Immunohistochemistry For Podoplanin Research Articles

Abstract PD2-09: Characterization of the lymphovascular invasion microenvironment reveals immune response dichotomy

Abstract Background: Metastasis is the leading cause of cancer related deaths in breast cancer patients. Lymphovascular invasion represents one of the earliest stages of metastasis wherein the cells are introduced to a very different and distinct microenvironment. Methods: We leveraged spatial techniques developed for limited specimens in archival tissue to study patient matched cross-sectional tumor samples from different stages of breast neoplasia including normal breast, ductal carcinoma in situ (DCIS), primary invasive carcinoma (IBC), lymphovascular invasion (LVI) and regional lymph node metastasis. We selected a set of 21 patients with ER+ breast cancer to generate cross-sectional samples of each of these stages, for a total of 331 samples. The areas of LVI were identified by a combination of H&E review and immunohistochemistry for podoplanin. We performed smart-3SEQ for gene expression profiling and light pass whole genome sequencing for DNA copy number alterations. Results: We profiled the spectrum of neoplasia for transcriptome-wide gene expression. Principal component analysis of all 252 DCIS, LVI, IBC, or metastasis samples using the top 500 genes with the highest variance demonstrated that clustering was roughly based on the diagnostic stage (i.e. DCIS, LVI, IBC, or metastasis). Differential gene expression profiling identified thousands of genes increased or decreased in expression across the transitional stages with the largest change in gene expression being the transition from normal breast to DCIS, dominated by gene expression down regulation. We next performed NMF clustering on 62 samples of LVI from 18 cases and identified two patterns of gene expression which define two subgroups. Gene ontology analysis revealed that one cluster was associated with increased proliferation and metabolism, whereas the second cluster was dominated by an immune response. When we analyzed the immune and proliferative LVI subgroups separately, we found that the immune profiles in the patient matched IBC and LVI samples from the LVI Immune cluster were similar, whereas the immune profiles in the patient matched IBC and LVI samples from the Proliferative cluster were significantly different. At the LVI stage, all immune cell populations estimated by CibersortX were decreased in the Proliferative LVI cluster. These changes were validated using immunofluorescence for proliferation (Ki67), T cells (CD3) and macrophages (CD68) on the same samples. Using the LVI centroids, we built a model that could predict the same clusters in the METABRIC IBC. Kaplan-Meier analysis showed a significant difference between groups, with the Proliferative-like IBC group having a worse prognosis than the Immune-like IBC group. Conclusions: We observed a dichotomy at the LVI stage with a more proliferative cluster that may escape the immune response and an immune cluster which has a microenvironment with a similar pattern to its primary IBC. The recognition of two groups of LVI, differing in immune association and proliferation, raises the possibility that the risk of metastasis could be different in these two groups, leading to different biological pathways of progression. Citation Format: Belén Rivero-Gutiérrez, Diego Mallo, Almudena Espín-Pérez, Sujay Vennam, Chunfang Zhu, Sushama Varma, Greg Scott, Joseph Foley, E Shelley Hwang, Carlo Maley, Robert West. Characterization of the lymphovascular invasion microenvironment reveals immune response dichotomy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-09.

Human collecting lymphatic glycocalyx identification by electron microscopy and immunohistochemistry

Blood flow is translated into biochemical inflammatory or anti-inflammatory signals based onshear stress type, by means of sensitive endothelial receptors. Recognition of the phenomenon is of paramount importance for enhanced insights into the pathophysiological processes of vascular remodeling. The endothelial glycocalyx is a pericellular matrix, identified in both arteries and veins, acting collectively as a sensor responsive to blood flow changes. Venous and lymphatic physiology is interconnected; however, to our knowledge, a lymphatic glycocalyx structure has never been identified in humans. The objective of this investigation is to identify glycocalyx structures from ex vivo lymphatic human samples. Lower limb vein and lymphatic vessels were harvested. The samples were analyzed by transmission electron microscopy. The specimens were also examined by immunohistochemistry. Transmission electron microscopy identified a glycocalyx structure in human venous and lymphatic samples. Immunohistochemistry for podoplanin, glypican-1, mucin-2, agrin and brevican characterized lymphatic and venous glycocalyx-like structures. To our knowledge, the present work reports the first identification of a glycocalyx-like structure in human lymphatic tissue. The vasculoprotective action of the glycocalyx could become an investigational target in the lymphatic system as well, with clinical implications for the many patients affected by lymphatic disorders.

