HGF and MET mutations in primary and secondary lymphedema

BACKGROUND: Lymphedema is the abnormal accumulation of protein-rich fluid in the interstitial space. Primary lymphedema is a rare genetic condition with both autosomal dominant and autosomal recessive modes of inheritance. Three genes, FLT4 (VEGFR3), FOXC2, and SOX18 cause varying forms of primary lymphedema. In industrialized countries, secondary lymphedema is usually associated with cancer therapy and/or trauma. Recent observations suggested that hepatocyte growth factor/high affinity hepatocyte growth factor receptor (HGF/MET) were new candidate lymphedema genes. METHODS AND RESULTS: The coding exons and flanking regions of HGF and MET were directly sequenced in 145 lymphedema probands, 59 unrelated women with secondary lymphedema following treatment for breast cancer, 21 individual patients with lymphedema and intestinal lymphangiectasia, and at least 159 unrelated ethnic matched control individuals. Mutations leading to truncation or missense changes in evolutionarily conserved residues of HGF and MET were identified. These mutations were not polymorphic in control individuals. CONCLUSIONS:The identification of HGF/MET mutations in primary lymphedema, lymphedema/lymphangiectasia, and breast cancer-associated secondary lymphedema suggests that the HGF/MET pathway is causal or alters susceptibility for a broad range of lymphedema phenotypes. The HGF/MET pathway provides a new target for the prevention and/or treatment of lymphedema.     

An experimental model for chronic lymphedema

Although a multitude of operations exist for the treatment of lymphedema, none is highly successful. An experimental model that reliably and easily produces chronic lymphedema in an extremity would be useful to study treatments in a controlled and comparative manner and would enhance our understanding of the physiology and treatment of lymphedema. Many models that simulate clinical lymphedema have been described, but they suffer from cumbersome protocols, high laboratory costs, and an inconsistent yield of permanent lymphedema. We describe an experimental model for chronic lymphedema in the lower extremity of the rat that creates a lymphatic block in the groin induced by radiation treatment and one operation--surgical division of the superficial and deep lymphatics. All animals develop stable chronic lymphedema of the lower extremity within days of operation, with swelling that persists for at least 9 months. A mortality rate of 8 percent was associated with this technique. Methods for quantification of limb swelling are described, as is analysis of the lymphatic block by lymphoscintigraphic imaging of lymph channels and nodes. This model has the advantages of simplicity of technique, cost-effective use of rodent subjects, reproducibility of lymphedema, and quantification of results.     

Feasibility and Effectiveness of Basic Lymphedema Management in Leogane,Haiti, an Area Endemic for Bancroftian Filariasis

Background

Approximately 14 million persons living in areas endemic for lymphatic filariasis have lymphedema of the leg. Clinical studies indicate that repeated episodes of bacterial acute dermatolymphangioadenitis (ADLA) lead to progression of lymphedema and that basic lymphedema management, which emphasizes hygiene, skin care, exercise, and leg elevation, can reduce ADLA frequency. However, few studies have prospectively evaluated the effectiveness of basic lymphedema management or assessed the role of compressive bandaging for lymphedema in resource-poor settings.

Methodology/Principal Findings

Between 1995 and 1998, we prospectively monitored ADLA incidence and leg volume in 175 persons with lymphedema of the leg who enrolled in a lymphedema clinic in Leogane, Haiti, an area endemic for Wuchereria bancrofti. During the first phase of the study, when a major focus of the program was to reduce leg volume using compression bandages, ADLA incidence was 1.56 episodes per person-year. After March 1997, when hygiene and skin care were systematically emphasized and bandaging discouraged, ADLA incidence decreased to 0.48 episodes per person-year (P<0.0001). ADLA incidence was significantly associated with leg volume, stage of lymphedema, illiteracy, and use of compression bandages. Leg volume decreased in 78% of patients; over the entire study period, this reduction was statistically significant only for legs with stage 2 lymphedema (P = 0.01).

