Pathologic assessment of tumor-associated macrophages and their histologic localization in invasive breast carcinoma.

BACKGROUND: Tumor-associated macrophages (TAMs) are important in regulating cross-talk between tumor cells and tumor microenvironment. TAMs are involved in multiple steps of tumor progression and invasion. This study aimed to compare CD163 expression with the widely used CD68 pan-macrophage marker in invasive breast carcinoma. Furthermore, it focused on assessing the significance of TAMs localization in relation to clinicopathological parameters. RESULTS: CD68 and CD163 immunohistochemical expressions within TAMs infiltrating both tumor nest (TN) and tumor stroma (TS) were evaluated in 60 specimens with invasive breast carcinoma. High CD68-positive stromal TAMs was significantly related to larger tumor, nodal metastasis and vascular invasion (p = 0.003, 0.037, 0.032, respectively), whereas high CD163-positive stromal TAMs was significantly related to larger tumors, nodal metastasis, stage III tumors, vascular invasion, estrogen receptor (ER) negativity, and triple-negative subtype (p = 0.023, < 0.001, 0.001, 0.022, 0.002, 0.017, respectively). On multivariate analysis, high CD68-positive TAMs infiltrating TS was significantly associated with larger tumor and positive nodal metastasis (p = 0.006 and 0.016, respectively), whereas high CD163 TAMs density within TS was significantly associated with positive vascular invasion, nodal metastasis, and molecular subtypes (p = 0.003, 0.001, and 0.009, respectively). CONCLUSION: TAMs within tumor stroma and tumor nest have different levels of association with poor prognostic parameters. So, it is of great importance to consider the histologic localization of TAMs in addition to the degree of TAMs infiltration.

Detailed assessment of microvasculature markers in non-small cell lung cancer reveals potentially clinically relevant characteristics.

Tumor angiogenesis is important for the progression of cancer and is orchestrated by various factors associated with tumor vessels, tumor cells, and stromal cells. Angiogenic signaling in non-small cell lung cancer (NSCLC) needs to be further clarified, especially regarding existing and upcoming therapeutic approaches. Expression of CD34, CD105, Mel-CAM, VE-cadherin, D2-40, VEGF, VEGFR1, and VEGFR2 was assessed immunohistochemically on a cohort of 371 well documented, surgically resected NSCLC using a standardized tissue microarray platform. Extensive clinical data and a postoperative follow-up period of up to 18 years allowed us to assess clinicopathological correlations in detail. Microvasculature in NSCLC was significantly denser at the tumor periphery as compared to the tumor center. Squamous cell carcinomas (SCC) were associated with a notably lower microvessel density (MVD) than adenocarcinomas (ACA). CD105 was present at significantly higher levels on stromal cells of ACA as compared to SCC. Expression of VE-cadherin by tumor cells (6% of cases, mainly ACA) as well as decreased MVD in the tumor centers was independently associated with poor prognosis in the entire cohort. Low MVD in SCC might be related to lower efficacy of and fatal bleeding during therapy with bevacizumab. In other NSCLC entities for which treatment with VEGF inhibitors is studied in clinical trials, the predictive value of MVD for therapy response merits to be prospectively examined. Our data suggest that patients with ACA may be candidates for therapies targeting CD105. VE-cadherin is another promising target for therapy, but its expression also provides independent prognostic information.

Microvessel Landscape Assessment in Pancreatic Ductal Adenocarcinoma: Unclear Value of Targeting Endoglin (CD105) as Prognostic Factor of Clinical Outcome.

OBJECTIVES: Tumor angiogenesis based on microvessel density assessment has been associated with poor prognosis in several studies of patients with pancreatic ductal adenocarcinoma (PDAC). Expression of endoglin (CD105), a tumor-induced vascularization marker, has been found to represent a negative prognostic factor in many malignant tumors. The aim of our study was to assess the value of tumoral microvascularity both with pan-endothelial markers and endoglin as well, in correlation with the clinical outcome of patients with PDAC. METHODS: Fifty-eight patients with PDAC, 36 males and 22 females, with a mean (SD) age of 65.4 (10.0) years were included in the study. Deparaffinized sections from formalin-fixed areas both from the center and periphery (invasion front) of the tumors were immunostained for CD105 as well as for the endothelial markers CD31 and CD34. Tumoral angiogenesis was assessed on the basis of microvessel density (number of vessels per square millimeter) and on microvascular area (square micrometers) as well. RESULTS: High intratumoral microvascular area, in endoglin-stained sections, was found to be of marginal prognostic significance for recurrence (log rank, P 0.05). Survival was also marginally associated with CD31 intratumoral microvascular area (log rank, P 0.05). CONCLUSIONS: Further studies are needed before endoglin replaces the conventional angiogenesis markers in PDCA.