What We Need to Learn When Exploring the Mixed Basal Cell Carcinoma of Head and Neck

Abstract Coexistence of different histopathological types of basal cell carcinomas (BCC) in the same anatomical localisation is rare, and, therefore, is engaging for histopathologists and clinicians. In many cases, the determination of a neoplasm type remains difficult, since BCC may consist of more than one histopathological subtype. Mixed BCCs often present with an aggressive course and recurrence when compared to other subtypes of a tumour. Furthermore, tumours of this type are associated with time-consuming treatment and not a very satisfactory cosmetic result, thus worsening the quality of the patient’s life. Several clinical studies have been published regarding the histopathologically diverse tumours developed in the same anatomical region; however, largely peculiarities of mixed BCCs are not explored sufficiently. The purpose of this study was to substantiate the use of dermoscopy and morphology, assessing mixed type BCC of the head and neck. The tumours were removed with a surgical excision of 1 cm margins, and the tumour sites were assessed in a 24-month-long follow-up period. The dermoscopic characteristics of mixed and aggressive BCC are analysed in this study. Finally, to better estimate the invading cone of the tumour, a complex morphology, which included collagen type IV and podoplanin immunohistochemistry, and electron microscopy were used.

Analysis of the inflammatory tumor microenvironment in meningeal neoplasms.

Properties of the inflammatory tumor microenvironment are associated with disease subtype, grade, and prognosis in various cancer entities. As immune-modulatory therapies are currently being explored in patients with meningeal neoplasms, we investigated their inflammatory microenvironment (meningiomas and solitary fibrous tumor/hemangiopericytoma (SFT/HPC)). 74 meningeal tumor specimens: (10/74 (13.5%) atypical meningioma; 8/74 (10.8%) anaplastic meningioma; 8/74 (10.8%) chordoid meningioma; 9/74 (12.2%) fibroblastic meningioma; 10/74 (13.5%) transitional meningioma; 3/74 (4.1%) rhabdoid meningioma; 7/74 (9.5%) meningothelial meningioma; SFT/HPC (19/74 (25.7%) were retrieved from the Neuro-Biobank, Medical University of Vienna, Austria. Tumor-infiltrating lymphocyte (TIL) infiltration could be observed in the majority of the investigated specimens (CD3+: 66/74 (89.2%); CD8+: 47/74 (63.5%); CD45RO+: 29/73 (39.2%); FOXP3+ 19/74 (25.7%); PD1+: 3/74 (4.1%). No difference in TIL infiltration was observed between SFT/HPC and meningioma cases. Higher density of FOXP3+ TILs was observed with increasing WHO grade in meningioma specimens (p = 0.005). Membranous programmed cell death ligand 1 (PD-L1) expression was observed in 4/74 (5.4%) specimens, with 3/74 (4.1%) presenting with 1% and 1/74 (1.4%) with 3% PD-L1 expressing tumor cells. Lymphatic vessels as identified by podoplanin immunohistochemistry were observed in 10/74 (13.5%) specimens and were significantly associated with presence of membranous PD-L1 expression on tumor cells (p = 0.003). Infiltration by various TIL subtypes can be observed in the majority of meningeal neoplasms, with enrichment of FOXP3-positive regulatory T-cells in higher-grade meningioma. PD-L1 expression on tumor cells was only infrequently found. A better understanding of the pathobiological role of the immune system in meningeal neoplasms may facilitate development of immunomodulatory treatment approaches in meningeal tumors.

