Magnetic resonance lymphangiography: Establishing normal.

BACKGROUND: Despite an increased interest in visualizing the lymphatic vessels with magnetic resonance lymphangiography (MRL), little literature is available describing their appearance in nonlymphedematous individuals. To determine lymphatic abnormalities, an understanding of how healthy lymphatic vessels appear and behave needs to be established. Therefore, in this study, MRL of individuals without a history of lymphatic disease was performed. METHODS: A total of 25 individuals (15 women) underwent MRL of their lower limbs using a 3.0 T Philips magnetic resonance imaging scanner (Philips Medical Systems). The first nine participants were recruited to establish the concentration of gadolinium-based contrast agent (GBCA) to administer, with the remainder imaged before and after interdigital forefoot GBCA injections at the optimized dose. Outcomes, including lymphatic vessel diameter, tortuosity, and frequency of drainage via particular drainage routes, were recorded. RESULTS: Healthy lymphatic vessels following the anteromedial pathway were routinely observed in post-contrast T1-weighted images (average tortuosity, 1.09 ± 0.03), with an average of 2.16 ± 0.93 lymphatic vessels with a diameter of 2.47 ± 0.50 mm crossing the anterior ankle. In six limbs, vessels following the anterolateral pathways were observed. No vessels traversing the posterior of the legs were seen. In a subset of 10 vessels, the lymphatic signal, measured at the ankle, peaked 29 minutes, 50 seconds ± 9 minutes, 29 seconds after GBCA administration. No lymphatic vessels were observed in T2-weighted images. CONCLUSIONS: Contrast-enhanced MRL reliably depicts the lymphatic vessels in the legs of healthy controls. Following interdigital contrast injection, anteromedial drainage appears dominant. Quantitative measures related to lymphatic vessel size, tortuosity, and drainage rate are readily obtainable and could be beneficial for detecting even subtle lymphatic impairment.

Erythematous capillary-lymphatic malformations mimicking blood vascular anomalies.

Superficial erythematous cutaneous vascular malformations are assumed to be blood vascular in origin, but cutaneous lymphatic malformations can contain blood and appear red. Management may be different and so an accurate diagnosis is important. Cutaneous malformations were investigated through 2D histology and 3D whole-mount histology. Two lesions were clinically considered as port-wine birthmarks and another 3 lesions as erythematous telangiectasias. The aims were (i) to demonstrate that cutaneous erythematous malformations including telangiectasia can represent a lymphatic phenotype, (ii) to determine if lesions represent expanded but otherwise normal or malformed lymphatics, and (iii) to determine if the presence of erythrocytes explained the red color. Microscopy revealed all lesions as lymphatic structures. Port-wine birthmarks proved to be cystic lesions, with nonuniform lymphatic marker expression and a disconnected lymphatic network suggesting a lymphatic malformation. Erythematous telangiectasias represented expanded but nonmalformed lymphatics. Blood within lymphatics appeared to explain the color. Blood-lymphatic shunts could be detected in the erythematous telangiectasia. In conclusion, erythematous cutaneous capillary lesions may be lymphatic in origin but clinically indistinguishable from blood vascular malformations. Biopsy is advised for correct phenotyping and management. Erythrocytes are the likely explanation for color accessing lymphatics through lympho-venous shunts.

Investigation of clinical characteristics and genome associations in the 'UK Lipoedema' cohort.

Lipoedema is a chronic adipose tissue disorder mainly affecting women, causing excess subcutaneous fat deposition on the lower limbs with pain and tenderness. There is often a family history of lipoedema, suggesting a genetic origin, but the contribution of genetics is currently unclear. A tightly phenotyped cohort of 200 lipoedema patients was recruited from two UK specialist clinics. Objective clinical characteristics and measures of quality of life data were obtained. In an attempt to understand the genetic architecture of the disease better, genome-wide single nucleotide polymorphism (SNP) genotype data were obtained, and a genome wide association study (GWAS) was performed on 130 of the recruits. The analysis revealed genetic loci suggestively associated with the lipoedema phenotype, with further support provided by an independent cohort taken from the 100,000 Genomes Project. The top SNP rs1409440 (ORmeta ≈ 2.01, Pmeta ≈ 4 x 10-6) is located upstream of LHFPL6, which is thought to be involved with lipoma formation. Exactly how this relates to lipoedema is not yet understood. This first GWAS of a UK lipoedema cohort has identified genetic regions of suggestive association with the disease. Further replication of these findings in different populations is warranted.

Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

Systematic Review of Magnetic Resonance Lymphangiography From a Technical Perspective.

BACKGROUND: Clinical examination and lymphoscintigraphy are the current standard for investigating lymphatic function. Magnetic resonance imaging (MRI) facilitates three-dimensional (3D), nonionizing imaging of the lymphatic vasculature, including functional assessments of lymphatic flow, and may improve diagnosis and treatment planning in disease states such as lymphedema. PURPOSE: To summarize the role of MRI as a noninvasive technique to assess lymphatic drainage and highlight areas in need of further study. STUDY TYPE: Systematic review. POPULATION: In October 2019, a systematic literature search (PubMed) was performed to identify articles on magnetic resonance lymphangiography (MRL). FIELD STRENGTH/SEQUENCE: No field strength or sequence restrictions. ASSESSMENT: Article quality assessment was conducted using a bespoke protocol, designed with heavy reliance on the National Institutes of Health quality assessment tool for case series studies and Downs and Blacks quality checklist for health care intervention studies. STATISTICAL TESTS: The results of the original research articles are summarized. RESULTS: From 612 identified articles, 43 articles were included and their protocols and results summarized. Field strength was 1.5 or 3.0 T in all studies, with 25/43 (58%) employing 3.0 T imaging. Most commonly, imaging of the peripheries, upper and lower limbs including the pelvis (32/43, 74%), and the trunk (10/43, 23%) is performed, including two studies covering both regions. Imaging protocols were heterogenous; however, T2 -weighted and contrast-enhanced T1 -weighted images are routinely acquired and demonstrate the lymphatic vasculature. Edema, vessel, quantity and morphology, and contrast uptake characteristics are commonly reported indicators of lymphatic dysfunction. DATA CONCLUSION: MRL is uniquely placed to yield large field of view, qualitative and quantitative, 3D imaging of the lymphatic vasculature. Despite study heterogeneity, consensus is emerging regarding MRL protocol design. MRL has the potential to dramatically improve understanding of the lymphatics and detect disease, but further optimization, and research into the influence of study protocol differences, is required before this is fully realized. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 2.

Development and physiological functions of the lymphatic system: insights from human genetic studies of primary lymphedema.

Primary lymphedema is a long-term (chronic) condition characterized by tissue lymph retention and swelling that can affect any part of the body, although it usually develops in the arms or legs. Due to the relevant contribution of the lymphatic system to human physiology, while this review mainly focuses on the clinical and physiological aspects related to the regulation of fluid homeostasis and edema, clinicians need to know that the impact of lymphatic dysfunction with a genetic origin can be wide ranging. Lymphatic dysfunction can affect immune function so leading to infection; it can influence cancer development and spread, and it can determine fat transport so impacting on nutrition and obesity. Genetic studies and the development of imaging techniques for the assessment of lymphatic function have enabled the recognition of primary lymphedema as a heterogenic condition in terms of genetic causes and disease mechanisms. In this review, the known biological functions of several genes crucial to the development and function of the lymphatic system are used as a basis for understanding normal lymphatic biology. The disease conditions originating from mutations in these genes are discussed together with a detailed clinical description of the phenotype and the up-to-date knowledge in terms of disease mechanisms acquired from in vitro and in vivo research models.

Axillary Web Syndrome: Evidence for Lymphatic Origin with Thrombosis.

