Microvessel Density Measurement Research Articles

Measurement of Microvessel Density Using CD105 (Endoglin) as a Marker in Prostatic Adenocarcinoma

Introduction Angiogenesis has been proposed as a promising prognostic marker in a variety of tumours. Microvessel density (MVD) measurement is the most reliable method for the semiquantitative evaluation of angiogenesis. Endoglin (CD105) is a recognised marker of proliferating endothelium and is expressed in intra-tumoral as well as peri-tumoral vessels. It is upregulated in various cancers including prostate cancer, therefore, it is an upcoming prime marker for prognosis, tumour imaging and antiangiogenesis therapy. Aim The study was done to evaluate microvessel density (MVD) by using CD105 (endoglin) as an IHC marker in prostatic adenocarcinoma and to find its relationship with Gleason score and grade groups. Materials and methods This was an observational cross-sectional study under which a total of 31 histopathologically diagnosed cases of prostatic adenocarcinoma on TURP chips were included and were graded according to Gleason score and grades.IHC using endoglin (CD105) was done to detect the highest-density areas of stained vessels (hot spots). The highest value obtained in three hot spot fields was reported as microvessel density (MVD) and its relationship with Gleason score and grade groups was analysed. Results Most common age group was 71-80 years (48.39% cases). Maximum cases (45.16%) had a Gleason score of 9. The maximum mean MVD CD105 score was found in the Gleason grade group 5 (45.32%) while the minimum was in grade group 1 (24%). Comparison of mean MVD CD105 scores in different Gleason grade groups revealed a significant association of MVD CD105 with Gleason grade groups. Conclusion Endoglin measures microvessel density and its expression correlated with Gleason score and grade group. Hence it can be used as a potential prognostic marker in adenocarcinoma prostate.

Modified microvessel density based on perfusion distance: a preferable NSCLC prognostic factor.

BackgroundDespite the vital role of blood perfusion in tumor progression, the prognostic value of typical blood perfusion markers, such as microvessel density (MVD) or microvessel area (MVA), in patients with non-small cell lung cancer (NSCLC) is still unclear. This study established a modified MVD (mMVD) measurement based on perfusion distance and determined its prognostic value in patients with NSCLC.MethodsA total of 100 patients with NSCLC were enrolled in this retrospective study. The intratumor microvessels of NSCLC patients were visualized using immunohistochemical staining for CD31. The blood perfusion distance was evaluated as the distance from each vessel to its nearest cancer cell (Dmvcc), and the cutoff value for prognosis was determined. Apart from the total MVD (tMVD), microvessels near cancer cells within the cutoff-Dmvcc were counted as mMVD. Predictive values for mortality and recurrence were evaluated and compared.ResultsThe Dmvcc ranged from 1.6 to 269.8 µm (median, 13.1 µm). The mMVD (range: 2–70; median 23) was counted from tMVD according to the cutoff-Dmvcc (~20 µm). Compared with tMVD, a larger fraction of mMVD (80% vs. 2.9%) played a significant role in overall survival, with an improved area under the receiver operating characteristic (ROC) curve (AUC) (0.74 vs. 0.56). A high mMVD was an independent positive indicator of overall survival (OS) and progression-free survival (PFS). In contrast, tMVD was only related to PFS at the optimal cutoff.ConclusionsPerfusion-distance-based mMVD is a promising prognostic factor for NSCLC patients with superior sensitivity, specificity, and clinical applicability compared to tMVD. This study provides novel insights into the prognostic role of tumor vessel perfusion in patients with NSCLC.

Evaluation of Intra-Tumoral Vascularization in Hepatocellular Carcinomas.

Intratumoral neovascularization has intricate effects on tumor growth, metastasis, and treatment. Over the last 30 years, Microvessel density (MVD) has been the standard method for laboratory and clinical evaluation of angiogenesis. Hepatocellular carcinoma (HCC) is a typical hypervascularized tumor, and the predictive value of MVD for prognosis is still controversial. According to previous viewpoints, this has been attributed to the determination of hotspot, counting methods, vascular endothelial markers, and different definitions of high and low vascular density; however, the heterogeneity of tumor angiogenesis patterns should be factored. The breakthroughs in artificial intelligence and algorithm can improve the objectivity and repeatability of MVD measurement, thus saving a lot of manpower. Presently, anti-angiogenesis therapy is the only effective systematic treatment for liver cancer, and the use of imaging technology-assisted MVD measurement is expected to be a reliable index for evaluating the curative effect. MVD in multinodular hepatocellular carcinoma represents a subject area with huge understudied potential, and exploring it might advance our understanding of tumor heterogeneity.

Endoglin Expression as a Measure of Microvessel Density in Oral Head and Neck Cancer by Immunohistochemical Study.

