S1 guidelines: Lipedema.

The present, revised guidelines on lipedema were developed under the auspices of and funded by the German Society of Phlebology (DGP). The recommendations are based on a systematic literature search and the consensus of eight medical societies and working groups. The guidelines contain recommendations with respect to diagnosis and management of lipedema. The diagnosis is established on the basis of medical history and clinical findings. Characteristically, there is a localized, symmetrical increase in subcutaneous adipose tissue in arms and legs that is in marked disproportion to the trunk. Other findings include edema, easy bruising, and increased tenderness. Further diagnostic tests are usually reserved for special cases that require additional workup. Lipedema is a chronic, progressive disorder marked by the individual variability and unpredictability of its clinical course. Treatment consists of four therapeutic mainstays that should be combined as necessary and address current clinical symptoms: complex physical therapy (manual lymphatic drainage, compression therapy, exercise therapy, and skin care), liposuction and plastic surgery, diet, and physical activity, as well as psychotherapy if necessary. Surgical procedures are indicated if - despite thorough conservative treatment - symptoms persist, or if there is progression of clinical findings and/or symptoms. If present, morbid obesity should be therapeutically addressed prior to liposuction.

S1-Leitlinie Lipödem.

Die vorliegende überarbeitete Leitlinie zum Lipödem wurde unter der Federführung der Deutschen Gesellschaft für Phlebologie (DGP) erstellt und finanziert. Die Inhalte beruhen auf einer systematischen Literaturrecherche und dem Konsens von acht medizinischen Fachgesellschaften und Berufsverbänden. Die Leitlinie beinhaltet Empfehlungen zu Diagnostik und Therapie des Lipödems. Die Diagnose ist dabei auf der Basis von Anamnese und klinischem Befund zu stellen. Charakteristisch ist eine umschriebene, symmetrisch lokalisierte Vermehrung des Unterhautfettgewebes an den Extremitäten mit deutlicher Disproportion zum Stamm. Zusätzlich finden sich Ödeme, Hämatomneigung und eine gesteigerte Schmerzhaftigkeit der betroffenen Körperabschnitte. Weitere apparative Untersuchungen sind bisher besonderen Fragestellungen vorbehalten. Die Erkrankung ist chronisch progredient mit individuell unterschiedlichem und nicht vorhersehbarem Verlauf. Die Therapie besteht aus vier Säulen, die individuell kombiniert und an das aktuelle Beschwerdebild angepasst werden sollten: komplexe physikalische Entstauungstherapie (manuelle Lymphdrainage, Kompressionstherapie, Bewegungstherapie, Hautpflege), Liposuktion und plastisch-chirurgische Interventionen, Ernährung und körperliche Aktivität sowie ggf. additive Psychotherapie. Operative Maßnahmen sind insbesondere dann angezeigt, wenn trotz konsequent durchgeführter konservativer Therapie noch Beschwerden bestehen bzw. eine Progredienz des Befundes und/oder der Beschwerden auftritt. Eine begleitend zum Lipödem bestehende morbide Adipositas sollte vor einer Liposuktion therapeutisch angegangen werden.

Characterisation of Prognosis and Invasion of Cutaneous Squamous Cell Carcinoma by Podoplanin and E-Cadherin Expression.

BACKGROUND: Around 5% of all cutaneous squamous cell carcinoma (cSCC) metastasise. Metastases usually locate in regional skin and lymph nodes, suggesting collective cancer invasion. The cellular level of tumour invasion and prognostic parameters remain to be characterised. METHODS: We performed immunohistochemical analyses of E-cadherin (marker for collective cancer invasion) and podoplanin (marker for epithelial-mesenchymal transition [EMT], single-cell invasion) expression in 102 samples of metastatic and non-metastatic cSCC and 18 corresponding skin and lymph node metastases to characterise the invasion of cSCC. Immunohistochemical results were retrospectively correlated with clinical data. RESULTS: E-cadherin was highly expressed in metastatic and non-metastatic cSCC and skin metastases. This suggests collective cancer invasion. However, E-cadherin was downregulated in poorly differentiated cSCC and lymph node metastases, suggesting partial EMT. Podoplanin was significantly upregulated in metastatic (p = 0.002) and poorly differentiated (p = 0.003) cSCC. Overexpression of podoplanin represented a statistically independent prognostic factor for disease-free survival (p = 0.014). CONCLUSION: Collective cancer invasion is likely in cSCC. In lymph node metastases and poorly differentiated cSCC, partial EMT is possible. Podoplanin is an independent prognostic parameter for metastasis.