Conclusions/Significance

Basic lymphedema management, which emphasized hygiene and self-care, was associated with a 69% reduction in ADLA incidence. Use of compression bandages in this setting was associated with an increased risk of ADLA. Basic lymphedema management is feasible and effective in resource-limited areas that are endemic for lymphatic filariasis.     

The resolution of lymphedema by interstitial flow in the mouse tail skin

Lymphangiogenesis is considered a promising approach for increasing fluid drainage during secondary lymphedema. However, organization of lymphatics into functional capillaries may be dependent upon interstitial flow (IF). The present study was undertaken to determine the importance of lymphangiogenesis for lymphedema resolution. We created a lymphatic obstruction that produces lymphedema in mouse tail skin. The relatively scar-free skin regeneration that occurred across the obstruction allowed the progression of lymphangiogenesis to be observed and compared with the evolution of lymphedema. The role of vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3 signaling in lymphedema resolution was investigated by exogenous administration of VEGF-C or neutralizing antibodies against VEGFR-3. VEGF-C protein improved lymphedema at 15 days [reducing dermal thickness from 742 +/- 105 to 559 +/- 141 microm with 95% confidence intervals (CIs), P < 0.05] without increasing lymphatic capillary coverage (11.6 +/- 6.4% following VEGF-C treatment relative to 9.6 +/- 6.2% with 95% CIs, P > 0.50). Blocking VEGFR-3 signaling did not inhibit lymphedema resolution at 25 days (dermal thickness of 462 +/- 127 microm following VEGFR-3 inhibition relative to 502 +/- 87 microm with 95% CIs) or inhibit IF, although VEGFR-3 blocking prevented lymphangiogenesis (reducing lymphatic coverage to 0.2 +/- 0.7% relative to 8.7 +/- 7.3% with 95% CIs, P < 0.005). A second mouse tail lymphedema model was employed to investigate the ability of VEGF-C to increase fluid drainage across a scar. We found that neither neutralization of VEGFR-3 nor administration of VEGF-C affected the course of skin swelling over 25 days. These findings suggest that resolution of lymphedema in the mouse tail skin may be more dependent upon IF and regeneration of the extracellular matrix across the obstruction than lymphatic capillary regeneration.     

Overview of surgical treatments for breast cancer-related lymphedema

Breast cancer-related upper extremity lymphedema is an unsolved iatrogenic complication with a reported incidence ranging from 9 to 41 percent. The increase in volume and recurrent cellulitis of the affected limb cause both physical and mental distress to many breast cancer survivors. However, postmastectomy lymphedema has received little attention, and no curative treatment is available. Conservative treatment with decongestive therapy has been the primary choice for lymphedema treatment, but it is cumbersome and has limited benefits. To date, there is no consensus on surgical procedure and protocol. However, refinements in microsurgical techniques and improved examination devices may lead to the establishment of a standard surgical treatment for lymphedema. This review of surgical procedures for the treatment of postmastectomy lymphedema focuses on microsurgical lymphovenous shunt operations and discusses current issues in surgical treatment and the need for uniform treatment standards.     

Candidate gene analysis in primary lymphedema

BACKGROUND: Primary lymphedema, the accumulation of protein-rich fluid in the interstitial space, is the clinical manifestation of mutations involved in lymphatic development and function. Mutations in three genes, VEGFR3, FOXC2, and SOX18, cause primary lymphedema. However, mutations in these three genes only account for a fraction of primary lymphedema. To identify other genes mutated in primary lymphedema, we resequenced twenty-five biologically plausible candidate genes for lymphedema in a large collection of primary lymphedema families. METHODS AND RESULTS: Candidate genes were selected on the basis of gene expression in lymphatic endothelial cells, differential antigenic expression in lymphatics, and mouse studies of lymphatic development. The gene sequence was downloaded from GenBank and sequence primers designed to amplify 1 Kb of the 5' sequence, exons and flanking intron-exon boundaries, and 500 bp of the UTR of each gene. No common causative mutations were observed among the 25 genes screened. Single mutations were observed in elastin microfibril interfacer (EMILIN1), lymphocyte cytosolic protein 2 (LCP2), fatty acid binding protein 4 (FABP4), protein tyrosine kinase SYK (SYK), neuropilin-2 (NRP2), SpSRY-box 17 (SOX17), vascular cell adhesion molecule 1 (VCAM1), ROR orphan receptor C (RORC), and vascular endothelial growth factor B (VEGFB). Among these, the mutations in EMILIN1, RORC, LCP2, SYK, and VEGFB failed to segregate with lymphedema. The mutations in FABP4 (2), NRP2, SOX17, and VACM1 are consistent with being causative mutations, but occur in families too small to convincingly confirm cosegregation of mutation and phenotype. CONCLUSION: We excluded mutation in 21 biological candidate genes as a common cause of primary lymphedema. Mutations in FABP4, NRP2, SOX17 and VCAM1 are consistent with causality and follow up of these four genes are warranted. The evidence for FABP4 harboring lymphedema mutations is discussed.     

