Pathogenic variants in HGF give rise to childhood-to-late onset primary lymphoedema by loss of function.

Developmental and functional defects in the lymphatic system are responsible for primary lymphoedema (PL). PL is a chronic debilitating disease caused by increased accumulation of interstitial fluid, predisposing to inflammation, infections and fibrosis. There is no cure, only symptomatic treatment is available. Thirty-two genes or loci have been linked to PL, and another 22 are suggested, including Hepatocyte Growth Factor (HGF). We searched for HGF variants in 770 index patients from the Brussels PL cohort. We identified ten variants predicted to cause HGF loss-of-function (six nonsense, two frameshifts, and two splice-site changes; 1.3% of our cohort), and 14 missense variants predicted to be pathogenic in 17 families (2.21%). We studied co-segregation within families, mRNA stability for non-sense variants, and in vitro functional effects of the missense variants. Analyses of the mRNA of patient cells revealed degradation of the nonsense mutant allele. Reduced protein secretion was detected for nine of the 14 missense variants expressed in COS-7 cells. Stimulation of lymphatic endothelial cells with these 14 HGF variant proteins resulted in decreased activation of the downstream targets AKT and ERK1/2 for three of them. Clinically, HGF-associated PL was diverse, but predominantly bilateral in the lower limbs with onset varying from early childhood to adulthood. Finally, aggregation study in a second independent cohort underscored that rare likely pathogenic variants in HGF explain about 2% of PL. Therefore, HGF signalling seems crucial for lymphatic development and/or maintenance in human beings and HGF should be included in diagnostic genetic screens for PL.

The VASCERN PPL working group patient pathway for primary and paediatric lymphoedema.

Lymphoedema is caused by an imbalance between fluid production and transport by the lymphatic system. This imbalance can be either caused by reduced transport capacity of the lymphatic system or too much fluid production and leads to swelling associated with tissue changes (skin thickening, fat deposition). Its main common complication is the increased risk of developing cellulitis/erysipelas in the affected area, which can worsen the lymphatic function and can be the cause of raised morbidity of the patient if not treated correctly/urgently. The term primary lymphoedema covers a group of rare conditions caused by abnormal functioning and/or development of the lymphatic system. It covers a highly heterogeneous group of conditions. An accurate diagnosis of primary lymphoedema is crucial for the implementation of an optimal treatment plan and management, as well as to reduce the risk of worsening. Patient care is diverse across Europe, and national specialised centres and networks are not available everywhere. The European Reference Network on Rare Multisystemic Vascular Diseases (VASCERN) gathers the best expertise in Europe and provide accessible cross-border healthcare to patients with rare vascular diseases. There are six different working groups in VASCERN, which focus on arterial diseases, hereditary haemorrhagic telangiectasia, neurovascular diseases, lymphoedema and vascular anomalies. The working group Paediatric and Primary Lymphedema (PPL WG) gathers and shares knowledge and expertise in the diagnosis and management of adults and children with primary and paediatric lymphoedema. The members of PPL WG have worked together to produce this opinion statement reflecting strategies on how to approach patients with primary and paediatric lymphoedema. The objective of this patient pathway is to improve patient care by reducing the time to diagnosis, define the best management and follow-up strategies and avoid overuse of resources. Therefore, the patient pathway describes the clinical evaluation and investigations that lead to a clinical diagnosis, the genetic testing, differential diagnosis, the management and treatment options and the patient follow up at expert and local centres. Also, the importance of the patient group participation in the PPL WG is discussed.

Current Data and Future Perspectives on Patients with Atrial Fibrillation and Cancer.