Angiogenesis in superficial esophageal squamous cell carcinoma: assessment of microvessel density based on immunostaining for CD34 and CD105.

OBJECTIVE: The esophagus is the only organ where changes in the superficial microvasculature from normal squamous epithelium to invasive cancer are evident by magnifying endoscopy. We investigated in detail the features of angiogenesis in early-stage esophageal cancer using CD34 and CD105 immunostaining, and also the correlation between angiogenesis and mononuclear cell infiltration. MATERIALS AND METHODS: Using 10 samples of normal squamous epithelium, 7 samples of low-grade intraepithelial neoplasia, and 45 samples of superficial esophageal cancer, we determined the microvessel density at hot spots showing positive staining for CD34 and CD105. We observed the histological features of CD34- and CD105-positive microvessels that corresponded to observations made by magnifying endoscopy. We then investigated the correlation between microvessel density and each histological situation or the grade of mononuclear cell infiltration. RESULTS: The histological features of CD34- and CD105-positive microvessels were able to explain the morphological changes in the microvasculature during cancer progression observed by magnifying endoscopy. The microvessel density for CD34 or CD105 was significantly correlated with each of the histological types (P < 0.001, rS = 0.51 and 0.76, respectively). Mononuclear cell infiltration at CD105 hot spots was most frequent in M1 and M2 cancer (94.7%). The correlation between the degree of mononuclear cell infiltration and microvessel density for CD105 staining was also significant (P < 0.001, rS = 0.49). CONCLUSIONS: The microvessel density based on CD34 and CD105 immunostaining can be used to corroborate observations of superficial esophageal squamous cell carcinoma made by magnifying endoscopy. Mononuclear cell infiltration may play an important role in angiogenesis at the early stage of cancer progression.

Endoglin and CD-34 immunoreactivity in the assessment of microvessel density in normal pituitary and adenoma subtypes.

UNLABELLED: Vascularization is a prerequisite of tumor growth, invasion and metastasis. In the present work, microvessel density was assessed by quantitating using two different endothelial cell biomarkers, endoglin (CD-105) and CD-34. Fifty endocrinologically active and 36 clinically nonfunctioning pituitary adenomas, all surgically resected, as well as 10 autopsy-derived normal adenohypophyses were investigated by immunohistochemistry. The results showed that in every pituitary adenoma type endoglin, an assumed biomarker of proliferating endothelial cells, immunostained fewer vessels than CD-34 which revealed immunopositivity in all capillaries. Differences in endoglin versus CD-34 immunoexpression indicate varying degrees of vascularity in pituitary adenoma subtypes. The low levels of endoglin immunoexpression in pituitary tumors exposed to long-acting somatostatin analogs and dopamine agonists are consistent with the view that these agents inhibit angiogenesis. KEYWORDS: immunohistochemistry, endoglin, CD34, microvascular density, angiogenesis, pituitary.

Microvessel density assessment in benign and malignant endometrial changes.