High reproducibility is attainable in assessing histoprognostic parameters of pT1 colorectal cancer using routine histopathology slides and immunohistochemistry analyses

Assessment of the risk of lymph node invasion and tumour recurrence is critical to determine whether additional surgery is required in patients with endoscopically-removed pT1 colorectal cancer (CRC). A reproducible assessment of this risk of recurrence based on histopathological parameters is crucial for relevant therapeutic decisions. The inter-observer reproducibility of these parameters was the subject of our study. Two pathologists independently analysed 163 endoscopically-removed pT1 CRC recorded in a local digestive cancer registry database (Finistère, France). Using haematoxylin-eosin-saffron (HES) and immunohistochemistry slides, they evaluated several parameters related to the risk of tumour recurrence according to the international pT1 CRC-dedicated guidelines. Based on Kappa and intra-class correlation coefficients, good to very good inter-observer agreement was obtained by analysing vertical and lateral margins, submucosal invasion, tumour differentiation and lymphovascular invasion. The reproducibility of tumour budding quantification was only fair on the basis of HES slides but reached a very good agreement using cytokeratin immunohistochemistry. Dual colour cytokeratin and podoplanin immunohistochemistry also improved inter-observer agreement for the detection of lymphovascular invasion. All patients with loco-regional nodal metastases (7 of 101 who underwent complementary surgery) or distant metastases (3 patients) were diagnosed as having a high risk of recurrence and requiring an additional surgery by the two observers. Our study showed that good to very good inter-observer agreement is achievable in evaluating the pathological parameters of recurrence risk in endoscopically-removed pT1 CRC. In addition to HES slides, the detection of lymphovascular invasion and tumour budding can benefit with more reproducible immunohistochemical analyses.

Immunohistochemical expression of podoplanin (D2-40), lymphangiogenesis, and neoangiogenesis in tooth germ, ameloblastomas, and ameloblastic carcinomas.

Ameloblastoma is a benign but locally aggressive odontogenic tumor, while ameloblastic carcinoma is its malignant counterpart. Angiogenesis and lymphangiogenesis in malignancies have been correlated with higher aggressiveness and poor prognosis, as well as greater expression of podoplanin by tumoral cells. Immunohistochemical expression of podoplanin, CD34, and CD105 (endoglin) was evaluated in 53 ameloblastomas and three ameloblastic carcinomas; additionally, immunohistochemistry for podoplanin was also performed in 10 tooth germs. Microvessel density of blood and lymphatic vessels was calculated and compared between ameloblastomas and ameloblastic carcinomas. Immunoexpression of podoplanin by ameloblastic cells was evaluated in tooth germs, ameloblastomas, and ameloblastic carcinomas. Podoplanin was similarly expressed by odontogenic epithelial cells of tooth germs and ameloblastomas, while its expression was lower in ameloblastic carcinomas. There was no difference in microvessel density assessed by CD34 between ameloblastomas and ameloblastic carcinomas; nevertheless, the latter presented higher amounts of lymphatic and new formed blood vessels. Results suggest that podoplanin does not seem to be involved in invasion mechanisms of ameloblastic carcinomas, as its expression was decreased in the malignant tumoral cells. On the other hand, the increased lymphatic microvessel density and neoangiogenesis found in ameloblastic carcinomas could be related to its aggressiveness and potential for metastasis.

Prevention of Postsurgical Lymphedema by 9-cis Retinoic Acid

To determine the effect of 9-cis retinoic acid (9-cis RA) on postsurgical lymphedema. 9-cis RA promotes lymphangiogenesis in vitro and in vivo and has promise as a therapeutic agent to limit the development of postsurgical lymphedema. Lymphedema was induced in the right hind limb after a single fraction of 20 Gy radiation, popliteal lymphadenectomy, and lymphatic vessel ablation. Postoperatively, mice were randomly divided in to 2 groups that received daily intraperitoneal injections of either (1) an oil-based vehicle solution (control) or (2) 0.08 mg/kg of 9-cis RA dissolved in a vehicle solution. Outcome measures included paw thickness, lymphatic drainage, and lymphatic vessel density as measured by podoplanin immunohistochemistry and whole mount skin analysis. Using our combined injury protocol, postsurgical lymphedema was observed 89% of the time. 9-cis RA-treated animals had less early postsurgical edema and significantly less paw lymphedema compared with vehicle-treated animals at all time-points (P < 0.001). 9-cis RA-treated animals had significantly faster lymphatic drainage as measured by indocyanine green clearance and increased lymphatic vessel density as measured by podoplanin immunohistochemistry (P < 0.001) and whole mount skin analysis (P < 0.05). We have developed a highly reproducible model of secondary lymphedema and have demonstrated that 9-cis RA significantly prevents postsurgical lymphedema. Treatment with 9-cis RA is associated with increased lymphatic clearance and lymphangiogenesis. Because 9-cis RA (alitretinoin) is already approved for clinical use by the US Food and Drug Administration for other conditions, it has the potential to be repurposed as a preventative agent for postsurgical lymphedema in humans.