Atrial fibrillation (AF) is an increasingly recognized comorbidity in patients with cancer. Indeed, cancer patients have a significantly higher incidence of AF than that observed in the general population. A reciprocal relationship between these two diseases has been observed, as much as some assume AF to be a marker for occult cancer screening, especially in older adults. The pathophysiological mechanisms are many and varied, including the underlying pro-inflammatory state, specific treatments (chemo- and radiotherapy), and surgery. The therapeutic management of patients with cancer and AF involves the same rhythm and frequency control strategies as the general population; however, the numerous interactions with chemotherapeutics, which lead to a significant increase in side effects, as well as the extreme fragility of the patient, should be considered. Anticoagulant therapy is also a complex challenge to address, as bleeding and stroke risk scores have not been fully assessed in this subpopulation. Furthermore, in large studies establishing the efficacy of direct oral anticoagulants (DOACs), cancer patients have been underrepresented. In this review, we elaborate on the mechanisms linking AF to cancer patients with a particular focus on the therapeutic challenges in this population.

Ureteropelvic junction obstruction with primary lymphoedema associated with CELSR1 variants.

BACKGROUND: Primary lymphoedema (PL) is a chronic, debilitating disease caused by developmental and functional defects of the lymphatic system. It is marked by an accumulation of interstitial fluid, fat and tissue fibrosis. There is no cure. More than 50 genes and genetic loci have been linked to PL. We sought to study systematically cell polarity signalling protein Cadherin Epidermal Growth Factor Laminin G Seven-pass G-type Receptor 1 (CELSR1) variants linked to PL. METHODS: We investigated 742 index patients from our PL cohort using exome sequencing. RESULTS: We identified nine variants predicted to cause CELSR1 loss of function. Four of them were tested for nonsense-mediated mRNA decay, but none was observed. Most of the truncated CELSR1 proteins would lack the transmembrane domain, if produced. The affected individuals had puberty/late-onset PL on lower extremities. The variants had a statistically significant difference in penetrance between female patients (87%) and male patients (20%). Eight variant carriers had a kidney anomaly, mostly in the form of ureteropelvic junction obstruction, which has not been associated with CELSR1 before. CELSR1 is located in the 22q13.3 deletion locus of the Phelan-McDermid syndrome. As variable renal defects are often seen in patients with the Phelan-McDermid syndrome, CELSR1 may be the long-sought gene for the renal defects. CONCLUSION: PL associated with a renal anomaly suggests a CELSR1-related cause.

Paediatric lymphoedema: An audit of patients seen by the paediatric and primary lymphoedema group of vascular European Reference Network (VASCERN).

Little is known about the overall prevalence of lymphoedema in children and the types of paediatric lymphoedema seen by specialist centres. Therefore, this study was aimed to provide a profile of children with primary or secondary lymphoedema seen by the expert centres of the paediatric and primary lymphoedema working group (PPL-WG) of VASCERN and to compare the profile between the different countries. A retrospective review of all children (aged up to 18 years) seen for the first time by the expert centres over one year (2019) was carried out. Lymphoedema-, patient- and genetics-related data was collected and described for the whole group and compared between the different European countries/UK. In 2019, a total of 181 new children were seen by eight expert centres. For primary lymphoedema, the phenotype was based on the St George's classification of lymphatic anomalies. The percentages diagnosed according to each category were: 7.2% for syndromic lymphoedema, 2.8% for systemic/visceral involvement, 30.4% for congenital, 35.9% for late-onset lymphoedema and 19.3% for vascular/lymphatic malformations. 4.4% had secondary lymphoedema. Nearly 10% of all children had had at least one episode of cellulitis. The median delay from onset of symptoms to being seen by an expert centre was 2.4 years. In 44.4% of the children with primary lymphoedema a genetic test was performed, of which 35.8% resulted in a molecular diagnosis. Across the different centres, there was a wide variety in distribution of the different categories of paediatric lymphoedema diagnosed and the frequency of genetic testing. In conclusion, this paper has demonstrated that there is a large delay between the onset of paediatric lymphoedema and the first visit in the expert centres and that an episode of cellulitis is a relatively common complication. Diagnostic variation across the centres may reflect different referral criteria. Access to genetic testing was limited in some centres. It is recommended that these issues are addressed in the future work of the PPL-WG to improve the referral to the expert centres and the consistency in service provision for paediatric lymphoedema in Europe.