Tumor angiogenesis is believed to be a prognostic indicator associated with tumor growth and metastasis. Microvessel density (MVD) assessment with common endothelial markers such as CD34 has been found to influence prognosis among endometrial carcinoma patients. The CD105/endoglin antibody has been reported to preferentially bind to proliferated endothelial cells in tissues participating in angiogenesis. The aim of this study was to evaluate the quantification of angiogenesis by assessing MVD in endometrial lesions when comparing the performance of anti-CD34 and anti-CD105 in women with benign and malignant endometrial changes. The study included 58 women (37 postmenopausal) with normal, hyperplastic and malignant endometrium in which preoperative transvaginal sonography was performed. Histological results of the removed endometrium were correlated with MVD assessed in "hot areas" where high densities of microvessels were detected within tumoral tissue. Endometrial cancer was confirmed in 37 women (3 premenopausal). Benign hyperplasia (14 cases), secretory or proliferative endometrium (5 cases) or endometrial atrophy (2 cases) was found in the remaining women. Malignant changes were mostly noted as FIGO stage I and II (28 cases) and had a low (1 or 2) histological grade (29 cases). Median MVD's assessed with CD105 and CD34 were 10.4 and 32.3, respectively. Median MVD assessed with CD34 was almost twice higher in women with endometrial cancer than in women with benign endometrium (CD34 MVD = 41.8 vs. 27.6, p=0.004). In cases of CD105 MVD significant differences between women with benign and malignant endometrial changes were also found (CD105 MVD = 11.8, vs. 6.4; p=0.00007). The menopausal status, but not the clinical stage or histological grading was significantly correlated with both CD34 MVD (p=0.02) and CD105 MVD (p=0.0003). A significant correlation was also found between CD34 and CD105 measured MVD (p=0.000001). In conclusion, transition from endometrial hyperplasia to endometrial cancer appears to be accompanied by microvessel density changes. MVD assessed with both CD34 and CD105 antibodies could be used as a potential prognostic factor in women with endometrial cancer. Our study showed that endoglin, by staining the proliferating microvessels could be more specific and sensitive marker for tumor neoangiogenesis than the more commonly used marker, CD34.

Assessment of endoglin and calprotectin as potential biomarkers in ovarian carcinoma and borderline tumors of the ovary.

OBJECTIVE: The objective of the study was to analyze circulating endoglin concentration in ovarian carcinoma and evaluate a prognostic role for calprotectin and endoglin in effusions in advanced-stage disease. STUDY DESIGN: Preoperative plasma concentration of endoglin from women with benign ovarian tumors (n = 71), borderline ovarian tumors (BOT, n = 39), and ovarian carcinomas (n = 89) was analyzed with an enzyme-linked immunosorbent assay, as were endoglin and calprotectin concentrations in effusions from 164 women with advanced-stage ovarian carcinoma. RESULTS: Median endoglin plasma concentration was higher in the BOT group as compared with both control and invasive carcinoma groups (4.9 vs 4.5 and 4.3 ng/mL, P = .04 and P = .02), whereas the difference between the control and invasive group was not statistically significant (4.5 vs 4.3 ng/mL, P = .08). Endoglin and calprotectin effusion concentrations did not correlate with survival. CONCLUSION: Circulating endoglin is not elevated in advanced ovarian carcinoma. This is in contrast to the situation in breast and gastric cancer.

Assessment of vascularity as an index of angiogenesis in periradicular granulomas. Comparison with oral carcinomas and normal tissue counterparts.

AIM: To quantify vascularity in periradicular granulomas using different endothelial markers, and assess its value as an index of angiogenesis by comparing granulomas with healthy periodontal ligament (PDL). To use oral tumours, compared with adjacent normal mucosa, as positive controls. METHODOLOGY: Paraffin-embedded sections were stained with antibodies to von Willebrand factor (vWF), a pan-endothelial marker, and CD105, a putative marker for angiogenic vessels. Vascularity was quantified by different methods reflecting vessel volume and density. RESULTS: Irrespective of the marker or method used, vascularity values were similar in periradicular granuloma and PDL. Both tissues were highly vascularized, with levels similar to those found in oral squamous cell carcinoma. Vascularity was significantly higher in the latter than in normal mucosa. Fewer vessels were positive for CD105 than for vWF in the normal mucosa, whereas similar numbers were found in the other tissues examined. CONCLUSIONS: A comparison of vascularity in oral tumours and normal oral mucosa provided evidence of angiogenesis in the former. Staining with CD105 added limited value to staining with vWF in these tissues. In contrast, a comparison of periradicular granuloma and PDL failed to demonstrate evidence of angiogenesis in the granuloma. As all vessels were similarly stained with vWF and CD105 in granuloma and PDL, a possible hypothesis is that all vessels are newly formed in these tissues. A more plausible alternative is that CD105 expression may reflect the metabolic activity or intrinsic characteristics of the tissues, rather than the presence of angiogenic vessels.

A colorimetric assay for studying effector secretion through the bacterial type III secretion system.

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.

The in vivo assessment of a novel scaffold containing heparan sulfate for tissue engineering with human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.

Optimization of the cost and sensitivity of receptor- and enzyme-based assays.