Lymphatic vessel assessment by podoplanin (D2-40) immunohistochemistry in breast cancer.

Lymph node metastasis has an important bearing on the staging of breast cancer. Lymph node metastasis occurs by hematogenous and lymphatic spread. The hematogenous and lymphatic spread can be quantified by the blood vessel and lymphatic vessel density in the intra-tumoral and peri-tumoral zone by specific markers for blood vessels and lymph vessels. In this study, we are trying to study the localization of podoplanin in lymph vessels of invasive breast carcinoma, to quantify lymphangiogenesis in tissue sections of invasive breast carcinomas by podoplanin immunohistochemistry (IHC) by D2-40 antibody, and compare it with blood microvessel count using CD-31 antibody and correlating clinicopathologic parameters with the results of IHC. IHC for biomarkers D2-40 and CD-31 were performed on sections from 30 mastectomy specimens to assess blood vessel and lymphatic vessel density in intra-tumoral and peri-tumoral zone. The data were analyzed using Statistical Package for Social Sciences (SPSS) version 15.0 statistical analysis software. The results showed that both lymph vessel density and blood vessel density increased with the increase in lymph node ratio. Lymph node ratio is the ratio of positive lymph nodes to the total number of lymph nodes removed. Taking into account our small sample size, we conclude that a further large-sized study should be carried out to further prove the role of lymphatics in tumor dissemination. New therapeutic options can be developed targeting the lymphatic channels to arrest the lymphatic spread of the breast cancer.

Mobilization of lymphatic endothelial progenitor cells and lymphatic neovascularization in primary Sjögren’s syndrome

Primary Sjogren’s syndrome (pSS) is a systemic autoimmune disorder charac- terized by chronic lymphocytic infiltration of exocrine glands leading to progressive functional impairment. Lympangiogenesis is a common finding in chronic inflamma- tory diseases; however, its role in pSS remains to be elucidated. Inflammation induces the production of growth factors for lymphatic vessels, such as vascular endothelial growth factor-C (VEGF-C). Recent growing evidence indicates that bone marrowderived lymphatic endothelial progenitor cells (LEPCs) may differentiate into lymphatic endothelial cells via VEGF-C/VEGFR-3 signaling contributing to lymphangiogenesis. This study was designed to investigate the circulating levels of LEPCs and the occurrence of lymphangiogenesis in pSS. Pheripheral blood mononuclear cells were collected from 10 female pSS patients and 11 healthy females. LEPCs, defined as CD34+CD133+VEGFR-3+ cells, were identified by FACS using CD34-FITC, CD133-APC and VEGFR-3-PE antibodies. Results were expressed as percentage of CD133+VEGFR-3+ cells among CD34+ cells. Labial minor salivary gland (MSG) biop- sies were obtained from 12 female pSS patients and 16 sicca non-pSS control females. MSGs were evaluated by haematoxylin-eosin and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germi- nal center-like structures. Lymphatic vessels were identified by immunohistochemistry for podoplanin (D2-40), a mucin-type transmembrane protein expressed by lymphatic endothelial cells but not by blood vessels. VEGF-C/VEGFR-3 expression in MSGs was investigated by immunofluorescence. An average ten-fold increase in circulating levels of LEPCs was found in pSS (35.2±2.7%) compared with controls (3.4±0.8%) (p=0.0003). In control MSGs, lymphatic vessels were only detected around excretory ducts in the interlobular connective tissue. In pSS MSGs, the number of lymphatic vessels was increased around interlobular excretory ducts and a newly formed lymphatic capil lary network was found within inflammatory foci. A strong expression of VEGF-C was detected in ductal cells, vessels and inflammatory cells in pSS MSGs. VEGFR-3 expres- sion was observed in a subset of vessels and infiltrating mononuclear cells. Our find ings suggest that LEPC mobilization and MSG lymphatic vessel reorganization may take center stage in the chronic inflammatory process of pSS.