Recent Advances in Ultrasound Technology: Ultra-High Frequency Ultrasound for Reconstructive Supermicrosurgery.

BACKGROUND: Currently, microsurgeons are in the era of supermicrosurgery and perforator flap reconstruction. As these reconstructions frequently utilize vessels that are smaller than a single millimeter, understanding of location of lymphatic vessels and perforator anatomy preoperatively is essential. To change with the times, the role of ultrasound has changed from just an adjunct to primary imaging of the choice in reconstructive supermicrosurgery. Recently, a novel ultrasonographic technique involving the use of ultra-high frequency ultrasound (UHFUS) frequencies has entered the scene, and appears a promising tool in surgical planning. METHODS: The literatures on the applications of UHFUS in reconstructive supermicrosurgery were retrieved and reviewed from more than 60 literatures have been published on the surgical applications of UHFUS. RESULTS: Nine studies were retrieved from the literature on the applications of UHFUS in reconstructive supermicrosurgery. The articles report both application for lymphatic surgery and perforator flaps. CONCLUSION: UHFUS application involves an increasing number of reconstructive supermicrosurgery field. UHFUS is a valuable and powerful tool for any reconstructive surgeons who are interested in performing supermicrosurgery.

Intraoperative Real-Time Visualization of the Lymphatic Vessels Using Microscope-Integrated Laser Tomography.

BACKGROUND: Detection and selection of the lymphatic vessels are important for maximizing therapeutic efficacy of lymphaticovenular anastomosis (LVA). Some imaging modalities have been reported to be useful for intraoperative identification of the lymphatic vessels, but they have limitations. In this article, we present new capabilities of intraoperative laser tomography, which was used to evaluate the lumen of the lymphatic vessel and to validate the patency of anastomosis. METHODS: Fifty-two patients with upper extremity lymphedema secondary to breast cancer treatment underwent indocyanine green (ICG) lymphography and real-time laser tomography imaging of ICG-enhanced lymphatic vessels intraoperatively before transecting the vessels during LVA. The imaging findings of the lymphatic vessels in laser tomography were investigated. Time required for scanning of the lymphatic vessels was compared between laser tomography and ultrasonography. The correlation between the thickness of the lymphatic vessel wall measured with laser tomographic imaging and the histologically measured thickness of the lymphatic vessel wall was examined. The patency of anastomosis sites was determined based on the image using laser tomography immediately after establishment of LVA. RESULTS: A total of 132 ICG-enhanced lymphatic vessels were scanned with laser tomography showing clear lumen with surrounding vessel wall. The required time for lymphatic vessel scanning was significantly shorter with laser tomography than with ultrasonography (1.6 ± 0.3 vs. 4.8 ± 1.2 minutes; p = 0.016). Strong correlation was seen between the thickness of the lymphatic vessels wall measured using laser tomography and the histologically measured thickness of the lymphatic vessel wall (r = 0.977, 95% confidence interval: 0.897-0.992, p < 0.001). The quality of patency was evaluated immediately after anastomosis, which assisted in deciding whether reanastomosis was needed. CONCLUSION: Microscope-integrated laser tomography provides real-time images of the lymphatic vessels in extremely high resolution and enables evaluation of lymphatic lumen condition and objective post-LVA anastomosis status.

Bedside 3D Visualization of Lymphatic Vessels with a Handheld Multispectral Optoacoustic Tomography Device.