No human virus sequences detected by next-generation sequencing in benign verrucous skin tumors occurring in BRAF-inhibitor-treated patients.

Patients treated with BRAF inhibitors (e.g. vemurafenib), a novel targeted therapy for advanced melanoma harbouring certain BRAF mutations, develop numerous adverse cutaneous side effects, including skin tumors such as squamous cell carcinoma or non-malignant verruciform keratinocyte proliferations, termed 'BRAF-inhibitor-associated verrucous keratosis (BAVK) lesions'. These keratinocyte proliferations are believed to be caused by paradoxical hyperactivation of the MAPK pathway in cells with wild-type BRAF, but mutated RAS. However, due to the clinical and histological verruca-like appearance of these lesions, additional aetiologic cofactors, such as infectious agents (i.e. oncogenic viruses), might be suspected. Therefore, we performed 454 high-throughput sequencing of BAVK lesions from vemurafenib-treated patients on the transcript level to identify actively transcribed viral sequences of known [e.g. human papilloma viruses (HPV)] or even yet-unknown viruses. Next-generation sequencing did not identify transcripts of any human viruses out of 1 595 161 reads obtained from BAVK lesions of four patients. Nevertheless, all controls were recognized correctly, and the detection of sequences derived from the cutaneous microbiome (e.g. skin commensals and bacterial phages) confirmed the validity and sensitivity of the sequencing data. Our results are consistent with preliminary histological and immunohistochemical findings recently reported by others, who also failed to detect the expression of HPV proteins in BAVK. Although the patient number is limited and we cannot exclude the possibility of having missed a viral transcript of very low abundance, our study argues against a viral aetiology of BRAF-inhibitor-associated verruciform keratoses occurring under vemurafenib.

Collagen-based cell migration models in vitro and in vivo.

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.

Hyperforin and aristoforin inhibit lymphatic endothelial cell proliferation in vitro and suppress tumor-induced lymphangiogenesis in vivo.

The phloroglucinol derivative hyperforin, a major bioactive constituent of St. John's wort, is increasingly recognized as being able to regulate a variety of pathobiological processes and, thus, to possess potential therapeutic properties. In the context of cancer, hyperforin induces the apoptosis of cancer cells, inhibits angiogenesis and suppresses metastasis formation. Here, we report a new pharmacological function of hyperforin and its stabilized derivative aristoforin, namely the suppression of lymphatic endothelial cell (LEC) growth and lymphangiogenesis. At concentrations less than 10 microM, we found that these compounds induce cell cycle arrest of LECs, and at higher concentrations induce apoptosis. The loss of mitochondrial membrane potential and the activation of caspase-9 during the induction of apoptosis indicate that the intrinsic pathway of apoptosis is stimulated by these compounds, similar to the situation in tumor cells. In thoracic duct ring outgrowth assays, hyperforin and aristoforin both inhibited lymphangiogenesis, as evidenced by the suppression of lymphatic capillary outgrowth. In an in vivo animal model, both compounds were able to inhibit tumor-induced lymphangiogenesis. Together these data substantiate a new role for hyperforin and its derivatives as suppressors of lymphangiogenesis, and support their further investigation as potential anticancer drugs that target tumor growth and metastasis at multiple levels.

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.

First Association of Interleukin 12 Receptor Beta 1 Deficiency with Sjögren's Syndrome.