Analysis of lymphatic and blood vessel invasion biomarkers in T1 esophagogastric adenocarcinomas for improved patient prognostication

Lymphovascular invasion (LVI) in T1 esophagogastric adenocarcinoma may predict risk of recurrence despite definitive treatment with surgery or endoscopic resection. Podoplanin and CD34 are emerging biomarkers of lymphatic and blood vessel invasion, respectively, and could be adopted to refine LVI assessment. A consecutive series of 65 patients with T1 adenocarcinomas diagnosed at Nottingham University Hospitals were investigated. T1 tumors from 43/65 patients who received primary surgery only were suitable for LVI evaluation by hematoxylin and eosin (H&E) staining as well as by CD34 and Podoplanin immunohistochemistry. LVI was correlated to clinicopathological features and recurrence free survival. H&E staining detected LVI in 11.6% (5/43) of T1 tumors. CD34 and Podoplanin immunohistochemistry significantly improved LVI detection to 25.6% (11/43). Compared with LVI by H&E, immunohistochemical evaluation of blood vessel invasion (CD34) or lymphatic vessel invasion (Podoplanin) was significantly associated with higher grade (P = 0.005), submucosal invasion (T1b) (P = 0.018), lymph node positivity (N1) (P = 0.029) and poor recurrence free survival (P = 0.0003). Our study provides evidence that CD34 and Podoplanin immunohistochemistry could improve LVI detection and allow better prognostication of patients and optimum selection of definitive treatment. Larger multicenter studies are required for further validation that could have significant clinical implications.

Abstract 5526: Lymphangiogenesis in regional lymph nodes is an independent prognostic marker in rectal cancer patients after neoadjuvant treatment

Abstract One of the major prognostic factors in rectal cancer is lymph node metastasis. The formation of lymph node metastases is dependent on the existence of a premetastatic niche. An important factor preceding metastasis are lymph vessels which are located in the lymph node. Accordingly, the occurrence of intranodal lymphangiogenesis is thought to indicate distant metastasis and worse prognosis. To evaluate the significance of lymph node lymphangiogenesis, we studied formalin fixed, paraffin embedded adenocarcinomas and regional lymph nodes of 203 rectal cancer patients who were treated with neoadjuvant radiochemotherapy and consecutive surgery. Regional lymph node lymph vessels were detected by immunohistochemistry for podoplanin (D2-40). Our results show that the presence of lymphatic vessels in regional lymph nodes significantly affects the disease-free survival in univariate and multivariate analyses. In contrast, there was no correlation between peritumoral or intratumoral lymph vessel density and prognosis. Indeed, our study demonstrates the importance of lymphangiogenesis in regional lymph nodes after neoadjuvant radiochemotherapy and consecutive surgery as an independent prognostic marker. Staining for intranodal lymphangiogenesis and methods of intravital imaging of lymphangiogenesis and lymphatic flow may be a useful strategy to predict long-term outcome in rectal cancer patients. Furthermore, addition of VEGF-blocking agents to standardized neoadjuvant treatment schemes might be indicated in advanced rectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5526. doi:1538-7445.AM2012-5526

Lymphatic vessels in osteoarthritic human knees

The distribution and function of lymphatic vessels in normal and diseased human knees are understood incompletely. This study aimed to investigate whether lymphatic density is associated with clinical, histological or radiographic parameters in osteoarthritis (OA). Sections of synovium from 60 knees from patients with OA were compared with 60 post mortem control knees (from 37 individuals). Lymphatic vessels were identified using immunohistochemistry for podoplanin, and quantified as lymphatic vessel density (LVD) and lymphatic endothelial cell (LEC) fractional area. Effusion status was determined by clinical examination, radiographs were scored for OA changes, and inflammation grading used haematoxylin and eosin stained sections of synovium. Lymphatic vessels were present in synovia from both disease groups, but were not identified in subchondral bone. Synovial lymphatic densities were independent of radiological severity and age. Synovia from patients with OA displayed lower LVD (z=-3.4, P=0.001) and lower LEC fractional areas (z=-4.5, P<0.0005) than non-arthritic controls. In patients with OA, low LVD was associated with clinically detectable effusion (z=-2.2, P=0.027), but not with histological evidence of synovitis. The negative associations between lymphatics and OA/effusion appeared to be independent of other measured confounders. Lymphatic vessels are present in lower densities in OA synovia. Abnormalities of synovial fluid drainage may confound the value of effusion as a clinical sign of synovitis in OA.