Identification of lymphatics by Indocyanine Green (ICG) lymphography in patients with severe lymphedema is limited due to the overlying dermal backflow. Nor can the method detect deep and/or small vessels. Multispectral optoacoustic tomography (MSOT), a real-time three- dimensional (3D) imaging modality which allows exact spatial identification of absorbers in tissue such as blood and injected dyes can overcome these hurdles. However, MSOT with a handheld probe has not been performed yet in lymphedema patients. We conducted a pilot study in 11 patients with primary and secondary lymphedema to test whether lymphatic vessels could be detected with a handheld MSOT device. In eight patients, we could not only identify lymphatics and veins but also visualize their position and contractility. Furthermore, deep lymphatic vessels not traceable by ICG lymphography and lymphatics covered by severe dermal backflow, could be clearly identified by MSOT. In three patients, two of which had advanced stage lymphedema, only veins but no lymphatic vessels could be identified. We found that MSOT can identify and image lymphatics and veins in real-time and beyond the limits of near-infrared technology during a single bedside examination. Given its easy use and high accuracy, the handheld MSOT device is a promising tool in lymphatic surgery.

The Application of Virtual Reality for Preoperative Planning of Lymphovenous Anastomosis in a Patient with a Complex Lymphatic Malformation.

The management of lymphatic malformations (LMs) is challenging, particularly for large and complex lesions involving anatomical structures in the adjacent tissue. While lymphovenous anastomosis (LVA) has been reported as an effective treatment for lymphedema, it has hardly been described as a treatment for LM. Virtual reality has the ability to visualize human structures in three dimensions and can be used for the preoperative planning of complex cases. Here, we describe the first case of the management of an LM by LVA preoperatively planned with virtual reality. A young woman presented with an LM previously treated by gross excision. Following persistent complaints of swelling, a minimally invasive microsurgical intervention was planned. The results of the single photon emission tomography with computed tomography (SPECT-CT) and lymphoscintigraphy were analyzed using a virtual reality program, and a 3D patient-specific model was constructed. Based on the combined findings of this 3D model and lymphography with a fluorescent marker, a precise skin incision could be determined and one lymph vessel was anastomosed to a nearby vein. The swelling of the thigh reduced and the discomfort disappeared. Although more reports are needed to confirm its efficacy, LVA planned with virtual reality constructed images appears to be a valuable treatment option for complex lesions, including LMs.

Ultra High-frequency Ultrasonographic Imaging with 70 MHz Scanner for Visualization of the Lymphatic Vessels.

BACKGROUND: Identification and localization of functional lymphatic vessels are important for lymphaticovenular anastomosis. Conventional high-frequency ultrasound (CHFUS) has been reported to be useful for them, but it has some disadvantages. In this article, we present new capabilities of ultra high-frequency ultrasound (UHFUS) for imaging of the lymphatic vessels, which may overcome the weakness of CHFUS. METHODS: Thirty unaffected extremities in 30 unilateral secondary lymphedema patients (13 upper limbs and 17 lower limbs) were examined. Identification of the lymphatic vessels using UHFUS and CHFUS were performed at 3 sites in each unaffected extremity. Number and diameter of the detected lymphatic vessels were compared between UHFUS and CHFUS groups. At the same time, new characteristics of the lymphatic vessels seen with UHFUS were investigated. RESULTS: One hundred sixty-nine lymphatic vessels were detected with UHFUS, and 118 lymphatic vessels with CHFUS. The number of lymphatic vessels found in upper and lower extremities was significantly larger with UHFUS than with CHFUS. The diameter of lymphatic vessels found in upper and lower extremities was significantly smaller with UHFUS than with CHFUS. All lymphatic vessels that were detected in UFHUS were less likely to collapse when the transducer was against the skin of the examined sites. CONCLUSIONS: Detection rate of the lymphatic vessels in nonlymphedematous extremities with UHFUS was higher than that with CHFUS. UHFUS provides images with extremely high resolution, demonstrating new characteristics of the lymphatic vessels.

Successful treatment of breast cancer-related breast lymphedema by lymphovenous anastomosis in a male patient.