Proteomic profiling of lymphedema development in mouse model

The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham‐operated and lymphedema‐operated mice for 3 days, using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced‐right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A‐1 preprotein, alpha‐actinin‐3, mCG21744, parkinson disease, serum amyloid P‐component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham‐operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up‐regulation of serum amyloid P‐component precursor, parkinson disease, and apolipoprotein A‐1 preprotein in lymphedema model over sham‐operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd.     

Computed tomographic symptomatology of lymphedema of the lower extremities]

CT potentialities in the diagnosis and differential diagnosis of lower limb lymphedema in 40 patients (11 men and 29 women), aged 16 to 56, are illustrated. Eighty studies on the limbs were performed to 20 patients with primary lymphedema, 6 with congenital lymphedema, 26 with secondary lymphedema, and 28 with unaffected limbs. The data were statistically processed according to the following parameters: leg diameter, skin and subcutaneous fat thickness and density, the presence of fibrosis in subcutaneous fat, its localization, shape and spreading, fascial thickness. CT permitted objective assessment of disease and a right choice of a type of surgical intervention.     

Reduction of postoperative lymphedema after oral tumor surgery with sodium selenite

The objective of this double-blind, randomized study was to establish whether sodium selenite administered orally or intravenously reduces postoperative lymphedema after oral tumor surgery and to study the effect of sodium selenite on glutathione peroxidase (GPX) activity and oxygen radical production. Twenty patients were enrolled in the study. Each of the participants received 1000 μg sodium selenite intravenously or orally daily for 3 wk during the pre-, intra-, and postoperative period. The extent of lymphedema was measured for 2 wk and the plasma and whole-blood selenium concentration, GPX, reactive oxygen species (ROS), NO, and malonic dialdehyde were measured for 1 yr postoperatively. There was an inverse correlation between the severity of the lymphedema and the wholeblood/plasma selenium concentration and GPX activity. In addition, a positive correlation between the ROS concentration and the extent of lymphedema was observed. A significant reduction of lymphedema occurred in the sodium selenite-treated group. It is concluded that sodium selenite represents a suitable adjuvant treatment of secondary lymphedema in surgically treated patients with tumors in the oral and maxillofacial areas. Treatment with sodium selenite is especially advantageous as it can be instituted immediately after surgery prior to wound healing when manual lymphatic decongestion therapy cannot be applied.     

Yellow nail syndrome: treatment of lymphedema using low pressure compression

The present report describes a case with the triad of yellow nail syndrome (YNS) and the use of low-pressure compression pump as treatment of lymphedema in YNS. A 71-year-old woman presented with bilateral lower extremity lymphedema, yellow nails, and recurrent bilateral pleural effusion. In this case, we specifically focused on lymphedema treatment of the legs besides other recommendations for YNS.     