The extent of retraction clefts correlates with lymphatic vessel density and VEGF-C expression and predicts nodal metastasis and poor prognosis in early-stage breast carcinoma

Although the earliest feature of disseminated disease in breast cancer is regional lymph node involvement, little is known about the mechanisms whereby cancer cells interact with lymphatic endothelial cells and enter the lymphatic system. We have previously reported that the extensive presence of retraction clefts in breast carcinomas highly significantly correlates with lymphatic tumor spread and predicts poor outcome, suggesting that retraction clefts are not just fixation artifacts, but real potential spaces that are exaggerated by tissue processing and may reflect an early stage of lymphatic invasion. In this study, we examined the correlation between the extent of retraction clefts and lymphangiogenesis, as assessed by lymphatic vessel density and vascular endothelial growth factor-C (VEGF-C) expression in a series of 256 early-stage breast carcinomas. The presence and extent of retraction clefts around tumor cell nests was determined by review of all hematoxylin- and eosin-stained tumor sections. Lymphatic vessels were detected by podoplanin immunohistochemistry and lymphatic vessel density was measured using the hot-spot method. The expression of VEGF-C in the tumor cells was determined by immunohistochemistry and analyzed semiquantitatively on a four-tiered scale. High levels of retraction clefts, peritumor lymphatic vessel density and VEGF-C expression at the invasive edge in breast carcinomas significantly correlated with tumor size, histological grade, lymphatic invasion and nodal metastasis. Breast carcinomas showing extensive retraction clefts (>20% of tumor volume) were found to have significantly higher lymphatic vessel density and VEGF-C expression levels compared to tumors without this feature. High retraction clefts, peritumor lymphatic vessel density and VEGF-C expression predicted poor outcome in breast carcinomas. Our results support the hypothesis that retraction clefts are real potential spaces that may represent ‘pre-lymphatic spaces' facilitating initial lymphatic invasion and that growth factors secreted by the tumor cells may stimulate tumor-associated lymphangiogenesis by promoting the endothelialization of these ‘pre-lymphatic channels'.

Lymphangiogenesis in Regional Lymph Nodes Is an Independent Prognostic Marker in Rectal Cancer Patients after Neoadjuvant Treatment

One of the major prognostic factors in rectal cancer is lymph node metastasis. The formation of lymph node metastases is dependent on the existence of a premetastatic niche. An important factor preceding metastasis are lymph vessels which are located in the lymph node. Accordingly, the occurrence of intranodal lymphangiogenesis is thought to indicate distant metastasis and worse prognosis. To evaluate the significance of lymph node lymphangiogenesis, we studied formalin fixed, paraffin embedded adenocarcinomas and regional lymph nodes of 203 rectal cancer patients who were treated with neoadjuvant radiochemotherapy and consecutive curative surgery with cancer free surgical margins (R0). Regional lymph node lymph vessels were detected by immunohistochemistry for podoplanin (D2-40). Our results show that the presence of lymphatic vessels in regional lymph nodes significantly affects the disease-free survival in univariate and multivariate analyses. In contrast, there was no correlation between peritumoral or intratumoral lymph vessel density and prognosis. Indeed, our study demonstrates the importance of lymphangiogenesis in regional lymph nodes after neoadjuvant radiochemotherapy and consecutive surgery as an independent prognostic marker. Staining for intranodal lymphangiogenesis and methods of intravital imaging of lymphangiogenesis and lymphatic flow may be a useful strategy to predict long-term outcome in rectal cancer patients. Furthermore, addition of VEGF-blocking agents to standardized neoadjuvant treatment schemes might be indicated in advanced rectal cancer.