Abstract Background and Aim: Tumor growth is limited by the balance between the need for oxygen and nutrients and diffusion of these substances from vessels around tumors. Therefore, angiogenesis, development of new blood vessels from previous blood vessels, is necessary for tumor growth and metastasis. This study was carried out with the aim of evaluating the angiogenesis in oral cancer by assessing microvessel density (MVD) using Endoglin (CD105). This work also correlated the expressions of marker with histopathological grades of oral squamous cell carcinoma. Material and Methods: Immunohistochemical staining with Streptavidin peroxidase was used to analyze the CD105 expression in 25 cases of oral squamous cell carcinoma. Results: All studied cases showed positive of CD105 immunoreactivity. The study revealed a highly significant increase of CD105-MVD in poorly differentiated OSSC than well differentiated OSCC. Conclusion: Increased expression of CD 105 in high grade tumors suggests that Endoglin is likely to contribute significantly to resistance in response to therapy.

MGMT Promoter Methylation Status Is Not Related to Histological or Radiological Features in IDH Wild-type Glioblastomas.

O6-methylguanine DNA methyltransferase (MGMT) promoter methylation is an important favorable predictive marker in patients with glioblastoma (GBM). We hypothesized that MGMT status could be a surrogate marker of pretreatment tumor biology observed as histopathological and radiological features. Apart from some radiological studies aiming to noninvasively predict the MGMT status, few studies have investigated relationships between MGMT status and phenotypical tumor biology. We have therefore aimed to investigate such relationships in 85 isocitrate dehydrogenase (IDH) wild-type GBMs. MGMT status was determined by methylation-specific PCR and was assessed for associations with 22 histopathological features, immunohistochemical proliferative index and microvessel density measurements, conventional magnetic resonance imaging characteristics, preoperative speed of tumor growth, and overall survival. None of the investigated histological or radiological features were significantly associated with MGMT status. Methylated MGMT status was a significant independent predictor of improved overall survival. In conclusion, our results suggest that MGMT status is not related to the pretreatment phenotypical biology in IDH wild-type GBMs. Furthermore, our findings suggest the survival benefit of MGMT methylated GBMs is not due to an inherently less aggressive tumor biology, and that conventional magnetic resonance imaging features cannot be used to noninvasively predict the MGMT status.

Pro-Angiogenic and Pro-Inflammatory Regulation by lncRNA MCM3AP-AS1-Mediated Upregulation of DPP4 in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) represents the most common type of renal cell carcinoma (RCC) in adults, in addition to the worst prognosis among the common epithelial kidney tumors. Inflammation and angiogenesis seem to potentiate tumor growth and metastasis of the malignancy. The current study explored the contributions of the lncRNA MCM3AP-AS1 in tumor-associated inflammation and angiogenesis in ccRCC with a specific focus on its transcriptional regulation and its interactions with transcription factor E2F1 and DPP4. Tumor tissues and matched adjacent non-tumor tissues were collected from 78 ccRCC patients. Methylation-specific PCR and ChIP assays were applied to detect the methylation at the promoter region of MCM3AP-AS1. Dual-luciferase reporter assay, RIP, RNA pull-down, and ChIP assays were employed to confirm the interactions between MCM3AP-AS1, E2F1, and DPP4. Nude mice were subcutaneously xenografted with human ccRCC cells. Cell proliferation was evaluated by CCK-8 assays and EDU staining in ccRCC cells in vitro and by immunohistochemical staining of Ki67 in vivo. Inflammation was examined by detecting the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Pro-angiogenic ability of ccRCC cells was assessed by the co-culture with human umbilical vein endothelial cells (HUVEC) in vitro and by microvessel density (MVD) measurements and angiogenesis in the chicken chorioallantoic membrane. MCM3AP-AS1 was highly-expressed in ccRCC and associated with poor patient survival. Demethylation of MCM3AP-AS1 was noted in ccRCC tissues and cells. Over-expression of MCM3AP-AS1 enhanced cell proliferation, the release of pro-inflammatory cytokines, and the tube formation of HUVECs in cultured human Caki-1 and 786-O cells. MCM3AP-AS1 was shown to enhance the E2F1 enrichment at the DPP4 promoter, to further increase the expression of DPP4. Knockdown of DPP4 could abate pro-angiogenic and pro-inflammatory abilities of MCM3AP-AS1 in ccRCC cells. Pro-angiogenic and pro-inflammatory abilities of MCM3AP-AS1 in vivo were confirmed in mice subcutaneously xenografted with human ccRCC cells. Our findings demonstrate a novel mechanism by which lncRNA MCM3AP-AS1 exerts pro-angiogenic and pro-inflammatory effects, highlighting the potential of MCM3AP-AS1 as a promising target for treating ccRCC.

Development of an Automated Image Analyzer for Microvessel Density Measurement in Bone Marrow Biopsies.

Angiogenesis is important for the proliferation and survival of multiple myeloma (MM) cells. Bone marrow (BM) microvessel density (MVD) is a useful marker of angiogenesis and an increase in MVD can be used as a marker of poor prognosis in MM patients. We developed an automated image analyzer to assess MVD from images of BM biopsies stained with anti-CD34 antibodies using two color models. MVD was calculated by merging images from the red and hue channels after eliminating non-microvessels. The analyzer results were compared with those obtained by two experienced hematopathologists in a blinded manner using the 84 BM samples of MM patients. Manual assessment of the MVD by two hematopathologists yielded mean±SD values of 19.4±11.8 and 20.0±11.8. The analyzer generated a mean±SD of 19.5±11.2. The intraclass correlation coefficient (ICC) and Bland-Altman plot of the MVD results demonstrated very good agreement between the automated image analyzer and both hematopathologists (ICC=0.893 [0.840–0.929] and ICC=0.906 [0.859–0.938]). This automated analyzer can provide time- and labor-saving benefits with more objective results in hematology laboratories.

