 14 days later. Decellularization was best accomplished with the detergent sodium dodecyl sulfate (SDS), resulting in negligible residual cellular material but maintenance of LN architecture. Implantation of decellularized LNs into syngeneic or allogeneic mice did not elicit a significant antigenic response. In addition, repopulation of decellularized LNs with splenocytes resulted in successful in vivo cellular delivery. CONCLUSIONS: We show, for the first time, that LNs can be successfully decellularized and that these matrices have preserved extracellular matrix architecture and the potential to deliver leukocytes in vivo. Future studies are needed to determine if tissue engineered lymph nodes maintain immunologic function.

Lymphaticovenous bypass decreases pathologic skin changes in upper extremity breast cancer-related lymphedema.

INTRODUCTION: Recent advances in microsurgery such as lymphaticovenous bypass (LVB) have been shown to decrease limb volumes and improve subjective symptoms in patients with lymphedema. However, to date, it remains unknown if these procedures can reverse the pathological tissue changes associated with lymphedema. Therefore, the purpose of this study was to analyze skin tissue changes in patients before and after LVB. METHODS AND RESULTS: Matched skin biopsy samples were collected from normal and lymphedematous limbs of 6 patients with unilateral breast cancer-related upper extremity lymphedema before and 6 months after LVB. Biopsy specimens were fixed and analyzed for inflammation, fibrosis, hyperkeratosis, and lymphangiogenesis. Six months following LVB, 83% of patients had symptomatic improvement in their lymphedema. Histological analysis at this time demonstrated a significant decrease in tissue CD4(+) cell inflammation in lymphedematous limb (but not normal limb) biopsies (p<0.01). These changes were associated with significantly decreased tissue fibrosis as demonstrated by decreased collagen type I deposition and TGF-ß1 expression (all p<0.01). In addition, we found a significant decrease in epidermal thickness, decreased numbers of proliferating basal keratinocytes, and decreased number of LYVE-1(+) lymphatic vessels in lymphedematous limbs after LVB. CONCLUSIONS: We have shown, for the first time, that microsurgical LVB not only improves symptomatology of lymphedema but also helps to improve pathologic changes in the skin. These findings suggest that the some of the pathologic changes of lymphedema are reversible and may be related to lymphatic fluid stasis.

Sterile inflammation after lymph node transfer improves lymphatic function and regeneration.

BACKGROUND: The aim of this study was to determine whether sterile inflammatory reactions can serve as a physiologic means of augmenting lymphangiogenesis in transplanted lymph nodes using a murine model. METHODS: The authors used their previously reported model of lymph node transfer to study the effect of sterile inflammation on lymphatic regeneration. Mice were divided into three groups: group 1 (controls) underwent lymphadenectomy followed by immediate lymph node transplantation without inflammation; group 2 (inflammation before transfer) underwent transplantation with lymph nodes harvested from donor animals in which a sterile inflammatory reaction was induced in the ipsilateral donor limb; and group 3 (inflammation after transfer) underwent transplantation with lymph nodes and then inflammation was induced in the ipsilateral limb. Lymphatic function, lymphangiogenesis, and lymph node histology were examined 28 days after transplantation and compared with those of normal lymph nodes. RESULTS: Animals that had sterile inflammation after transplantation (group 3) had significantly improved lymphatic function (>2-fold increase) on lympho scintigraphy, increased perinodal lymphangiogenesis, and functional lymphatics compared with the groups with no inflammation and inflammation before transplantation (p<0.01). Inflammation after transplantation was associated with a more normal lymph node architecture, expansion of B-cell zones, and decreased percentage of T cells compared with the other experimental groups. CONCLUSIONS: Sterile inflammation is a potent method of augmenting lymphatic function and lymphangiogenesis after lymph node transplantation and is associated with maintenance of lymph node architecture. Induction of inflammation after transplantation is the most effective method and promotes maintenance of normal lymph node B- and T-cell architecture.

Obesity increases inflammation and impairs lymphatic function in a mouse model of lymphedema.

