Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska‐Okolow M
(2011) Histopathology  59, 1249–1260
Podoplanin expression by cancer‐associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma Aims: It has recently been shown that podoplanin, a mucin‐type glycoprotein, is expressed by cancer cells and cancer‐associated fibroblasts (CAFs), and promotes cancer cell migration and invasiveness. The biological role of podoplanin expression in tumour stroma of invasive ductal carcinoma of the breast (IDC) has not been determined. Methods and results: Podoplanin expression was analysed in 117 cases of IDC and 27 cases of fibrocystic change, as well as in breast cancer cell lines, with the use of immunohistochemistry and real‐time polymerase chain reaction. In 82.1% of analysed tumours, podoplanin was found only in CAFs. Only two of 117 IDC cases (1.7%) were characterized by expression of this glycoprotein in cancer cells. None of the fibrocystic changes or stroma surrounding normal ducts showed podoplanin expression. Podoplanin‐positive CAFs correlated with tumour size (P = 0.0125), grade of malignancy (P = 0.0058), lymph node metastasis (P = 0.0149), lymphovascular invasion (LVI) (P = 0.0486) and Ki67 expression in cancer cells (P = 0.0128). High‐level podoplanin expression (>50% of positive stroma) in the tumour stroma was significantly associated with a negative oestrogen status (P = 0.0201). Univariate, but not multivariate, analysis showed that podoplanin expression by CAFs was associated with poor patient outcome (P = 0.0202). Conclusions: Our results suggest that podoplanin expression by CAFs could be an unfavourable prognostic marker for IDC.     

Concordant podoplanin expression in cancer-associated fibroblasts and tumor cells is an adverse prognostic factor in esophageal squamous cell carcinoma

There is growing evidence that cancer-associated fibroblasts (CAFs) interact with tumor cells and play important roles in tumor progression and invasion. Podoplanin is a type-1 transmembrane glycoprotein expressed in a variety of normal human tissues, including lymphatic endothelium. Tumor cell expression of podoplanin correlates with nodal metastasis and poor prognosis in squamous cell carcinoma (SCC) of oral cavity and esophagus. Recently, podoplanin-positive CAFs have been shown to exert adverse or beneficial prognostic effect on different cancer types. However, the significance of podoplanin-positive CAFs in esophageal SCC has not been investigated. This is the first study to investigate podoplanin expression in CAFs and tumor cells by immunohistochemistry in 59 cases of surgically resected esophageal SCC. We found significant association of podoplanin expression between CAFs and tumor cells (P = 0.031). Although the abundance of podoplanin-positive CAFs per se had no prognostic effect, concordant podoplanin expression in CAFs and tumor cells (both high or both low) was strongly associated with short survival (P = 0.00088). Multivariate analysis showed that concordant podoplanin expression was the strongest independent adverse prognostic factor (hazard ratio: 3.62; 95% confidence interval: 1.69-7.77; P = 0.00094). Our data suggest that interaction between podoplanin-positive CAFs and tumor cells is important in tumor biology of esophageal SCC.     

食管鳞状细胞癌肿瘤细胞和间质细胞中podoplanin的表达及临床意义

目的探讨食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)中podoplanin在肿瘤细胞和间质细胞的表达及其临床意义。方法应用免疫组化EnVision法检测150例ESCC中podoplanin的表达,分析肿瘤细胞、间质细胞中podoplanin表达以及两者共表达与临床病理特征之间的关系。结果 150例ESCC中,肿瘤细胞podoplanin阳性者50例,其表达与淋巴结转移、脉管侵犯和TNM分期呈正相关(P<0.05);与患者性别、年龄、肿瘤大小、组织学分级、浸润深度和神经侵犯均无关(P>0.05)。podoplanin在间质细胞中的阳性率为54%(81/150),其表达与浸润深度、淋巴结转移、脉管侵犯和TNM分期均有关(P<0.05);与患者性别、年龄、肿瘤大小、组织学分级及神经侵犯无关(P>0.05)。ESCC肿瘤细胞和间质细胞中podoplanin共阳性者44例,肿瘤细胞和间质细胞中podoplanin共阳性与淋巴结转移、脉管侵犯、神经侵犯和TNM分期密切相关(P<0.05)。相关分析显示肿瘤细胞和间...     