In detecting receptor antagonists or enzyme inhibitors, there are three parameters that often affect the outcome in a predictable quantitative manner: concentrations of the receptors (enzyme), labeled ligand (substrate), and antagonist (inhibitor). The usual goal of assay optimization is to maximize the ability of the assay to detect low concentrations of the analyte. Another question of practical importance, especially in screening of large numbers of samples, would be minimization of the reagent cost. Although the mathematical theory of optimization of the receptor binding assay was developed a long time ago, the resulting formulas (in the general case of unequal affinities of ligand and competitor) were not well suited for practical use. The current availability of computational programs, such as Mathematica, makes possible an efficient solution, both for receptor- and enzyme-based assays. We use a graphical approach to assay optimization and apply it to the following problems: (1) optimization of assay sensitivity, (2) optimization of the reagent cost, and (3) analysis of the entire range of the parameter values since the mathematically optimal values may sometimes be impractical. The computation is extremely simple and the problem can sometimes be solved in several minutes.

Endothelial Tie growth factor receptor provides antigenic marker for assessment of breast cancer angiogenesis.

Breast cancer prognosis has previously been linked to the degree of tumour vascularisation. In order to establish additional markers for tumour angiogenesis, we have used monoclonal antibodies against the endothelial Tie receptor tyrosine kinase to study the degree of vascularisation of breast carcinomas and the regulation of Tie expression in the vascular endothelial cells. Antibodies were used for Tie detection and the results were correlated with other prognostic markers. Of four monoclonal antibodies directed against different epitopes of the Tie extracellular domain, two reacted against Tie in unfixed histopathological sections of breast carcinomas. One of these antibodies (clone 7e8) was specific for the endothelial cells whereas the other (clone 10f11) also reacted with basement membranes and occasional carcinoma cells. When Tie expression was studied with the antibody clone 7e8, all 27 carcinomas, two in situ carcinomas, samples of histologically normal breast tissue (n = 16) or normal skin or lymph node tissue (n = 5) showed staining. Microvessel counts were higher in carcinomas (median 14; range 3-27) than in fibrodenomas (median 10; range 5-18) or histologically normal breast tissue (median 7; range 3-15, P = 0.0006). A similar result was obtained using antibodies against the CD31 (PECAM) antigen. Microvessel counts in 7e8 staining were not significantly associated with primary tumour size, axillary nodal status, histological grade or staining for oestrogen receptor, progesterone receptor, Ki-67 proliferation marker or p53 oncoprotein.

Usefulness of technetium-99m-galactosyl human serum albumin liver scintigraphy for assessment of severity of alcoholic hepatitis.

We performed a liver scintigraphy using technetium-99m diethylene-triaminepentaacetic acid-galactosyl human serum albumin (99mTc-GSA), which images the functional liver mass through its binding to the specific receptor asialoglycoprotein receptor in patients with severe alcoholic hepatitis. Receptor index (LHL 15) was significantly lower in patients with alcoholic hepatitis, compared with controls with normal liver. Difference in the isotope uptake patterns between liver and heart varied according to the severity of liver disease, and made it possible to categorize 5 grades. Grading score could discriminate between the eventual outcome of the patients. Furthermore, single photon emission computed tomography showed the variable uptake patterns in the hepatic lobule, wherein there were no evident findings in macroscopic view at autopsy. The results of this study show the usefulness of 99mTc-GSA scintigraphy in the evaluation and prognosis of alcoholic hepatitis.

Biotinylation of lipoprotein lipase and hepatic triglyceride lipase: application in the assessment of cell binding sites.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HL) were biotinylated using N-hydroxysuccinamide ester of biotin (25-fold molar excess) which was incorporated into the lysine amino groups of the enzyme protein. By assessing enzyme activity and heparin-agarose affinity a biotinylation protocol which did not denature lipases was developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that biotinylated LPL (bLPL) has the same mobility as that of unlabeled or iodinated LPL. Receptor binding activity of bLPL was studied in (i) cell binding experiments using cultured bovine aortic endothelial cells and (ii) ligand blotting experiments using endothelial cell plasma membranes. Endothelial cells in culture bound similar amounts of bLPL and 125I-LPL. We previously described a 116-kDa heparin-releasable LPL binding protein (hrp-116) on endothelial cells. Using biotinylated lipases in ligand blotting experiments we now demonstrate that both bLPL and biotinylated HL can bind to hrp-116. bLPL in addition also bound to low-density lipoprotein receptor related protein in ligand blotting. Thus, our protocol has produced biotinylated lipases which are both chemically and biologically active and can be used instead of iodinated lipases.

An assessment of the ability of human bone marrow cultures to generate osteoclasts.

