Sebastian F. Schoppmann

Tumor-Associated Macrophages Express Lymphatic Endothelial Growth Factors and Are Related to Peritumoral Lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.

Prognostic value of lymphangiogenesis and lymphovascular invasion in invasive breast cancer

Objective:The aim of this study was to investigate the prognostic relevance of lymphangiogenesis and lymphovascular invasion in a large cohort of breast cancer patients. Introduction:Invasion of tumor cells into blood and lymphatic vessels is one of the critical steps for metastasis. The presence or absence of lymph node metastasis is one of the main decision criteria for further therapy. One shortcoming of previous morphologic studies was the lack of specific markers that could exact discriminate between blood and lymphatic vessels. The aim of this study was to evaluate the prognostic relevance of lymphangiogenesis and lymphovascular invasion in breast cancer patients. Methods:We investigated 374 tissue specimens of patients suffering from invasive breast cancer by immunostaining for the lymphatic endothelial specific marker podoplanin. Lymphangiogenesis, quantified by evaluating the lymphatic microvessels density (LMVD), and lymphovascular invasion (LVI) were correlated with various clinical parameters and prognostic relevance. Results:LMVD correlated significantly with LVI (P = 0.001). LVI was associated significantly with a higher risk for developing lymph-node metastasis (P = 0.004). Calculating the prognostic relevance, LVI presented as an independent prognostic parameter for disease free as well as overall survival (P = 0.001, and P = 0.001, respectively). Conclusion:Our data provide evidence that the biologic system of lymphangiogenesis constitutes a potential new target for development of anti-breast cancer therapeutic concepts. Our results further suggest that young, premenopausal patients with low differentiated breast tumors and high LMVD and LVI would, in particular, benefit from lymphangiogenesis-associated therapeutic strategies.

Overexpression of Id-1 is associated with poor clinical outcome in node negative breast cancer

Id‐1 is an important regulator of cellular growth and differentiation and controls malignant progression of breast cancer cells. The aim of our study was to assess the clinical impact of Id‐1 expression in breast cancer, i.e., its potential impact on prognosis and prediction of treatment response. Id‐1 protein expression was determined immunohistochemically in 191 patients with lymph‐node negative breast cancer, and univariate and multivariate survival analysis was carried out. Fifteen (7.9%) specimens showed strong expression, 75 (39.3%) moderate, 55 (28.8%) weak expression and 46 (24.1%) cases no expression of Id‐1. Patients with strong or moderate Id‐1 expression had a significant shorter overall (p = 0.003, Cox regression) and disease‐free survival (p = 0.01, Cox regression) compared to those with absent or low expression. Progesterone receptor density was significantly higher in breast cancers with absent/low Id‐1 expression compared to those with moderate/strong expression (p < 0.001, t‐test). Id‐1 expression was significantly stronger in cases positive for p16INK4a expression compared to those negative for p16 (p = 0.049, Mann‐Whitney test). The influence of Id‐1 on clinical outcome seems much stronger in patients with negative estrogen receptor status compared to those with positive status, who received receptor antagonists as adjuvant therapy in most cases. Overexpression of Id‐1 protein represents a strong independent prognostic marker in node negative breast cancer, and future therapies inhibiting Id‐1 expression might be beneficial for these patients. Our results also suggest that due to the apparent interaction of Id‐1 with the steroid‐receptor system in breast cancer, hormonal therapies might influence Id‐1 expression and its impact on clinical outcome.

Increase in podoplanin-expressing intestinal lymphatic vessels in inflammatory bowel disease

Ulcerative colitis (UC) and Crohns disease (CD) are two presentations of inflammatory bowel diseases (IBDs). Whereas the inflammation in UC is confined to the mucosa/submucosa, CD is considered a transmural disease with characteristic lymphoid aggregates with or without epithelioid granulomas in the subserosa. Here we examined and quantified the distribution of lymphatic capillaries in small- and large-bowel resection specimens (non-IBD n=8; CD n=20 and UC n=13) using immunohistochemical staining with anti-human podoplanin antibody, an established marker for lymphatic endothelium. In normal small intestine, the lymphatic network originated in the capillaries beneath the surface epithelial cells, whereas it started in the lower third of the mucosa of the large intestine. Lymphatic microvessel counts revealed a statistically highly significant increase (P<0.005 in the muscularis mucosae, P=0.012 in the tunica submucosa and P=0.012 in the tunica subserosa) in IBDs when compared with normal intestine. Numerical differences between CD and UC samples were not significant. Prominence of lymphatic capillaries could also be observed in areas where fibrosis replaced chronic inflammation. These findings suggested that lymph-vessel proliferation in IBDs may be triggered by chronic inflammation irrespective of its organization and is maintained in fibrotic end-stage disease.