Intratumoral as well as peritumoral lymphatic vessel invasion correlates with lymph node metastasis and unfavourable outcome in colorectal cancer

The aim of this study was to assess the intratumoral and peritumoral distribution of lymphatic vessel density (LVD) and lymphatic vessel invasion (LVI) in colorectal cancer and their relationships with patients’ clinicopathological characteristics and survival. Paraffin sections of 81 primary colorectal cancers were examined by immunohistochemical staining using monoclonal antibody D2-40. Peritumoral LVD was significantly higher than intratumoral LVD (P = 0.000). Both intratumoral LVD and peritumoral LVD were correlated with the presence of LVI (P = 0.006 and P = 0.003, respectively). LVI, intratumoral LVI and peritumoral LVI were identified, respectively in 38, 28 and 32% of the samples investigated. Both intratumoral LVI and peritumoral LVI were correlated with lymph node metastasis (P = 0.030 and P = 0.014, respectively). Lymph node metastasis, the presence of intratumoral LVI and peritumoral LVI were adversely associated with the 5-year overall survival in a univariate analysis (P = 0.001, P = 0.011 and P = 0.017, respectively). Multivariate analysis using Cox proportional hazard model showed that neither intratumoral LVI nor peritumoral LVI was an independent prognostic factor of overall survival. The results of this study demonstrated that intratumoral as well as peritumoral LVI was associated with lymph node metastasis and adverse outcome in colorectal cancer.     

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2-40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF-C), VEGF-D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF-D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF-C or VEGF-D expression. High LVD, induced by VEGF-C or VEGF-D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.     

Reduced expression of Raf-1 kinase inhibitory protein predicts regional lymph node metastasis and shorter survival in esophageal squamous cell carcinoma

Raf-1 kinase inhibitory protein (RKIP), a suppressor of metastasis, is associated inversely with the progression and metastasis of human malignancies. The present study evaluated relationships between RKIP expression and metastatic potential, clinicopathological characteristics and patient outcome in esophageal squamous cell carcinoma (ESCC). We examined tissue specimens from 138 patients with thoracic ESCC. Using immunohistochemistry, RKIP expression was detected in ESCC in situ, primary ESCC and nodal metastatic ESCC. RKIP expression was reduced in 28.9% (13/45) of ESCC in situ, in 50.0% (69/138) of primary ESCC and in 71.4% (65/91) of nodal metastatic ESCC. These levels of RKIP down-regulation differed significantly. RKIP expression was associated inversely with histological grade (P=0.008), pathological T stage (P=0.044), lymphatic invasion (P=0.019), regional lymph node metastasis (LNM; P=0.002) and stage (P=0.041). Pathological T stage (P=0.001), lymphatic invasion (P<0.001) and reduced RKIP expression (P=0.039) were independent predictors of regional LNM in ESCC. In addition, the postoperative survival of patients with RKIP-reduced ESCC was significantly shorter than for patients with RKIP-positive ESCC (P=0.004). Reduced RKIP expression in ESCC correlated with advanced disease, regional LNM and poor prognosis. RKIP expression may serve as a novel clinical biomarker in patients with ESCC.     