Background: The axillary web syndrome (AWS) occurs in the axilla and on the frontal side of the upper arm and sometimes along the forearm to the thumb. The cord is painful, particularly on movement, and can therefore be very distressing for the patient. Although the phenomenon has been examined and discussed for decades, no evidence for the origin has been found until now. The aim of this study was to perform a histopathologic analysis of cords taken between 1996 and 1998 in the Surgical Clinic, Skane University Hospital, Lund, Sweden. Methods and Results: In seven patients, biopsies of the AWS cords were obtained 4-5 weeks after axillary node surgery for breast cancer and examined with standard hematoxylin and eosin and D2-40 (lymphatic endothelial cell) staining. In one biopsy, there was a dilated vessel with a thickened wall, which was confirmed by D2-40 immunostaining to represent a lymphatic vessel. The lumen was occluded by organized thrombus, within which new vessels were being formed, indicating recanalization. In two other biopsies, similar lymphatic vessels with thickened walls were present, although the lumen of the vessels was not visualized in the planes of the section. The other four biopsies do not show specific features. Conclusion: Although only one case, this is the first pathological evidence of thrombosis within a confirmed lymphatic vessel from a case of cording. We propose that the axillary cord represents lymphatic vessel thrombosis. Recanalization of the thrombus may eventually restore lymphatic flow consistent with the transient nature of the condition.

Lymphoscintigraphic Abnormalities Associated with Milroy Disease and Lymphedema-Distichiasis Syndrome.

Background: Primary lymphedema is genetically heterogeneous. Two of the most common forms of primary lymphedema are Milroy disease (MD) and lymphedema-distichiasis syndrome (LDS). This study aims to look further into the pathogenesis of the two conditions by analyzing the lymphoscintigram images from affected individuals to ascertain if it is a useful diagnostic tool. Methods and Results: The lymphoscintigrams of patients with MD and LDS were analyzed, comparing the images and transport parameters of the two genotypes against a control population. Lymphoscintigrams were available for 12 MD and 16 LDS patients (all genetically proven diagnoses). Eight of the 12 (67%) lymph scans performed on patients with MD demonstrated little or no uptake from the initial lymphatics and poor visualization of the inguinal lymph nodes. These changes were consistent with a "functional aplasia," that is, the lymphatic vessels were present but appeared to be ineffective in absorbing the interstitial fluid into the lymphatic system. In patients with LDS the lymphoscintigraphic appearances were different. In 12 of the 16 scans (75%), the lymph scans were highly suggestive of lymphatic collector reflux. Quantification revealed a significantly reduced uptake of tracer within the inguinal lymph nodes and a higher residual activity in the feet at 2 hours in MD compared with LDS and compared with controls. Conclusion: Lymphoscintigraphic imaging and quantification can be characteristic in specific genetic forms of primary lymphedema and may be useful as an additional tool for in-depth phenotyping, leading to a more accurate diagnosis and providing insight into the underlying mechanism of disease.

Author Correction: Novel mutations in PIEZO1 cause an autosomal recessive generalized lymphatic dysplasia with non-immune hydrops fetalis.

This Article contains an error in the last sentence of the 'Variant analysis suggests they are pathogenic' section of the Results, which incorrectly reads 'No truncated PIEZO1 protein products were identified in western blot analysis in GLD1:II.3 and GLD2:II.2 (Fig. 2, Supplementary Fig. 6), suggesting that the truncated protein is not stable and therefore degraded.' This should read 'No full-size PIEZO1 protein products were identified in western blot analysis in GLD1:II.3 and GLD2:II.2 (Fig. 2, Supplementary Fig. 6); the three nonsense mutations are predicted to lead to premature termination of the protein, hence it is possible that those truncated proteins will be non-functional or even unstable and degraded.' The error has not been fixed in the PDF or HTML versions of the Article.

A Case-Control Study of the Lymphatic Phenotype of Yellow Nail Syndrome.