Men, as well as women may develop breast lymphedema following breast cancer treatment. Microsurgically performed lymphovenous anastomosis (LVA), an effective treatment for lymphedema of the extremities, has also been successfully applied to breast lymphedema. Here we report the first case of breast lymphedema secondary to male breast cancer, treated with supermicrosurgical LVA. A 48-year-old man presented with breast lymphedema following mastectomy, axillary lymph node dissection, and adjuvant radiotherapy. After the oncological treatments, the patient reported a sensation of tension, pain, and swelling of the left breast. The diagnosis of breast lymphedema was confirmed by lymphoscintigraphy. Since conservative treatment with manual lymphdrainage was ineffective, we performed LVAs at the left breast region. In total, two lymph vessels were anastomosed to two nearby veins. Immediately following this intervention, the left breast and lateral thorax region decreased in size and the sensation of tension disappeared. One year postoperative there was no recurrence of the swelling and the patient was very satisfied with the result. Although more reports are needed to confirm its efficacy, supermicrosurgical LVA appears to be a valuable treatment option for breast lymphedema in both women and men.

Lymphatic supermicrosurgery for the treatment of recurrent lymphocele and severe lymphorrhea.

BACKGROUND: Lymphocele and lymphorrhea are frequent complications after lymph node excision. Recurrent lymphoceles and intractable lymphorrhea are particularly difficult to treat conservatively. We describe the outcomes of four patients with recurrent lymphocele and nine patients with persistent lymphorrhea that were treated by supermicrosurgery. METHODS: Four patients with recurrent lymphoceles with a size between 7 and 21 cm and located in the groin (n = 1) or upper leg (n = 3), were referred for surgical treatment between 2013 and 2017 after unsuccessful conservative therapy. Nine patients with lymphorrhea from the groin (n = 7), scrotum (n = 1), or axilla (n = 1) after lymph node or lipoma excision were referred for surgical treatment. Of these, five patients presented with a drainage system and two had a lymphocutaneous fistula. Indocyanine green (ICG) lymphography was used to visualize the lymphatic flow toward the lymphocele, to detect ruptured lymph vessels causing lymphorrhea and for preoperative lymphatic mapping. RESULTS: All 13 patients were successfully treated by one or more (mean: 3, range 1-4) lymphaticovenous anastomoses without perioperative complications. The lymphoceles resolved in all four patients, and no recurrence was recorded during follow-up. The lymphorrhea was cured in all patients by means of lymphaticovenous anastomosis performed distal to the site of leakage. No recurrence was observed during follow-up. The patency of the lymphaticovenous anastomosis was confirmed intraoperatively by means of ICG lymphography in all cases. CONCLUSION: Lymphaticovenous anastomosis is a minimally invasive and effective procedure for the treatment of recurrent lymphocele and persistent lymphorrhea.

Supermicrosurgical lymphaticovenous anastomosis for a patient with breast lymphedema secondary to breast cancer treatment.

Breast lymphedema (BLE) has been reported as a complication following breast cancer treatment. As for extremity lymphedema treatment, supermicrosurgical lymphaticovenous anastomosis (LVA) is considered an option for the treatment of progressive BLE refractory to conservative treatments, but no case has been reported so far. We report the first case of BLE successfully treated with supermicrosurgical LVA. A 55-year-old female presented with left BLE after breast conserving surgery and axillary lymph node dissection and adjuvant radiotherapy. After the cancer treatments, the patient suffered from pronounced swelling, sensation of tension, and pain of the left breast with frequent episodes of breast cellulitis. Conservative treatments had been performed for 7 years, but were not effective, and the patient was referred for further surgical treatment. Supermicrosurgical LVA was performed at the left breast. LVA surgery resulted in three anastomoses, in which three lymphatic vessels were anastomosed to three nearby veins in an intima-to-intima coaptation manner. After LVA, the left breast decreased in size, and the sensation of tension disappeared. One year postoperatively, the patient had no distress nor cellulitis episode, and was satisfied with the results of normal breast conditions. Although further clinical studies are required to confirm efficacy, supermicrosurgical LVA has the potential to be an option for the treatment of progressive BLE refractory to conservative treatments.