Increased density and diameter of lymphatic microvessels correlate with lymph node metastasis in early stage invasive colorectal carcinoma

To determine whether lymphangiogenesis was associated with the development of colorectal carcinoma and whether the mean maximal diameter of lymphatic microvessels (LMMMD) or lymphatic microvessel density (LMVD) is associated with lymph node metastasis in early stage invasive colorectal carcinoma (T1 carcinoma), we used immunohistochemical staining with podoplanin to measure LMMMD and LMVD in intratumoral (LMMMDit, LMVDit) and peritumoral areas (LMMMDpt, LMVDpt) of T1 carcinomas (n=87). By comparing the LMMMD and LMVD in normal large intestine (n=10), adenoma (n=15), and Tis carcinoma (n=15), we found out that the LMVDpt in T1 carcinoma with lymphatic vessel invasion (LVI) was significantly high (P<0.001), and there was a significant decrease in LMMMDpt in T1 carcinoma (P=0.031). Both LMMMDpt and LMVDpt were significantly increased in the T1 carcinomas, with LVI compared with the T1 carcinomas without LVI (P=0.018, P=0.003). Multivariate analysis revealed that LVI and combined greater LMMMDpt and greater LMVDpt were associated with lymph node metastases (P=0.005, P=0.036). These results indicate that lymphangiogenesis might be induced in the surrounding tumor areas of the T1 colorectal carcinoma with LVI; thus, evaluation of the diameter and density of lymphatic microvessels is important in T1 colorectal carcinoma to predict lymph node metastases.     

Blood vessel invasion and other variables as predictors of long-term survival in Japanese and British patients with primary invasive breast cancer

This study was undertaken to investigate the associations of blood vessel invasion (BVI), lymphatic vessel invasion (LVI) or other variables and long-term survival in 173 Japanese and 184 British patients with primary invasive breast cancer, and whether they are associated with survival differences between Japanese and British patients. BVI was detected by objective methods, using both factor VIII-related antigen (F-VIII) staining and elastica van Gieson (E v G) staining. BVI was classified into three subtypes. 1) BVI e, BVI detected by E v G staining alone, 2) BVI f, BVI detected by F-VIII staining alone, 3) BVIef, BVI evaluated by combining BVIf and BVIe. LVI was also detected by objective methods, using lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) staining alone. There was a borderline significance between the frequencies for BVIef of British patients and those of Japanese patients (8.2% vs 3.5%; P = 0.06) but not for LVI (P = 0.36). British patients had a significantly worse relapse-free survival (RFS) and overall survival (OS) than Japanese patients (P < 0.01, P < 0.01, respectively) even though their tumors were smaller and more ER-positive with a similar prevalence of lymph-node involvement. LVI was not significantly associated with RFS and OS, however, BVIef positive tumors had a significantly worse RFS and OS compared with BVIef negative patients, after statistical adjustment for the other variables (P = 0.02, P = 0.01, respectively). The present study shows that BVIef variability might contribute to the Japanese and British disparities in breast cancer outcomes.     

Prognostic value of podoplanin expression in intratumoral stroma and neoplastic cells of uterine cervical carcinomas

OBJECTIVE:

To investigate the clinicopathological significance of podoplanin expression in the intratumoral stroma and neoplastic cells of early stage uterine cervical cancer.

MATERIALS AND METHODS:

A total of 143 patients with clinical stage I and IIA uterine cervical carcinomas underwent surgery between 2000 and 2007. Clinicopathological data and slides associated with these cases were retrospectively reviewed. Immunodetection of podoplanin expression in histologic sections of tissue microarray blocks was performed using the monoclonal antibody D2‐40.

RESULTS:

Expression of podoplanin was detected in neoplastic cells in 31/143 (21.6%) cases, with 29/31 (93.5%) of these cases diagnosed as squamous carcinoma. For all of the cases examined, the strongest signal for podoplanin expression was observed at the proliferating edge of the tumor nests. The rate of positive podoplanin expression for node‐positive cases was lower than that of node‐negative (18.9% vs. 22.6%, respectively). Furthermore, the rate of positive podoplanin expression in fatal cases was 10.5% vs. 21.6%, respectively. In 27/143 (18.8%) cases, podoplanin expression was detected in fibroblasts of the intratumoral stroma, and this expression did not correlate with patient age, clinical stage, tumor size, histologic type, depth of infiltration, or vascular involvement. Moreover, expression of podoplanin in intratumoral stroma fibroblasts was only negatively associated with nodal metastasis. A greater number of fatal cases was observed among negative intratumoral stroma fibroblasts (15.5% vs. 3.7%, respectively), although this difference was not significant.