ALK gene translocations and amplifications in brain metastases of non-small cell lung cancer.

BACKGROUND Increased incidence of brain metastases (BM) in non-small cell lung cancer (NSCLC) with ALK translocations was postulated, however, ALK gene aberrations in NSCLC-BM have not been investigated so far. METHODS We investigated ALK and EML4 gene aberrations (amplifications, translocations, inversions) by fluorescent in situ hybridization (FISH) (n=175) and ALK and EML4 protein expression by immunohistochemistry (n=221) in NSCLC BM and corresponding primary tumors. RESULTS ALK translocations were found in 4/151 (2.6%; 3 of them involving EML4) of BM of adenocarcinomas (AC), 1/9 (11.1%) of adenosquamous carcinomas (ASC), 0/5 of squamous cell carcinomas (SCC) and 0/10 of large cell carcinomas (LCC). Rearrangement of ALK without involvement of EML4 was seen in 1 AC-BM and rearrangement of EML4 without involvement of ALK in 3 AC-BM, 1 ASC-BM and 1 LCC. ALK amplifications without gene rearrangements were found in BM of 16/151 (10.6%) AC, 2/5 (40%) SCC, 0/9 ASC and one LCC. ALK translocation status was constant between BM and primary tumors in 16 evaluable cases including two cases with ALK-EML4 translocations Among these 16 cases ALK amplification was seen in two BM and none of the primary tumors. All cases with translocations but not with amplifications of ALK showed protein expression. We found no association of ALK gene status with patient age, gender or overall survival time. CONCLUSIONS ALK translocations and amplifications are found in approximately 3% and 11% of NSCLC-BM, respectively. While ALK translocations appear to be constant between primary tumors and BM, amplifications seem to be more prevalent in BM. ALK translocation, but not ALK amplification is associated with ALK protein overexpression. Further studies are needed to determine whether NSCLC-BM patients with ALK gene aberrations may benefit from specific inhibitor therapy.

Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

Summary.  Background:  Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis.

HER2/neu expression correlates with vascular endothelial growth factor-C and lymphangiogenesis in lymph node-positive breast cancer

BACKGROUND Vascular endothelial growth factor-C (VEGF-C) is the main inducer of lymphangiogenesis. VEGF-C overexpression is associated with lymphovascular tumor cell invasion, an increased rate of lymph node metastasis and adverse prognosis in various human cancers. However, little is known about the upstream inducers of VEGF-C expression. Recent studies have shown that human epidermal growth factor receptor 2 (HER2/neu) overexpression is associated with high VEGF-C levels in human breast cancer cells. In addition to blocking of HER2/neu, tyrosine kinase significantly decreased VEGF-C expression in vitro. PATIENTS AND METHODS VEGF-C expression, lymphatic microvessel density (LMVD), lymphovascular invasion (LVI) and HER2/neu expression were evaluated with immunohistochemical/FISH methods in a collective of 150 lymph node-positive human breast cancers with long-term follow-up. RESULTS Cases with 3+ HER2/neu protein expression showed a significantly stronger VEGF-C expression than all others cases (P = 0.006). In addition, we found a significant correlation between VEGF-C expression and LMVD (P = 0.012) and a strong positive association between LMVD and LVI (P < 0.001). CONCLUSION Our data provide evidence for a clinically relevant association between HER2/neu and VEGF-C expression in human breast cancer. Inhibiting HER2/neu may reduce tumor progression by blocking VEGF-C-mediated tumor cell proliferation and lymphogenic metastasis.

Lymphangiogenesis and lymphovascular invasion diminishes prognosis in esophageal cancer.