INTRODUCTION: Interleukin 12 receptor beta 1 (IL12Rß1) deficiency is a primary immunodeficiency resulting mainly in susceptibility to opportunistic infection by non-tuberculous, environmental mycobacteria and severe infection caused by Salmonella spp. Till now, less than 300 patients with IL12Rß1 deficiency have been reported. Among them, only three have been described to develop autoimmunity. CASE PRESENTATION: We present the case of a 50-year-old male with IL12Rß1 deficiency due to compound heterozygosity [c. 1623_1624delGCinsTT (pGln542Stop) and c.1791 + 2T > C (donor splice site)], who-18 months after diagnosis of disseminated BCGitis-presented with recurrent fever and sicca syndrome. No indication of an infectious origin of these symptoms could be found at that point. The diagnosis of a Sjögren's syndrome (SS) was made on the basis of fulfilled American-European consensus classification criteria, including a positive minor salivary gland biopsy. CONCLUSION: Apart from persistent antigenic stimulation, which may drive autoimmune inflammation in primary immunodeficiency, evidence on the involvement of interleukin 12 in pathogenesis of SS suggests that the same immunological mechanism may underlie both defense against infection and the maintenance of tolerance. To our knowledge, this is the first report of a case of autoimmunity in the form of SS in a patient with a primary immunodeficiency and one of the rare cases of IL12Rß1 deficiency with manifested autoimmunity.

Allele frequencies of BRAFV600 mutations in primary melanomas and matched metastases and their relevance for BRAF inhibitor therapy in metastatic melanoma.

BACKGROUND: The detection of BRAFV600 mutations in patients with metastatic melanoma is important because of the availability of BRAF inhibitor therapy. However, the clinical relevance of the frequency of BRAFV600 mutant alleles is unclear. PATIENTS AND METHODS: Allele frequencies of BRAFV600 mutations were analyzed by ultra-deep next-generation sequencing in formalin-fixed, paraffin-embedded melanoma tissue (75 primary melanomas and 88 matched metastases). In a second study, pretreatment specimens from 76 patients who received BRAF inhibitors were retrospectively analyzed, and BRAFV600 allele frequencies were correlated with therapeutic results. RESULTS: Thirty-five patients had concordantly BRAF-positive and 36 (48%) patients had concordantly BRAF-negative primary melanomas and matched metastases, and four patients had discordant samples with low allele frequencies (3.4-5.2%). Twenty-six of 35 patients with concordant samples had BRAFV600E mutations, three of whom had additional mutations (V600K in two patients and V600R in one) and nine patients had exclusively non-V600E mutations (V600K in eight patients and V600E -c.1799_1800TG > AA- in one patient). The frequency of mutated BRAFV600 alleles was similar in the primary melanoma and matched metastasis in 27/35 patients, but differed by >3-fold in 8/35 of samples. BRAFV600E allele frequencies in pretreatment tumor specimens were not significantly correlated with treatment outcomes in 76 patients with metastatic melanoma who were treated with BRAF inhibitors. CONCLUSIONS: BRAFV600 mutation status and allele frequency is consistent in the majority of primary melanomas and matched metastases. A small subgroup of patients has double mutations. BRAFV600 allele frequencies are not correlated with the response to BRAF inhibitors.

The lymphatic system in the dorsal skinfold chamber of the Syrian golden hamster in vivo.