CONCLUSIONS:

These preliminary results suggest that podoplanin may have a role in host‐tumor interactions and, as a result, may represent a favorable prognostic factor for squamous cervical carcinomas.     

Phosphorylated mTOR expression correlates with podoplanin expression and high tumor grade in esophageal squamous cell carcinoma

Mechanistic (or mammalian) target of rapamycin (mTOR) plays important roles in cell growth and proliferation. In esophageal squamous cell carcinoma (SCC), high expression of phosphorylated (activated) mTOR (p-mTOR) has been reported as an adverse prognostic factor in some but not all studies. The signals of mTOR pathway and mitogen-activated protein kinase (MAPK) pathway converge on 4E-binding protein 1 (4EBP1), which drives the downstream proliferative signals. We previously found that high expression of phosphorylated 4EBP1 (p-4EBP1) is an adverse prognostic factor in esophageal SCC. Podoplanin is a type-1 transmembrane glycoprotein expressed in various normal human tissues, including lymphatic endothelium. Our previous study showed that high podoplanin expression correlates with clinical nodal metastasis, which is associated with short survival in esophageal SCC. In current study, we investigated p-mTOR expression by immunohistochemistry in 75 cases of surgically resected esophageal SCC. The result was correlated with p-4EBP1 expression, podoplanin expression, clinicopathologic features and patient survival. We found that high p-mTOR expression was significantly associated with high podoplanin expression (P = 0.0030) and high tumor grade (P = 0.0014). No correlation with p-4EBP1 expression, patient survival or other clinicopathologic features was found. Recently, podoplanin expression in astrocytic brain tumors was found to be regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT/activator protein-1 (AP-1) pathway. Similarly, mTOR is activated by a PI3K/AKT/mTOR pathway. The association of p-mTOR and podoplanin expression in our study could be due to a common upstream pathway. Since both mTOR and podoplanin are potential therapeutic targets, the possible benefit of combined targeted therapy warrants further investigation.     

Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors

Podoplanin is known as a lymphatic marker because its expression is detected in lymphatic but not vascular endothelium. Podoplanin is also expressed in several normal tissues including osteocytes or osteoblasts. A systematic examination of the podoplanin expression was conducted in normal skeletal tissues and some bone tumor cell lines, and the diagnostic value determined in primary bone tumors. Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7). Therefore, podoplanin may be a sensitive immunohistochemical marker of osteogenic and chondrogenic bone tumors.     

Podoplanin expressing cancer associated fibroblasts are associated with unfavourable prognosis in adenocarcinoma of the esophagus

Overexpression of the mucin-type sialoglycoprotein podoplanin in cancer associated fibroblasts (CAFs) was recently shown to be associated with tumor progression, metastasis and poor prognosis in lung and breast cancer. Here we investigate the role of podoplanin expressing CAFs in esophagal adenocarcinoma (AC), its precursor lesions and metastases. Podoplanin expression was investigated immunohistochemically in 200 formalin-fixed, paraffin embedded specimens of invasive esophagal ACs, their corresponding metastases and 35 precursor lesions. Podoplanin expressing CAFs (CAF+) were observed in 22 % of patients with invasive AC, but not in precursor lesions. CAF+ correlated with tumor stage (p = 0.004), lymphovascular tumor invasion (p = 0.018) and lymph node metastasis (p = 0.0016). Patients with CAF+ had a significant shorter disease free and overall survival (p < 0.05, Cox regression). Podoplanin expressing CAFs were only rarely observed in lymph node and distant metastases, as well as in local recurrences of ACs. Podoplanin expression in AC tumor cells was seen in only four cases. In around 20 % of patients with esophagal AC, podoplanin expressing CAFs are evident, defining a high risk subgroup. In these patients, podoplanin expressing CAFs might represent new therapeutical targets.     

Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium

Angiosarcomas apparently derive from blood vessel endothelial cells; however, occasionally their histological features suggest mixed origin from blood and lymphatic endothelia. In the absence of specific positive markers for lymphatic endothelia the precise distinction between these components has not been possible. Here we provide evidence by light and electron microscopic immunohistochemistry that podoplanin, a approximately 38-kd membrane glycoprotein of podocytes, is specifically expressed in the endothelium of lymphatic capillaries, but not in the blood vasculature. In normal skin and kidney, podoplanin colocalized with vascular endothelial growth factor receptor-3, the only other lymphatic marker presently available. Complementary immunostaining of blood vessels was obtained with established endothelial markers (CD31, CD34, factor VIII-related antigen, and Ulex europaeus I lectin) as well as podocalyxin, another podocytic protein that is also localized in endothelia of blood vessels. Podoplanin specifically immunolabeled endothelia of benign tumorous lesions of undisputed lymphatic origin (lymphangiomas, hygromas) and was detected there as a 38-kd protein by immunoblotting. As paradigms of malignant vascular tumors, poorly differentiated (G3) common angiosarcomas (n = 8), epitheloid angiosarcomas (n = 3), and intestinal Kaposi's sarcomas (n = 5) were examined for their podoplanin content in relation to conventional endothelial markers. The relative number of tumor cells expressing podoplanin was estimated and, although the number of cases in this preliminary study was limited to 16, an apparent spectrum of podoplanin expression emerged that can be divided into a low-expression group in which 0-10% of tumor cells contained podoplanin, a moderate-expression group with 30-60% and a high-expression group with 70-100%. Ten of eleven angiosarcomas and all Kaposi's sarcomas showed mixed expression of both lymphatic and blood vascular endothelial phenotypes. By double labeling, most podoplanin-positive tumor cells coexpressed endothelial markers of blood vessels, whereas few tumor cells were positive for individual markers only. From these results we conclude that (1) podoplanin is a selective marker of lymphatic endothelium; (2) G3 angiosarcomas display a quantitative spectrum of podoplanin-expressing tumor cells; (3) in most angiosarcomas, a varying subset of tumor cells coexpresses podoplanin and endothelial markers of blood vessels; and (4) all endothelial cells of Kaposi's sarcomas expressed the lymphatic marker podoplanin.     

Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas

We studied tumor lymphatic and vascular densities and lymphovascular invasion (LVI) as prognostic markers in 48 cases of invasive breast cancer treated with partial or total mastectomy and lymph node dissection. All cases were immunostained with D2-40 and CD31. Positively stained microvessels were counted in densely vascular/lymphatic foci (hot spots) at x400. The mean+/-SD peritumoral lymphatic microvessel density (LMD) was significantly higher than intratumoral LMD (9+/-7 vs 4+/-6; P< .01). There was a positive correlation of D2-40 LMD (peritumoral and intratumoral) and CD31 microvessel density counts with lymph node metastasis (r=0.35, 0.5, and 0.38), nuclear grade (r=0.36, 0.28, and 0.3), and stage (r=0.42, 0.56, and 0.49), respectively. Peritumoral and intratumoral D2-40 LMD correlated significantly with the presence of angiolymphatic invasion (detected by D2-40; r=0.54 and 0.54, respectively). D2-40 detected more LVI than H&E- and CD31-detectable vascular invasion (18/48, 5/48, 11/48, respectively). Increased D2-40 detected LVI, and high CD31 microvessel counts showed significant adverse effect on survival status.     

Low podoplanin expression in pretreatment biopsy material predicts poor prognosis in advanced-stage squamous cell carcinoma of the uterine cervix treated by primary radiation.