Functional recovery of fluid drainage precedes lymphangiogenesis in acute murine foreleg lymphedema

Secondary lymphedema in humans is a common consequence of axillary lymph node dissection (ALND) to treat breast cancer. It is commonly hypothesized that lymphatic growth is required to increase fluid drainage and ameliorate lymphedema. Although there is a pronounced alteration in the balance of interstitial forces regulating fluid transport that sustains the chronic form of lymphedema, it is presently unknown whether changes occur to the balance of interstitial forces during acute lymphedema that may play a role in the recovery of fluid drainage. Here, we compared the relative importance of lymphangiogenesis of lymphatic vessels and interstitial flows for restoring fluid drainage and resolving acute lymphedema in the mouse foreleg after ALND. We found that removal of the axillary lymph nodes reduced lymph drainage in the foreleg at days 0 and 5 postsurgery, with fluid tracer spreading interstitially through subcutaneous tissues. Interstitial fluid drainage returned to normal by day 10, whereas functional regrowth of lymphatic vessels was first detected by indocyanine green fluorescence lymphography at day 15, demonstrating that the recovery of interstitial fluid drainage preceded the regrowth of lymphatic vessels. This was confirmed by the administration of VEGF receptor-3-neutralizing antibodies, which completely blocks lymphatic regrowth. It was found that the recovery of interstitial fluid drainage and the natural resolution of acute lymphedema produced by ALND were not hindered by VEGF receptor-3 neutralization, demonstrating that interstitial fluid drainage recovery and the resolution of acute lymphedema are lymphangiogenesis independent. The data highlight the central role of the interstitial environment in adapting to lymphatic injury to increase fluid drainage.     

Evaluating the Burden of Lymphedema Due to Lymphatic Filariasis in 2005 in Khurda District,Odisha State,India

BackgroundOver 1.1 billion people worldwide are at risk for lymphatic filariasis (LF), and the global burden of LF-associated lymphedema is estimated at 16 million affected people, yet country-specific estimates are poor.SignificanceThese data highlight the magnitude of lymphedema in LF-endemic areas and emphasize the need to develop robust estimates of numbers of individuals with lymphedema in order to identify the extent of lymphedema management services needed in these regions.     

Impact of a Community-Based Lymphedema Management Program on Episodes of Adenolymphangitis (ADLA) and Lymphedema Progression - Odisha State,India

Background

Lymphedema management programs have been shown to decrease episodes of adenolymphangitis (ADLA), but the impact on lymphedema progression and of program compliance have not been thoroughly explored. Our objectives were to determine the rate of ADLA episodes and lymphedema progression over time for patients enrolled in a community-based lymphedema management program. We explored the association between program compliance and ADLA episodes as well as lymphedema progression.

Methodology/Principal Findings

A lymphedema management program was implemented in Odisha State, India from 2007–2010 by the non-governmental organization, Church''s Auxiliary for Social Action, in consultation with the Centers for Disease Control and Prevention. A cohort of patients was followed over 24 months. The crude 30-day rate of ADLA episodes decreased from 0.35 episodes per person-month at baseline to 0.23 at 24 months. Over the study period, the percentage of patients who progressed to more severe lymphedema decreased (P-value  = 0.0004), while those whose lymphedema regressed increased over time (P-value<0.0001). Overall compliance to lymphedema management, lagged one time point, appeared to have little to no association with the frequency of ADLA episodes among those without entry lesions (RR = 0.87 (0.69, 1.10)) and was associated with an increased rate (RR = 1.44 (1.11, 1.86)) among those with entry lesions. Lagging compliance two time points, it was associated with a decrease in the rate of ADLA episodes among those with entry lesions (RR = 0.77 (95% CI: 0.59, 0.99)) and was somewhat associated among those without entry lesions (RR = 0.83 (95% CI: 0.64, 1.06)). Compliance to soap was associated with a decreased rate of ADLA episodes among those without inter-digital entry lesions.

Conclusions/Significance

These results indicate that a community-based lymphedema management program is beneficial for lymphedema patients for both ADLA episodes and lymphedema. It is one of the first studies to demonstrate an association between program compliance and rate of ADLA episodes.     