Prognostic value of lymphangiogenesis in supraglottic laryngeal carcinoma

Metastasis to regional lymph nodes via lymphatic microvessels plays a key role in cancer progression, and is an important prognostic factor in many cancers. Recent evidence suggests that tumour lymphangiogenesis promotes lymphatic metastasis. To investigate whether tumour lymphatic microvessel density correlates with clinicopathological factors and serves as a prognostic indicator of supraglottic laryngeal carcinoma progression. The lymphatics of 84 supraglottic laryngeal carcinoma cases were investigated by immunohistochemical staining for podoplanin (also termed D2-40). The relationships between (intra- and peritumoural) lymphatic microvessel density, clinicopathological parameters and clinical prognosis were analysed. There was a significant relationship between high intratumoural lymphatic microvessel density and aggressive tumour node stage (p < 0.0001), distant metastasis (p = 0.037) and poor prognosis (p = 0.011), and between high peritumoural lymphatic microvessel density and node stage (p = 0.004) and poor prognosis (p = 0.029). Patients with high lymphatic microvessel density also had significantly worse disease-free survival (p = 0.003) and overall survival (p = 0.005). Intratumoural lymphatic microvessel density was found to be an independent prognostic factor for overall survival (p = 0.008) and disease-free survival (p = 0.005) (multivariate analysis). Lymphatic microvessel density (detected by podoplanin immunohistochemistry), especially intratumoural density, may be an independent predictor of lymphatic tumour spread and survival in supraglottic laryngeal carcinoma patients, and may be useful to guide decisions regarding additional surgery.

Podoplanin expression correlates with sentinel lymph node metastasis in early squamous cell carcinomas of the oral cavity and oropharynx

In patients with early head and neck squamous cell carcinoma (HNSCC), occult lymph node metastasis is difficult to predict by clinical or pathological parameters. However, such parameters are necessary to select patients either for elective neck dissection or the sentinel lymph node (SLN) procedure. The membrane glycoprotein podoplanin is normally expressed in lymphatic endothelial cells. Recently, expression of podoplanin by cancer cells was demonstrated to promote tumor cell motility and tumor lymphangiogenesis in vitro. The value of cancer cell-expressed podoplanin was to be determined as a predictive marker for SLN metastasis in early HNSCC of the oral cavity and oropharynx. One hundred twenty patients with HNSCC of the oral cavity and oropharynx undergoing a SLN biopsy were enrolled in this prospective clinical trial of SLN biopsy. Cancer cell-expressed podoplanin was determined by immunohistochemistry using tissue microarrays. Podoplanin expression was quantified by the intensity reactivity score and categorized into expression and nonexpression. SLN examination revealed occult metastasis in 45 patients (37.5%). Twenty-nine of 120 (24.2%) primary HNSCC showed podoplanin expression. Podoplanin expression correlated significantly with SLN metastasis (p = 0.029) and remained a significant predictor for lymph node status even after controlling for tumor stage (p = 0.028). As a predictive marker for SLN metastasis, however, podoplanin expression reached a sensitivity of a mere 36% and a specificity of 83%. Podoplanin expression is associated with metastasis to lymph nodes in vivo. Podoplanin immunohistochemistry in early HNSCC of the oral cavity and oropharynx may help to select patients for the SLN procedure and to identify patients with increased risk for presence of occult lymph node metastasis in the neck.

Podoplanin Expression in Cancerous Stroma Induces Lymphangiogenesis and Predicts Lymphatic Spread and Patient Survival

Podoplanin is a mucin-type glycoprotein and a lymphatic endothelial marker. Immunohistochemical staining for podoplanin is currently used as a routine pathologic diagnosis tool in Japan to identify lymphatic invasion of cancer cells. Recent reports suggest that podoplanin and other proangiogenic molecules are expressed in stromal fibroblasts and myofibroblasts. To analyze the distribution of podoplanin expression in tumor stroma and its clinical and biologic significance. We performed immunohistochemistry for podoplanin on tissue microarrays from 1350 cases of 14 common cancer types. Two hundred eighty-seven of 662 cases (43%) showed podoplanin expression in the stromal cells within cancer nests. Stromal podoplanin expression in 14 common cancer types was significantly associated with tumor stage (P < .001), lymph node metastases (P < .001), lymphatic invasion (P = .02), and venous invasion (P < .001). The stromal cells positive for podoplanin were also positive for α-smooth muscle actin but negative for desmin, confirming a myofibroblasts phenotype. In contrast, myofibroblasts in inflammatory fibrotic lung diseases were podoplanin negative. Lymphatic vessel density was greater in the stromas with podoplanin expression than in the stroma lacking podoplanin-expressing stromal cells (P = .01). Survival data were available for non-small cell lung cancer. Stromal podoplanin expression was associated with poorer prognosis in adenocarcinoma (P < .001) and remains statistically significant after adjustment for sex, age, and stage (P = .01). Our data indicate that podoplanin expression in stromal myofibroblasts may function as a proangiogenic biomarker and may serve as a predictive marker of lymphatic/vascular spread of cancer cells and a prognostic marker of patient survival.