Several groups have successfully generated osteoclasts in cultures of murine haemopoietic cells. This approach would clearly be useful in the analysis of mechanisms of regulation of human osteoclast formation if analogous results could be obtained in cultures of human bone marrow. This communication describes independent attempts by three groups to generate unequivocally defined osteoclasts from bone marrow obtained from human iliac crest, femoral neck, rib, and from foetuses. The haemopoietic tissue was incubated using techniques described by others for production of osteoclast-like cells, and with variants of this technique using strategies based on our experiences with murine osteoclastogenesis. Haemopoietic cells were incubated with calcium regulating hormones, cytokines, osteoblastic supernatants, and osteoblastic or bone marrow stromal cell layers. Formation of cells capable of excavation of bone slices was rarely seen. Despite the paucity of bone resorbing cells, multinucleate cells (MNCs) developed with similar characteristics to the MNCs that have been interpreted as osteoclast-like in human bone marrow cultures. The MNCs were, however, calcitonin-receptor (CTR) negative, and did not show the typical pattern of reactivity with osteoclast-specific antibodies. They possessed instead an antigenic profile characteristic of macrophage polykaryons. We conclude that the MNCs which consistently generate in human bone marrow cultures do not possess phenotypic characteristics specific for osteoclasts and appear to be macrophage polykaryons. The conditions required for osteoclast generation in cultures of human haemopoietic cells remain to be defined.

Means for the assessment of radioligand quality and its importance in receptor-binding studies. Observations with radiolabelled formylmethionyl-leucyl-phenylalanine.

Various methods for testing the quality of radioligands were applied to two different radiolabelled forms of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The purpose of the study was both to examine the value of these methods for assessing radioligand quality and to determine the suitability of these particular radioligands for studying the chemotactic formylpeptide receptors on the rabbit neutrophil. It is useful in this context to distinguish two different aspects of radioligand quality: these are purity and equivalence to the native ligand. The two methods described for measuring receptor-reactivity (or 'bindability'), by measuring binding to an increasing excess of receptors and by a re-incubation procedure, provide a reliable measure of purity that should readily be applicable to other radioligands. Equivalence to the native ligand is more difficult to establish, and any uncertainty about the specific radioactivity of the radioligand can pose serious problems with this assessment. Commercial preparations of both tritiated and 35S-labelled fMet-Leu-Phe were found to be inadequately pure for detailed receptor studies. Repurification by t.l.c., however, consistently yielded radioligand preparations of high purity and close equivalence to the native ligand. Other radioligands may often also require a suitable repurification step before use for detailed receptor studies; this is especially important whenever a complex receptor-binding pattern is envisaged.

Assessment of mammary lactogenic receptor changes in pregnant rabbits.

Lactogenic receptor was purified from rabbit mammary tissue and used to generate an antiserum in goats. The purified lactogenic receptor material bound lactogenic hormones specifically and reversibly. Antiserum generated in a goat bound a labeled human growth hormone/receptor complex; this was displaced by nonlabeled solubilized receptor preparations. This was used as a radioimmunoassay and was able to detect 0.037 fmol of lactogenic receptor. The specificity of the radioimmunoassay for lactogenic receptor was supported by three lines of evidence; first, the ligand used in the radioimmunoassay was an iodine 125-labeled human growth hormone/receptor combination; therefore, only membrane protein with structural homology to the protein which bound 125I-labeled human growth hormone competed for binding to the antiserum; second, depletion of radioreceptor binding sites by affinity chromatography with ovine prolactin as the fixed ligand was detected; third, an increase in breast lactogenic receptor during pregnancy was detected by both radioreceptor assay and the radioimmunoassay. We found a progressive increase in lactogenic receptors by radioimmunoassay which corresponded to parallel increases by radioreceptor assay in rabbit mammary tissue during pregnancy.

[Assessment of prognostic criteria in uterine carcinomas (author's transl)]. / Die Bewertung prognostischer Kriterien bei Karzinomen des Uterus.

Prognostic criteria of cervical and uterine carcinomas can be important for both physician and patient. Compared with patients not subjected to surgical treatment, a multitude of prognostic criteria must be investigated in operated women via the systemic examination of the surgical specimen, reaching beyond the weight attached to the clinically assumed staging. These criteria enable quantitative and qualitative characterization of the carcinomas allowing individualisation of therapy adapted to the tumour.