Lymphatic invasion and nodal metastasis are predictors of poor outcome in cervix carcinoma. We have recently found that low podoplanin immunoreactivity in cervix carcinoma correlated with the presence of lymphatic invasion and nodal metastasis. In the current study, we examined whether podoplanin expression in pretreatment cervical biopsies can predict the presence lymphatic invasion, nodal metastasis, and outcome in advanced-stage tumors treated by nonsurgical means. Podoplanin expression was analyzed by immunohistochemistry in 48 cervical biopsies and corresponding hysterectomy specimens of early-stage invasive squamous cell carcinoma and in 74 pretreatment biopsies from advanced-stage tumors treated with primary radiation. We found a highly significant correlation between podoplanin expression obtained in biopsy and corresponding hysterectomy materials (r = 0.8962, P < 0.0001). Low podoplanin expression showed a significant correlation with lymphatic invasion (P < 0.0001) and nodal metastasis (P = 0.0058). Low podoplanin expression in pretreatment biopsy material showed a significant correlation with poor disease-free (P = 0.0009) and overall (P = 0.0002) survival in advanced-stage tumors. Our results suggest that in advanced-stage cervix carcinomas treated by radiation, when traditional prognostic indicators are not available and treatment decisions are based on biopsy material and clinical staging parameters, examination of podoplanin expression in pretreatment biopsy material may be a useful marker to predict lymphatic metastasis and patient outcome. Prospective studies involving larger numbers of patients are needed to further evaluate the clinical utility of examination of podoplanin expression in patients with cervix carcinoma.     

Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma

Lymph node metastasis is a frequent reason for adverse clinical outcome in many epithelial neoplasms, including head and neck squamous cell carcinoma. The mechanisms underlying the capability of epithelial neoplasms to metastasize via lymphatic vessels have not yet been fully elucidated. There is great debate about whether cancer cells can metastasize by expansion and invasion of pre-existing peritumoral lymphatics or by the formation and invasion of new lymphatics within tumours (lymphangiogenesis). In order to investigate this issue, we examined 81 tissue specimens from patients with head and neck squamous cell carcinoma, using immunostaining for the specific lymphatic endothelium marker podoplanin, and assessed intratumoral and peritumoral lymphatic density. We also quantified lymphatic invasion and examined the possible associations of all the above parameters with clinicopathological features and outcome. Finally, we used double staining with podoplanin and the cell proliferation marker Ki-67 in order to evaluate lymphangiogenesis. High intratumoral and peritumoral lymphatic density were both significantly associated with the presence of lymph node metastasis at the time of diagnosis (chi2 test, p < 0.001 and p = 0.007, respectively) and there was a significant correlation between high intratumoral lymphatic density and lymphatic invasion. Patients with higher intratumoral lymphatic density exhibited shorter overall survival (log rank p < 0.001) and this correlation remained significant after multivariate analysis (Cox p = 0.04), indicating that intratumoral lymphatic density is an independent prognostic factor for mortality. Peritumoral lymphatic density had no influence on outcome. Double staining revealed the existence of proliferating intratumoral lymphatics, in which tumour emboli were occasionally observed. These results indicate that lymphangiogenesis indeed occurs in head and neck squamous cell carcinoma; that newly formed vessels are targets of invasion by cancer cells; and that intratumoral lymphatic density might be used as a criterion to separate patients at higher risk of an adverse clinical outcome.     