BACKGROUND Tumor-associated lymphatic networks are the primary routes for tumor cell dissemination and metastasis. Behind the background of possible lymphangiogenesis-associated therapies, the clinical impact of lymphangiogenesis (measured by lymphatic microvessel density [LMVD]) and specific lymphovascular invasion (LVI) in esophageal cancer remains unclear. The aim of this study was to evaluate the clinical role of lymphangiogenesis and LVI in a large cohort of esophageal cancer. METHODS For the specific assessment of LMVD and LVI, 393 tissue samples from a prospective tissue databank of esophageal adenocarcinomas, squamous cell carcinomas, and their precursor lesions were included into this study. LMVD and LVI were assessed by immunostaining for podoplanin, a selective marker of lymphatic endothelium. In addition the peritumoral inflammatory stroma reaction (ISR) was assessed. RESULTS Patients with high LMVD had a significant increased risk to develop LVI (P = .00123; coefficient of regression [CR] 0.27) and lymph node metastasis (P = .00233), independent of the tumors histology. During a follow-up of 52 months, patients with high LMVD had a significantly reduced overall survival (OS; P < .001; 5-year OS 30% vs 54%) and disease-free survival (DFS; P < .001; 5-year DFS 28% vs 48%). OS (P < .001; 5-year OS 14% vs 60%) and DFS (P < .001; 5-year DFS 14% vs 49%) were significantly reduced in patients with present LVI. In invasive cancer, LMVD was significantly increased compared with precursor lesions (P = .008). The amount of ISR correlated significantly with LMVD. CONCLUSION Our data provide evidence for a clinically significant role of specific lymphangiogenesis in esophageal cancer. Patients with high lymphangiogenic tumor activity represent candidates for lymphangiogenesis-associated therapies.

Telomerase‐Immortalized Lymphatic and Blood Vessel Endothelial Cells are Functionally Stable and Retain Their Lineage Specificity

Objective: Microvasculature plays an important role in a variety of physiological and pathological processes. The authors have previously shown that primary cultures of human microvascular endothelial cells consist of 2 distinct populations of blood vascular and lymphatic endothelial cells. These subtypes are ephemeral and lose purity through passaging. To generate reproducible in vitro and in vivo experiments stable blood and lymphatic endothelial cell lines are an essential prerequisite.

Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E

BRAF V600E mutation in serrated lesions of the colon has been implicated as an important mutation and as a specific marker for the serrated carcinogenic pathway. Recent findings point to microvesicular hyperplastic polyps that have similar histologic and molecular features to sessile serrated adenomas/polyps, as potential colorectal carcinoma precursors. The aim of this study was to evaluate BRAF V600E mutation status by immunohistochemistry in serrated lesions of the colon with regard to histomorphology. We investigated 194 serrated lesions of the colon, comprising 42 sessile serrated adenomas/polyps, 16 traditional serrated adenomas, 136 hyperplastic polyps and 20 tubular/tubulovillous adenomas (conventional adenomas) with the novel BRAF V600E mutation-specific antibody VE1. In addition, BRAF exon 15 and KRAS exon 2 status was investigated by capillary sequencing in selected cases. All sessile serrated adenomas/polyps (42/42, 100%), 15/16 (94%) traditional serrated adenomas and 84/136 (62%) hyperplastic polyps were VE1+. None of the VE1− serrated lesions showed BRAF V600E mutation. Forty out of 42 (95%) sessile serrated adenomas/polyps displayed areas with microvesicular hyperplastic polyp-like features. In microvesicular hyperplastic polyps, VE1 positivity was significantly associated with nuclear atypia (P=0.003); however, nuclear atypia was also present in VE1− cases. Immunostaining with VE1 allows not only the detection of BRAF V600E mutation but also the correlation with histomorphology on a cellular level in serrated lesions. VE1 enables a subclassification of microvesicular hyperplastic polyps according to the mutation status. This improved classification of serrated lesions including immunohistochemical evaluation of BRAF V600E mutation may be the key to identify lesions with higher potential to progression into sessile serrated adenoma/polyp, and further to BRAF V600E-mutated colorectal cancer.