Design of lymphedema ultrasound phantom with 3D-printed patient-specific subcutaneous anatomy: A-mode analysis approach for early diagnosis

The secondary lymphedema is mostly caused due to injury of lymphatic system during cancer treatment and its psychological and cosmetic issues are very critical for patients since it can cause severe thickening and swelling of lesions, mostly upper and lower limbs. Therefore, early diagnosis of the secondary lymphedema is more important to treat the symptoms in advance. The amplitude-mode (A-mode) ultrasound is suggested as an early diagnostic modality because it is relatively more cost-effective, portable, and easy to use than other previous diagnostic modalities. In order to see features of the A-mode ultrasound forearly diagnosis of lymphedema, ultrasound lymphedema phantoms were designed and fabricated with patient-specific subcutaneous honeycomb structures at the sub-stages of the international society of lymphedema (ISL) stage II and gelatin- or gelatin-salt based phantom materials. The patent-specific honeycomb structures were segmented from computed tomography (CT) venography images using various image process technologies and printed using a three dimensional (3D) printer for which its printing material shows similar acoustic impedance range with human subcutaneous tissues. The lymphedema phantoms showed similar subcutaneous anatomical features to those of patient's imagesin brightness mode (B-mode) ultrasound examination, and acoustic information originated from the stage-specific honeycomb structures was well represented in A-mode ultrasound examination. In particular, the A-mode wave form well represented stage-specific honeycomb information even with higher impedance value of fibrous fat region. Such stage-specific wave form information of A-mode ultrasound for the corresponding stage-specific lymphedema phantoms at the ISL stage II can be useful for further development of an A-mode ultrasound applications for early diagnosis of the secondary lymphedema.     

Creation of distal canine limb lymphedema

A canine model of distal limb lymphedema was established in order to study the treatment of this condition by lymph node transfer. This model was more difficult to establish than whole-limb lymphedema. Significant edema was achieved by a combination of preoperative irradiation and circumferential removal of skin from the irradiated areas followed by removal of the contents of the popliteal fossa. Despite these measures, it was not possible to produce lymphedema in every case, possibly because of the presence of lymphaticovenous shunts and panvascular compensation mechanisms.     

Contribution of fungi to chronic dermatitis and lymphangitis in patients with filariasis]

Adenolymphangitis is a common occurrence in filarial lymphedema. Damage to the lymphatics by F. bancrofti is followed by obliteration of lymph vessel and lymph stasis. Obstruction of lymphatics prevents the microbs skin penetration. Presented studies were performed to evaluate the role of fungi colonization of the skin among patients with filarial lymphedema. The fungal colonization of skin in patients with filarial lymphedema may be an important reason for chronic inflammatory disorders.     

A historical literature review on the role of posterior axillary boost field in the axillary lymph node coverage and development of lymphedema following regional nodal irradiation in breast cancer

To elucidate whether (1) a posterior axillary boost (PAB) field is an optimal method to target axillary lymph nodes (LNs); and (2) the addition of a PAB increases the incidence of lymphedema, a systematic review was undertaken. A literature search was performed in the PubMed database. A total of 16 studies were evaluated. There were no randomized studies. Seven articles have investigated dosimetric aspects of a PAB. The remaining 9 articles have determined the effect of a PAB field on the risk of lymphedema. Only 2 of 9 articles have prospectively reported the impact of a PAB on the risk of lymphedema development. There are conflicting reports on the necessity of a PAB. The PAB field provides a good coverage of level I/II axillary LNs because these nodes are usually at a greater depth. The main concern regarding a PAB is that it produces a hot spot in the anterior region of the axilla. Planning studies optimized a traditional PAB field. Prospective studies and the vast majority of retrospective studies have reported the use of a PAB field does not result in increasing the risk of lymphedema development over supraclavicular-only field. The controversies in the incidence of lymphedema suggest that field design may be more important than field arrangement. A key factor regarding the use of a PAB is the depth of axillary LNs. The PAB field should not be used unless there is an absolute indication for its application. Clinicians should weigh lymphedema risk in individual patients against the limited benefit of a PAB, in particular after axillary dissection. The testing of the inclusion of upper arm lymphatics in the regional LN irradiation target volume, and universal methodology measuring lymphedema are all areas for possible future studies.     