The lymphatic network contributes to maintaining tissue homeostasis and immunological function by transporting fluid, plasma protein and cells from peripheral tissue via the lymph nodes into the blood vascular system. In contrast to the blood circulatory system, little is known about the lymphatic system. In particular, suitable animal models are lacking. Therefore, the dorsal skinfold chamber model was used to investigate the existence of a lymphatic system. To analyze the lymphatic network Syrian golden hamsters (n=12) fitted with titanium chambers were used. FITC-dextran of different concentrations (5% or 25%) and different molecular weights (4, 40 or 150 kDa) was used to contrast lymphatic vessels and measure initial lymph flow velocity. Intravital fluorescence microscopy enabled the quantification of diameter, velocity and branching order. Histology and electron microscopy supported the in vivo findings. Immediately after intradermal injection of FITC-dextran the lymphatics including valves were visible. The diameters of the lymphatic vessels (n=189) ranged from 133+/-5.4 microm (branching order 1) to 26+/-4.0 microm (branching order 5). Using different molecular weights of FITC-dextran, no significant differences in velocity were measured (327+/-157 microm/s with 4 kDa, 391+/-126 microm/s with 40 kDa, and 378+/-175 microm/s with 150 kDa). Blood and lymphatic vessels could not be differentiated clearly by H&E staining. However, endothelial cells of vessels with an irregularly shaped lumen containing no erythrocytes in cross section showed a weaker signal for CD31 staining as compared to endothelial cells of vessels containing erythrocytes. Moreover, transmission electron microscopy identified the dye-containing vessels as lymphatics after intradermal injection of Berlin Blue. In conclusion, a lymphatic network was characterized in the dorsal skinfold chamber model of the Syrian golden hamster. Thus, this well-established animal model for intravital microscopy provides the opportunity to elucidate the physiological and pathological function of the lymphatic vascular system.

Apoptosis and leucocyte-endothelium interactions contribute to the delayed effects of cryotherapy on tumours in vivo.

In dermatology, cryotherapy is commonly used to treat benign and malignant skin lesions. However, studies investigating the time-course of the direct and delayed effects on the microvasculature and subsequent tissue destruction are lacking. Amelanotic melanomas (A-Mel-3) were implanted into the dorsal skinfold chamber of Syrian Golden hamsters (n=51). Tumour and normal tissue were frozen to -20 masculine C (cooling rate 150 masculine C/min). Intravital fluorescence microscopy was performed to assess microvascular changes and leucocyte-endothelium interactions up to 24 h. The relative degrees of necrosis and apoptosis and the accumulation of leucocytes were investigated by histology and immunohistochemistry. Apoptosis was quantified using the TUNEL assay in combination with computer-assisted image analysis. After cryotherapy, red blood cell velocity (RBCV) decreased in postcapillary venules and tumour vessels, whereas functional vessel density (FVD) was significantly reduced in tumour vessels as compared with postcapillary venules. Leucocyte-endothelium interaction increased first in normal and tumour tissue, and then after 24 h leucocytes accumulated in normal tissue close to the tumour margin. Necrosis was induced in the cryolesions directly after freezing and remained constant over the entire observation period. In contrast, apoptosis increased in the periphery of the tumour tissue up to 24 h following cryotherapy. In conclusion, tissue destruction by cryotherapy is not only induced by direct necrosis and microvascular stasis, but also by the inflammatory infiltrate and subsequent apoptosis. This could be an important finding regarding the generation of an immune response.

Role of kindlin-2 in fibroblast functions: implications for wound healing.

Kindlins are a protein family that regulates cell-matrix interactions. Although kindlin-2 is known to be essential for the early developmental processes, its role in the homeostasis of adult tissues has remained elusive. In this study, we used new domain-specific antibodies and small interfering RNA technology to uncover physiological functions of kindlin-2 in human dermal fibroblasts in vitro and in adult skin in situ. In primary dermal fibroblasts, kindlin-2 is essential for the integrin clustering and activation, and it regulates cell adhesion and directed migration by guiding formation and maturation of focal adhesions (FAs) and organization of the cytoskeleton. These functions are linked to the transmission of mechanical cues between cells and their microenvironment, typically in processes wherein fibroblasts differentiate to myofibroblasts under mechanical tension. In concert, kindlin-2 was shown to be required for the stabilization and maturation of FAs and stress fibers in activated fibroblasts and myofibroblasts. These findings implicated that in physiological wound closure and tissue repair, the prediction was validated by strong upregulation of kindlin-2 in contractile myofibroblasts during middle stages of wound healing in human skin. Taken together, the data reveal a physiological role for kindlin-2 in skin fibroblasts under normal steady-state conditions and during tissue regeneration.