Morphology of lymphatic regeneration in rat incision wound healing in comparison with vascular regeneration

In the wound healing process, angiogenesis is involved in the recovery of vasculature, and its process has been investigated. On the other hand, the reconstruction of lymphatic vessels in the injured subcutaneous tissue has not been studied in detail. We studied the recovery of lymphatic vessels using podoplanin immunohistochemistry in the paraffine section microscopy of the rat skin incision wound. Our result indicates a novel finding that subcutaneous tissue of the incised skin area does not show any recovery of lymphatic vessels up to 84 days after the skin incision. As the wound area shrunk, the surrounding subcutaneous tissue covered with the normal skin epithelial cells approached toward the center of the wound, and the lymphatic vessels in the surrounding tissue gradually reached the incision wound area. On the other hand, the regeneration of the vasculature occurred within the wound area as assessed by CD31 and von Willebrand Factor (vWF) immunohistochemistry. This difference was confirmed by the morphometric quantification of podoplanin- or vWF-positive vessels. Our results show that there is a clear difference in the recovery pattern of vascular and lymphatic vessels in the skin wound healing process.

Tumour necrosis factor blockade increases lymphangiogenesis in murine and human arthritic joints

Objective:To investigate the presence and regulation of lymphatic vessels in inflamed joints of mice with experimental arthritis as well as patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA).Methods:Lymphatic vessels and...

Podoplanin is a Better Immunohistochemical Marker for Sarcomatoid Mesothelioma Than Calretinin

Immunohistochemistry using a broad panel of markers is an invaluable tool for diagnosing sarcomatoid mesothelioma. Membranous podoplanin staining has been proposed as a specific and sensitive marker to distinguish epithelioid mesothelioma from adenocarcinoma. We found that cytoplasmic podoplanin staining was present in sarcomatoid mesotheliomas, and wanted to explore the reproducibility and specificity of this staining pattern. Immunohistochemistry for podoplanin, using 2 podoplanin antibodies (antipodoplanin and D2-40), was performed in 55 mesotheliomas (24 epithelioid, 18 sarcomatoid, and 13 biphasic), 80 pulmonary adenocarcinomas, 8 synovial sarcomas, and 16 sarcomatoid carcinomas. Expression of calretinin, vimentin, MOC31, and TTF-1 was also examined in all adenocarcinomas, sarcomatoid carcinomas, 7 synovial sarcomas, and 21 of the mesotheliomas. Calretinin staining performed previously on an additional 31 mesotheliomas was reviewed. Using membranous or cytoplasmic staining as indicative of positivity, we found that antipodoplanin and D2-40 each stained 84% of mesotheliomas (antipodoplanin: 46/55; D2-40: 38/44), including 72% of sarcomatoid mesotheliomas (antipodoplanin: 13/18; D2-40: 11/14). With antipodoplanin antibody, no staining was seen in the pulmonary adenocarcinomas (0/80, 0%) or the synovial sarcomas (0/8, 0%), and weak cytoplasmic staining was seen in only 1 sarcomatoid carcinoma (1/15, 7%). D2-40 showed similar results, staining 3% (2/80) of pulmonary adenocarcinomas, 13% (1/8) of synovial sarcomas, and 8% (1/13) of sarcomatoid carcinomas. Overall sensitivities and specificities were 84% and 99% for antipodoplanin, and 86% and 96% for D2-40. These findings suggest that cytoplasmic podoplanin expression may be useful in the diagnosis of sarcomatoid mesothelioma, although it should be used with caution on biopsy material.