Catarina Hadamitzky

Acquired lymphedema: an urgent need for adequate animal models.

In cancer patients, the removal of tumor-draining lymph nodes during tumor resection can lead to acquired lymphedema. This disease, which is characterized by tissue swelling and increased risk of infection due to restricted lymph flow, lacks an effective treatment. Limitations to the design and conduct of randomized trials to date have limited the evaluation of proposed surgical techniques. As a result, animal models have provided an important research base. This review summarizes work in canine, rabbit, and rodent models of acquired lymphedema, focusing on arising limitations and potential applications.

Age‐dependent histoarchitectural changes in human lymph nodes: an underestimated process with clinical relevance?

Experimental evidence indicates that lymph nodes in humans undergo alterations during ageing. This is clinically important because of the crucial role of these organs in the immune system and their lymph reabsorption and drainage function. Although some age‐related changes in lymph node histoarchitecture have been described, they are seldom taken into account in traditional depictions of lymph nodes. Recently introduced clinical procedures, such as intranodal vaccination or lymph node transplantation, have demonstrated the need for an accurate knowledge of these degenerative processes. In this study, superficial inguinal lymph nodes were obtained from 41 deceased patients between 17 and 98 years old. To minimize immunological influences, such as chronic diseases, specimens were only obtained from forensic pathology autopsies. An immunohistochemical analysis was carried out, on the basis of which lymph node degeneration was scored according to the numbers of lymphocytes and high endothelial venules, and degree of fibrosis and lipomatosis. We observed an age‐dependent tendency towards the replacement of areas populated with diverse immune cells by connective tissue. Paradoxically, these changes were also detected in some of the nodes from younger age groups. In conclusion, lymph nodes can display degenerative changes that are mainly age‐related and often diverge from the common description found in textbooks. These alterations should be taken into account when dealing with lymph nodes diagnostically and therapeutically in clinical practice.

Regeneration of autotransplanted avascular lymph nodes in the rat is improved by platelet-rich plasma.

The aim of this study was to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, in analogy to results obtained in other species. This rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. The results show for the first time that the rat is an adequate model to study the regeneration of transplanted lymph nodes. Lymph node fragmentation seems to affect transplant regeneration negatively. An immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. However, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. Lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. Acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. This disease causes lifelong disability due to chronic swelling and increased risk of infections. It currently lacks an effective treatment.

Comprehensive genetic and functional characterization of IPH-926: a novel CDH1-null tumour cell line from human lobular breast cancer†

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E‐cadherin. ILBCs also lack β‐catenin expression and show aberrant cytoplasmic localization of the E‐cadherin binding protein p120‐catenin. The lack of a well‐characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH‐926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH‐926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH‐926 expressed various epithelial cell markers but lacked expression of E‐cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH‐926 from this particular tumour. IPH‐926 also lacked β‐catenin expression and showed aberrant cytoplasmic localization of p120‐catenin. Array‐CGH analysis of IPH‐926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH‐926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH‐926 cells were anti‐cancer drug‐resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH‐926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH‐926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright

Improved Regeneration of Autologous Transplanted Lymph Node Fragments by VEGF-C Treatment

Secondary lymphedema is a common complication after removal of lymph nodes in combination with radiation therapy in the treatment of breast cancer, cervical cancer, and melanomas. Only symptomatic therapies are available at the moment, and lymphedema is for most patients a lifelong condition involving psychological and physical disabilities. Animal models exist to study the pathophysiology of lymphedema but not to study surgical treatments. The aim of this study was to show that regeneration of autologous transplanted lymph node fragments is possible in rats that were irradiated previously locally in the groin and to examine the effects of vascular endothelial growth factor (VEGF)‐C injections on the rate of regeneration of transplanted lymph nodes. In all of the animals, inguinal and popliteal lymph nodes and adjacent lymphatic vessels were unilaterally removed and the inguinal region irradiated by a single dose of 15 Gy. Afterward, lymph node fragments were transplanted subcutaneously in the irradiated region. Half of the animals were treated by local VEGF‐C injections after transplantation. Four weeks after transplantation, drainage of the leg was tested by injection of blue dye, and the transplanted fragments were removed and examined immunohistologically. We could show that regeneration of autologous transplanted lymph node fragments is possible in areas treated with radiotherapy in the rat. We also documented that transplants can achieve a connection to the lymphatic collectors of the leg. The results suggest that the outcome of regeneration can be improved by injection of VEGF‐C in the transplantation area. Thus, lymph node fragment regeneration may be relevant for lymphedema prevention and therapy. Anat Rec, 2012.

Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema.

Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

VEGF‐C improves regeneration and lymphatic reconnection of transplanted autologous lymph node fragments: An animal model for secondary lymphedema treatment

Secondary lymphedema occurs after for example breast cancer surgery and radiation in 20–50% of the patients. Due to the poor outcomes of surgical treatments in the past, the therapy often remains symptomatic. However, avascular transplantation of autologous lymph node fragments (LN‐Tx) combined with postoperative injections of vascular endothelial growth factor‐C (VEGF‐C) emerges as a potential surgical therapy. In this study, adult rats underwent LN‐Tx to investigate the following parameters of VEGF‐C application: time point, location and dosage. Furthermore, the influences of VEGF‐C on lymphatic reconnection and transplant regeneration were analyzed. The reconnection was investigated using intradermally injected blue dye and the regeneration was evaluated histologically using hematoxylin‐eosin (H&E) staining and immunohistochemistry. The higher dosage enhanced the reconnection rates significantly and showed a statistical tendency of improving regeneration. An application on early postoperative days and the injection into the medial thigh improved the reconnection significantly. However, these variables did not affect the regeneration statistically. This study confirms that LN‐Tx combined with lymphatic growth factor VEGF‐C is a possible approach in the therapy of secondary lymphedema and shows the important role of VEGF‐C application parameters.

Quantification of Lymphedema in a Rat Model by 3D-Active Contour Segmentation by Magnetic Resonance Imaging

Secondary lymphedema is a common complication after lymph node excision and radiotherapy in cancer therapy. Therapies are limited to symptomatic treatment. Adequate animal models to test potential surgical therapies are needed. The aim of this study was to induce a tissue environment in the hind leg of the rat similar to the one found in operated and irradiated patients. Quantification of edematous swelling was performed by an automatic 3D-contour segmentation (ITK- Snap ©) on MR- images. Swelling was induced by excision of superficial inguinal and popliteal lymph nodes and adjacent lymphatic vessels, followed by radiotherapy of the right groin with a single dose of 15 Gy. Four weeks after irradiation, the animals were examined with MRI of both hind legs. Fluid volumes around the joint line of the knee were calculated on T2-weighted images. We documented a significant higher volume of fluid in the legs following excision of lymph nodes and lymphatic vessels, combined with radiotherapy than in control legs.

Treatment options for head and neck lymphoedema after tumour resection and radiotherapy.

We read with great interest the recent publication of Mihara et al. 2011 entitled “lymphaticovenous anastomosis for facial lymphoedema after multiple courses of therapy for head and neck cancer”. Here, treatment of therapy resistant lymphoedema of the head and neck region with lymphaticovenous anastomosis (LVA) was described in one patient after multiple tumour resections and radiotherapy. This was an extremely rare case with five consecutive tumours of the neck. Only by collecting such rare cases can an improvement in the treatment of head and neck lymphoedema be established. This journal seems to be an adequate forum for this purpose. Lymphoedema of the facial area is functionally and aesthetically disturbing and in severe cases symptomatic treatment through complex decongestive therapy is not effective. Additionally, intracranial changes may occur as drainage of cerebrospinal fluid along cerebral nerves can be impaired.

Effect of cryopreservation on lymph node fragment regeneration after autologous transplantation in the minipig model

Abstract Introduction: Lymphoedema is a worldwide pandemic causing swelling of tissues due to dysfunctional transport of lymph fluid. Present management concepts are based in conservative palliation of symptoms through manual lymphatic drainage, use of compression garments, manual lymph drainage, exercise, and skin care. Nevertheless, some curative options as autologous lymph node transplantation were shown to reduce lymphoedema in selected cases. Lately, some concern has arisen due to reports of donor site morbidity. A possible solution could be the development of artificial lymph node scaffolds as niches of lymphatic regeneration. Engineering these scaffolds has included cryopreservation of lymph node stroma. However, the effects of cryopreservation on the regeneration capacities of these organs were unknown. Materials and methods: Here, we used the minipig animal model to assess lymphatic regeneration processes after cryopreservation of autologous lymph nodes. Superficial inguinal lymph nodes were excised and conserved at −80°C for 1 month. Thereafter, lymph node fragments were transplanted in the subcutaneous tissue. Results: Regeneration of the lymph nodes was assessed five months after transplantation. We show that lymph node fragment regeneration takes place in spite of former cryopreservation. Transplanted fragments presented typical histological appearance. Their draining capacity was documented by macroscopic transport of Berlin Blue dye as well as through SPECT-CT hybrid imaging. Discussion: In conclusion, our results suggest that processes of cryopreservation can be used in the creation of artificial lymph node scaffolds without major impairment of lymph node fragments regeneration.