BACKGROUND: Yellow nail syndrome (YNS) is a rare disease manifesting as a triad of yellow-green dystrophic nails, lymphedema, and chronic respiratory disease. The etiology of YNS is obscure and investigations are few. A single lymphatic pathogenesis has been proposed to account for all the associated features, and despite the lack of evidence for a unifying lymphatic mechanism, this hypothesis prevails. The objective was to explore the lymphatic phenotype in YNS and to establish whether lymphatic dysfunction could be a major contributing factor to the disease process. METHODS AND RESULTS: Four-limb lymphoscintigraphy was performed on patients with YNS and on healthy, age-matched controls. All 17 patients had lower limb swelling, and 14 (82%) had upper limb swelling also, including 5 (29%) with hand involvement. None of the YNS lymph scans was completely normal. Combined qualitative and quantitative assessment showed that 67% of YNS scans were clearly abnormal compared with 36% of healthy control scans. Mean axillary and ilio-inguinal nodal tracer uptakes were 41%-44% lower in the YNS group than in the controls (p < 0.0001). CONCLUSIONS: YNS is a lymphatic phenotype because lymphatic insufficiency was found to exist in all patients and the insufficiency was widespread (upper and lower limbs), with a common mechanistic fault of poor transport. The origin of the lymphatic fault is unclear. In healthy individuals, lymphatic abnormalities may be relatively common in the fifth decade of life onward.

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations.

BACKGROUND: Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS: VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy-based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS: Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION: VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING: Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003.

Magnetic Resonance Imaging-Based Assessment of Breast Cancer-Related Lymphoedema Tissue Composition.

OBJECTIVES: The aim of this study was to propose a magnetic resonance imaging acquisition and analysis protocol that uses image segmentation to measure and depict fluid, fat, and muscle volumes in breast cancer-related lymphoedema (BCRL). This study also aims to compare affected and control (unaffected) arms of patients with diagnosed BCRL, providing an analysis of both the volume and the distribution of the different tissue components. MATERIALS AND METHODS: The entire arm was imaged with a fluid-sensitive STIR and a 2-point 3-dimensional T1W gradient-echo-based Dixon sequences, acquired in sagittal orientation and covering the same imaging volume. An automated image postprocessing procedure was developed to simultaneously (1) contour the external volume of the arm and the muscle fascia, allowing separation of the epifacial and subfascial volumes; and to (2) separate the voxels belonging to the muscle, fat, and fluid components. The total, subfascial, epifascial, muscle (subfascial), fluid (epifascial), and fat (epifascial) volumes were measured in 13 patients with unilateral BCRL. Affected versus unaffected volumes were compared using a 2-tailed paired t test; a value of P < 0.05 was considered to be significant. Pearson correlation was used to investigate the linear relationship between fat and fluid excess volumes. The distribution of fluid, fat, and epifascial excess volumes (affected minus unaffected) along the arm was also evaluated using dedicated tissue composition maps. RESULTS: Total arm, epifascial, epifascial fluid, and epifascial fat volumes were significantly different (P < 0.0005), with greater volume in the affected arms. The increase in epifascial volume (globally, 94% of the excess volume) constituted the bulk of the lymphoedematous swelling, with fat comprising the main component. The total fat excess volume summed over all patients was 2.1 times that of fluid. Furthermore, fat and fluid excess volumes were linearly correlated (Pearson r = 0.75), with the fat excess volume being greater than the fluid in 11 subjects. Differences in muscle compartment volume between affected and unaffected arms were not statistically significant, and contributed only 6% to the total excess volume. Considering the distribution of the different tissue excess volumes, fluid accumulated prevalently around the elbow, with substantial involvement of the upper arm in only 3 cases. Fat excess volume was generally greater in the upper arm; however, the relative increase in epifascial volume, which considers the total swelling relative to the original size of the arm, was in 9 cases maximal within the forearm. CONCLUSIONS: Our measurements indicate that excess of fat within the epifascial layer was the main contributor to the swelling, even when a substantial accumulation of fluid was present. The proposed approach could be used to monitor how the internal components of BCRL evolve after presentation, to stratify patients for treatment, and to objectively assess treatment response. This methodology provides quantitative metrics not currently available during the standard clinical assessment of BCRL and shows potential for implementation in clinical practice.

EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.

Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate.

Constitutively Enhanced Lymphatic Pumping in the Upper Limbs of Women Who Later Develop Breast Cancer-Related Lymphedema.

BACKGROUND: It has previously been shown that the lymph drainage rate in both upper limbs is greater in women destined to develop breast cancer-related lymphedema (BCRL) than in those who do not develop BCRL, indicating a constitutive predisposition. We explored constitutive differences further by measuring the maximum lymphatic pump pressure (Ppump) and the rate of (99m)Tc-Nanocoll transport generated by the contractile upper limb lymphatics before and after breast cancer surgery in a group of women who were followed for 2 years to determine their eventual BCRL or non-BCRL status. METHODS AND RESULTS: Ppump and tracer transport rate were measured by lymphatic congestion lymphoscintigraphy in the ipsilateral upper limb in 26 women pre- and post-breast cancer surgery. BCRL occurred in 10/26 (38.5%) cases. Ppump in the women who later developed BCRL (40.0 ± 8.2 mmHg) was 1.7-fold higher than in those who did not develop BCRL (23.1 ± 10.8 mmHg, p = 0.001). Moreover, the rate of lymph tracer transport into the forearm was 2.2-fold greater in the women who later developed BCRL (p = 0.052). Surgery did not significantly reduce Ppump measured 21 weeks postsurgery, but impaired forearm tracer transport in pre-BCRL women by 58% (p = 0.047), although not in those who did not develop BCRL. CONCLUSIONS: Women destined to develop BCRL have higher pumping pressures and lymph transport, indicating harder-working lymphatics before cancer treatment. Axillary lymphatic damage from surgery appears to compromise lymph drainage in those women constitutively predisposed to higher lymphatic pressures and lymph transport.

Lymph proteins may access peripheral blood without entering thoracic duct in patients with lymphatic dysfunction.

OBJECTIVE: The objective was to investigate the hypothesis that lymphovenous communications, which allow lymph proteins to access peripheral blood without first entering the thoracic duct, open in patients with abnormal lymphatic function. METHODS: Routine lymphoscintigraphy of 182 patients, including 27 without clinical evidence of lymphedema (controls), was performed immediately and 45 and 150 minutes after subcutaneous injection of technetium Tc 99m nanocolloid into both feet. Counts per pixel in a region of interest over the liver (L) were divided by total counts in bilateral ilioinguinal nodes (N) at 45 minutes (L/N45) and 150 minutes (L/N150). If all activity leaving ilioinguinal lymph nodes entered the thoracic duct, these L/N ratios would be similar from patient to patient. RESULTS: Eight patients were excluded because of immediate liver activity suggesting inadvertent intravascular injection of tracer. In controls (group 1), L/N150 displayed a normal distribution with mean (± standard deviation) of 0.16 (0.09) × 10(-4) pixels(-1). Patients with L/N150 >0.34 × 10(-4) pixels(-1) (ie, 0.16 + 2 standard deviations) were assumed to have lymphovenous communications. Of 34 patients with clinical evidence of lymphedema but with normal findings on lymphoscintigraphy (group 2), 3 (9%) had lymphovenous communications; of 114 with abnormalities on lymphoscintigraphy (group 3), 43 (38%) had lymphovenous communications (P = .001). N45/150 was significantly higher than L45/150 in all four groups, indicating arrival of activity in nodes before the liver. Abnormal features of lymphoscintigraphy-lymph transport delay, popliteal node visualization, and diversion of lymph through the skin-showed no association with L/N ratios. CONCLUSIONS: Lymphovenous communications exist in about one-third of patients with abnormalities detected on lymphoscintigraphy. The timings of tracer arrival in the liver and lymph nodes is consistent with lymphovenous communication within lymph nodes themselves.

Novel mutations in PIEZO1 cause an autosomal recessive generalized lymphatic dysplasia with non-immune hydrops fetalis.