D2-40 immunoreactivity in penile squamous cell carcinoma: a marker of aggressiveness

D2-40 immunohistochemical expression was investigated in tissue specimens from 39 patients with squamous cell carcinoma of the penis who underwent partial or total penectomy between 1987 and 2008. Patient age, tumor size, and grade; D2-40-positive lymphatic vessel density in intratumoral, peritumoral, and normal tissue; cell positivity for D2-40 in intratumoral and normal tissue; and D2-40 staining intensity and distribution were analyzed and correlated with disease-specific survival. Analysis of D2-40-positive lymphatics disclosed that mean lymphatic vessel density was greater in peritumoral tissue than in intratumoral and normal tissue and lower in patients with lymph node metastasis than in those without lymph node metastasis. The receiver operating characteristic curve showed that an intratumoral lymphatic vessel density greater than 2.0 had 83.3% sensitivity and 78% specificity in predicting lymph node metastasis. Analysis of cell immunoreactivity showed cytoplasmic D2-40 positivity in intratumoral and normal tissue in 89.7% and 65.5% of patients, respectively. A strong correlation emerged between grade of cell differentiation and D2-40 immunoreactivity in intratumoral tissue; in particular, 88.9% of tumors with weak podoplanin expression were G1, whereas strong cellular immunoreactivity was detected in 83.3% of G3 patients (P = .003; χ(2) test). A significant correlation was also noted between pattern of reactivity and tumor grade because the basal layer was positive in patients with undifferentiated tumors (100% of G3) and in 72.2% of G1 tumors (P = .021; χ(2) test). D2-40 seems to be a useful marker for the development of node metastasis in squamous cell carcinoma of the penis, although validation in larger series is required to confirm its predictive value.     

D2‐40‐positive lymphatic vessel invasion is not a poor prognostic factor in stage I lung adenocarcinoma

The present study investigates whether lymphatic vessel invasion (LVI) detected by D2‐40 staining is a prognostic factor for stage I adenocarcinoma of the lung. We retrospectively reviewed 124 patients who underwent complete resection for stage I adenocarcinoma of the lung from January 1983 to June 2003. LVI was microscopically evaluated using D2‐40 immunostaining. The median follow‐up was 71 months. The LVI positive rate was 37%. The 5‐year cancer‐specific survival rates of the D2‐40 positive LVI and negative groups were 88.8% and 84.3%, respectively (P = 0.630). The stage I lung adenocarcinoma patients who were determined to be LVI positive based on D2‐40 immunostaining did not have a significantly poorer prognosis than the LVI negative cases. Thus, lymphatic microinvasion may not be a prognostic indicator in early lung cancer, although advanced LVI does appear to correlate with survival. It is therefore unnecessary to use D2‐40 immunostaining to diagnose LVI in practical settings, and Hematoxylin‐Eosin and Elastica van Gieson staining should continue to be used to predict the prognosis of patients with stage I lung adenocarcinoma.     

Angiogenesis and lymphangiogenesis in sporadic hepatic angiomyolipoma

Angiogenesis and lymphangiogenesis are critical processes for tumor growth, invasion, and metastasis. The present study aimed to investigate the distribution and clinical significance of angiogenesis and lymphangiogenesis in hepatic angiomyolipoma (AML). We performed immunohistochemical staining for endothelial cell markers (CD34 and podoplanin) on 80 cases of sporadic hepatic AMLs. Microvessel density (MVD) and lymphatic vessel density (LVD) were determined in intratumoral and peritumoral regions and adjacent non-tumorous liver tissues. All hepatic AMLs showed positive staining for CD34 and podoplanin. Intratumoral and peritumoral MVDs and LVDs were significantly higher than those in adjacent liver tissues (P < 0.001). No statistical difference in both MVD and LVD was found between intratumoral and peritumoral areas. Large tumors (>5 cm) had a significantly increased MVD and LVD as compared with smaller tumors. A significant positive correlation was found between average LVDs and MVDs (r = 0.567, P < 0.001), and LVDs were a relatively lower event as compared with MVDs. Double immunostaining revealed that no neoplastic cells positive for HMB-45, an antibody reacting with melanosome-associated antigen, were concurrently immunoreactive for endothelial cell markers. In conclusion, intratumoral and peritumoral angiogenesis and lymphangiogenesis commonly occur in hepatic AMLs, thus representing potential therapeutic targets for this disease.