Although obesity is a major clinical risk factor for lymphedema, the mechanisms that regulate this effect remain unknown. Recent reports have demonstrated that obesity is associated with acquired lymphatic dysfunction. The purpose of this study was to determine how obesity-induced lymphatic dysfunction modulates the pathological effects of lymphatic injury in a mouse model. We used a diet-induced model of obesity in adult male C57BL/6J mice in which experimental animals were fed a high-fat diet and control animals were fed a normal chow diet for 8-10 wk. We then surgically ablated the superficial and deep lymphatics of the midportion of the tail. Six weeks postoperatively, we analyzed changes in lymphatic function, adipose deposition, inflammation, and fibrosis. We also compared responses to acute inflammatory stimuli in obese and lean mice. Compared with lean control mice, obese mice had baseline decreased lymphatic function. Lymphedema in obese mice further impaired lymphatic function and resulted in increased subcutaneous adipose deposition, increased CD45(+) and CD4(+) cell inflammation (P < 0.01), and increased fibrosis, but caused no change in the number of lymphatic vessels. Interestingly, obese mice had a significantly increased acute inflammatory reaction to croton oil application. In conclusion, obese mice have impaired lymphatic function at baseline that is amplified by lymphatic injury. This effect is associated with increased chronic inflammation, fibrosis, and adipose deposition. These findings suggest that obese patients are at higher risk for lymphedema due to impaired baseline lymphatic clearance and an increased propensity for inflammation in response to injury.

IL-6 regulates adipose deposition and homeostasis in lymphedema.

Lymphedema (LE) is a morbid disease characterized by chronic limb swelling and adipose deposition. Although it is clear that lymphatic injury is necessary for this pathology, the mechanisms that underlie lymphedema remain unknown. IL-6 is a known regulator of adipose homeostasis in obesity and has been shown to be increased in primary and secondary models of lymphedema. Therefore, the purpose of this study was to determine the role of IL-6 in adipose deposition in lymphedema. The expression of IL-6 was analyzed in clinical tissue specimens and serum from patients with or without LE, as well as in two mouse models of lymphatic injury. In addition, we analyzed IL-6 expression/adipose deposition in mice deficient in CD4(+) cells (CD4KO) or IL-6 expression (IL-6KO) or mice treated with a small molecule inhibitor of IL-6 or CD4 depleting antibodies to determine how IL-6 expression is regulated and the effect of changes in IL-6 expression on adipose deposition after lymphatic injury. Patients with LE and mice treated with lymphatic excision of the tail had significantly elevated tissue and serum expression of IL-6 and its downstream mediator. The expression of IL-6 was associated with adipose deposition and CD4(+) inflammation and was markedly decreased in CD4KO mice. Loss of IL-6 function resulted in significantly increased adipose deposition after tail lymphatic injury. Our findings suggest that IL-6 is increased as a result of adipose deposition and CD4(+) cell inflammation in lymphedema. In addition, our study suggests that IL-6 expression in lymphedema acts to limit adipose accumulation.

Lymph node transplantation results in spontaneous lymphatic reconnection and restoration of lymphatic flow.

BACKGROUND: Although lymph node transplantation has been shown to improve lymphatic function, the mechanisms regulating lymphatic vessel reconnection and functional status of lymph nodes remains poorly understood. METHODS: The authors developed and used LacZ lymphatic reporter mice to examine the lineage of lymphatic vessels infiltrating transferred lymph nodes. In addition, the authors analyzed lymphatic function, expression of vascular endothelial growth factor (VEGF)-C, maintenance of T- and B-cell zone, and anatomical localization of lymphatics and high endothelial venules. RESULTS: Reporter mice were specific and highly sensitive in identifying lymphatic vessels. Lymph node transfer was associated with rapid return of lymphatic function and clearance of technetium-99 secondary to a massive infiltration of recipient mouse lymphatics and putative connections to donor lymphatics. T- and B-cell populations in the lymph node were maintained. These changes correlated with marked increases in the expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Newly formed lymphatic channels in transferred lymph nodes were in close anatomical proximity to high endothelial venules. CONCLUSIONS: Transferred lymph nodes have rapid infiltration of functional host lymphatic vessels and maintain T- and B-cell populations. This process correlates with increased endogenous expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Anatomical proximity of newly formed lymphatics and high endothelial venules supports the hypothesis that lymph node transfer can improve lymphedema by exchanges with the systemic circulation.

Obesity impairs lymphatic fluid transport and dendritic cell migration to lymph nodes.

INTRODUCTION: Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system. METHODS: Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8-10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up. RESULTS: High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages. CONCLUSIONS: Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.