Autologous stem cells for the treatment of post-mastectomy lymphedema: a pilot study

BACKGROUND AIMS. Lymphedema is a common complication with breast cancer treatment that does not have a definite cure. Our objective was to determine the efficacy of autologous stem cells (ASC) in the treatment of lymphedema secondary to mastectomy and axillary lymphadenectomy in comparison with traditional decongestive treatment with compression sleeves. METHODS. A prospective study including 20 women with lymphedema secondary to breast cancer surgery with axillary lymphadenectomy was conducted. Women were assigned at random to one of two groups. One group of 10 women was injected with ASC in the affected arm, whereas the other 10 women comprised the control group and received traditional compression sleeve therapy (CST). The follow-up for both groups was 12 weeks. Pain, sensitivity and mobility were assessed before and after therapy. RESULTS. There was improvement in the volume of lymphedema in both groups, with no significant difference. In the ASC group there was an overall volume reduction during the follow-up, whereas in the CST group lymphedema recurred after the compression sleeve was removed. CONCLUSIONS. Our findings suggest that ASC injection for patients with lymphedema can be an effective treatment. It reduces arm volume and associated co-morbidities of pain and decreased sensitivity. Traditional CST was also effective for lymphedema reduction, but it was dependent on continuous use of the treatment.     

Congenital, low penetrance lymphedema of lower limbs maps to chromosome 6q16.2–q22.1 in an inbred Pakistani family

Hereditary lymphedema is a rare, lymphatic disorder resulting in the chronic swelling of the extremities. It shows wide inter- and intra-familial clinical heterogeneity as well as variability in the age of onset. There are more than four genetically distinct lymphedema conditions known and mutations in three genes have been discovered in families with lymphedema. However, many other familial lymphedemas do not show linkage with the known loci, suggesting genetic heterogeneity. Here, we describe a large inbred Pakistani family with congenital, progressive lymphedema confined to the lower limbs, which fades away at 40–45 years of age. This condition segregates in an autosomal dominant fashion with reduced penetrance. The features are close to primary lymphedema I, Nonne–Milory type (MIM 153100). We exclude this condition for linkage to the known loci for lymphedema by employing highly polymorphic microsatellite markers from these intervals. Then, through a genome-wide linkage study we show that the malformation in our family maps to chromosome 6q16.2–q22.1. The highest pair-wise LOD score (Z _max = 3.19) was obtained with microsatellite marker D6S1671, and a multipoint score of 3.75 was obtained at 108 cM. Haplotype analysis indicated that the critical interval in this family flanks between markers D6S1716 and D6S303. Mutation analysis in FOXO3, a likely candidate within this interval, did not show any pathogenic change in the affected family subjects. Our study provides an evidence of a second locus for lymphedema type I. The discovery of the underlying gene could be helpful for the understanding of this heterogeneous hereditary condition.     

Stewart-Treves syndrome: Case report and literature review

Lymphangiosarcoma, or Stewart-Treves Syndrome (STS), is a very rare skin angiosarcoma with poor prognosis, which usually affects the upper limbs of patients who underwent breast cancer surgery, including axillary dissection followed by radiotherapy (RT). Cutaneous lymphangiosarcomas, which account for approximately 5% of all angiosarcomas, usually originate in the limb with chronic lymphedema. Lymphatic blockade is involved in the onset of STS. RT contributes indirectly to an increased risk of developing STS by causing axillary-node sclerosis and resulting in a lymphatic blockade and lymphedema. Chronic lymphedema causes local immunodeficiency, which indirectly leads to oncogenesis. Currently, axillary nodes are no longer routinely irradiated after axillary dissection, which is associated with a reduction in the incidence of chronic lymphedema from 40% to 4%. The use of sentinel lymph node biopsy technique is also widespread and the associated risk of lymphedema is further reduced. Thus, the incidence of STS decreased significantly with improved surgical and radiation techniques. The overall prognosis of STS patients is very poor. Only early radical surgical removal, including amputation or disarticulation of the affected limb, or wide excision at an early stage offers the greatest chance of long-term survival. Only a few case reports and series with a small number of patients with lymphangiosarcoma can be found in the literature. We present a case report of the first diagnosed STS at our department in an effort to highlight the need of the consideration of developing lymphangiosarcoma in patients with chronic lymphedema.