Generalized lymphatic dysplasia (GLD) is a rare form of primary lymphoedema characterized by a uniform, widespread lymphoedema affecting all segments of the body, with systemic involvement such as intestinal and/or pulmonary lymphangiectasia, pleural effusions, chylothoraces and/or pericardial effusions. This may present prenatally as non-immune hydrops. Here we report homozygous and compound heterozygous mutations in PIEZO1, resulting in an autosomal recessive form of GLD with a high incidence of non-immune hydrops fetalis and childhood onset of facial and four limb lymphoedema. Mutations in PIEZO1, which encodes a mechanically activated ion channel, have been reported with autosomal dominant dehydrated hereditary stomatocytosis and non-immune hydrops of unknown aetiology. Besides its role in red blood cells, our findings indicate that PIEZO1 is also involved in the development of lymphatic structures.

Quantitative Imaging In Vivo of Functioning Lymphatic Vessels Around Human Melanoma and Benign Nevi.

OBJECTIVES: The density of functioning human lymphatics in vivo and of immunohistochemically defined lymphatics was quantified around melanomas, benign nevi, and matched normal skin, to assess the current lymphangiogenesis paradigm. We investigated whether histological and functioning density increased around melanomas compared with benign nevi or matched skin; whether functioning and histological density increased similarly; and whether larger increases occurred around metastatic melanomas. METHODS: Functioning density was quantified in vivo as the total amount of human dermal microlymphatics taking up fluorescent marker injected at the lesion margin. After tissue excision, perilesion histological density was quantified using podoplanin marker D2-40. RESULTS: Histological density was raised similarly around metastasising and non-metastasising melanomas compared with normal skin (+71%, p < 0.0001, n = 32); but was also raised significantly around benign nevi (+17%, p = 0.03, n = 20). In contrast, functioning lymphatic density was substantially reduced around the margins of melanomas (both metastasising and non-metastasising) compared with benign nevi (by 65%, p = 0.02) or normal skin (by 53%, p = 0.0014). CONCLUSIONS: Raised perilesion histological lymphatic density is not unique to melanoma but occurs also around benign nevi. The findings indicated that the number of functioning lateral lymphatics around human melanomas in vivo but not benign nevi is reduced, despite histologically increased numbers of lymphatics.

Quantitative Contrast-Enhanced Magnetic Resonance Lymphangiography of the Upper Limbs in Breast Cancer Related Lymphedema: An Exploratory Study.

BACKGROUND: Contrast-Enhanced Magnetic Resonance Lymphangiography (CE-MRL) presents some limitations: (i) it does not quantify lymphatic functionality; and (ii) enhancement of vascular structures may confound image interpretation. Furthermore, although CE-MRL is well described in the published literature for the lower limbs, there is a paucity of data with regards to its use in the upper limbs. In this proof-of-principle study, we propose a new protocol to perform CE-MRL in the upper limbs of patients with breast cancer-related lymphedema (BCRL) which addresses these limitations. METHODS AND RESULTS: CE-MRL was performed using a previously published (morphological) protocol and the proposed protocol (quantitative) on both the ipsilateral (abnormal) and contralateral (normal) arms of patients with BCRL. The quantitative protocol employs contrast agent (CA) intradermal injections at a lower concentration to prevent T2*-related signal decay. Both protocols provided high-resolution three-dimensional images of upper limb lymphatic vessels. CA uptake curves were utilized to distinguish between lymphatic vessels and vascular structures. The quantitative protocol minimized venous enhancement and avoided spurious delays in lymphatic enhancement due to short T2* values, enabling correct CA uptake characterization. The quantitative protocol was therefore employed to measure the lymphatic fluid velocity, which demonstrated functional differences between abnormal and normal arms. The velocity values were in agreement with previously reported lymphoscintigraphy and near infra-red lymphangiography measurements. CONCLUSIONS: This work demonstrated the feasibility of CE-MRL of the upper limbs in patients with BRCL, introducing an advanced imaging and analysis protocol suitable for anatomical and functional study of the lymphatic system.