Disturbances in angiogenesis and vascular maturation in the skin are associated with diabetic kidney disease in type 1 diabetes

Introduction. The skin, as one of the most accessible tissues, is frequently used for investigations of microcirculation and angiogenesis. The aim of this study was to assess the relationship between the dermal microvessel density (MVD) and maturity and the presence of diabetic kidney disease (DKD) in adults with type 1 diabetes (T1D). Skin as the most accessible organ served as a model for the study of angiogenesis. Materials and methods. 148 consecutive T1D patients (87 men), median age of 41 [interquartile range (IQR): 31–49] years and diabetes duration of 21 (17–30) years, participated in the study. The patients were under the care of the Department of Internal Medicine and Diabetology, Poznan University of Medical Sciences. Diabetic kidney disease was diagnosed in patients with increased albuminuria and at least 10-year duration of diabetes or evidence of diabetic retinopathy. The skin biopsy was performed on distal part of lower leg, using a sterile, disposable 3 mm biopsy punch with plunger (Disposable Biopsy Punches, Integra™ Miltex ® ). In the immunohistochemical analyses, we used: anti-CD133, anti-CD34, anti-CD31, and anti-von Willebrand factor (vWF) autoantibodies. Microvessel density measurement in all specimens was performed using “hot spots technique”. Slides were scanned using the MiraxMidi scanner (Carl Zeiss) and were viewed using CaseViewer (3DHISTECH Ltd. Budapest, Hungary). Data were analyzed using Statistica v. 13 software. Results. In the study group 21 patients with diagnosis DKD+, as compared to 127 subjects withaout DKD–, had longer duration of diabetes [30 (IQR: 21–36) vs. 21 (16–28) years, p = 0.002], higher prevalence of hypertension [14 (67%) vs. 37 (29%), p = 0.002], lower estimated glomerular filtration rate (eGFR) [66 (55–88) vs. 94 (83–106) mL/min/1.73 m 2 , p < 0.001]. Median MVD compared between groups with and without DKD, was similar for CD34+ vessels/1 mm 2 [123 (100–170) vs. 121 (100–170), p = 0.775], CD133+ vessels/1 mm2 [79 (50–100) vs. 79 (63–93), p = 0.823], and for CD31+ vessels/ 1 mm 2 [29 (21–46) vs. 38 (17–58), p = 0.454]. Median MVD vWF+ vessels/1 mm 2 was lower in the group with than without DKD: 42 (25–54) vs. 54 (43–71), p = 0.009. The values given above were calculated for both layers of the dermis (papillary and reticular dermis). In multivariate logistic regression analysis presence of diabetic kidney disease was associated with lower median vWF+ MVD [odds ratio: 0.97 (95% confidence interval: 0.95–0.99), p = 0.017], with adjustment for age, gender, eGFR value, diabetes duration and presence of hypertension. MVD did not differ significantly between chronic kidney disease stages. Conclusion. In patients with type 1 diabetes and diabetic kidney disease the disturbances in the angiogenesis and vascular maturation are present. The number of mature blood vessels (vWF+) in the skin is reduced. Disturbances in the angiogenesis occur at early stages of diabetic kidney disease.

P04.35 Preoperative tumor growth and microvessel density measurements in patients with glioblastoma

P04.35 Preoperative tumor growth and microvessel density measurements in patients with glioblastoma

Vasoactive intestinal peptide is upregulated in women with endometriosis and chronic pelvic pain.

Chronic pelvic pain (CPP) causes compromised the quality of life in women with endometriosis and is often attributed to local inflammation and ingrowth of nerve fibers. In this pilot study, we aimed to investigate whether the inflammation-related vasoactive intestinal peptide (VIP) and interleukin (IL)-6 were increased in affected patients. Endometrial and endometriotic tissue biopsy specimens, and serum and peritoneal fluid (PF) samples, were obtained from 85 endometriosis patients and 53 controls. VIP and IL-6 analysis and measurement of microvessel density in tissue were performed using immunohistochemistry, Western blotting, RT-qPCR, and ELISA. Compared with controls, VIP transcript and protein levels were increased in endometrium from endometriosis patients and further elevated in patients with CPP. In addition, microvessel density, a measurement of angiogenic activity, was increased in the endometrium and in endometriosis lesions in the same subset of patients. Serum and PF levels of VIP and IL-6 were higher in women with endometriosis and CPP compared with endometriosis patients who reported no chronic pain. Vasoactive intestinal peptide is upregulated in endometriosis patients reporting chronic pain. Increased microvessel density in tissue and peritoneal fluid concentrations of IL-6 indicate an elevated inflammation in the pelvic microenvironment of these patients.

Effects of combined natural hirudin and hyperbaric oxygen therapy on survival of transplanted random-pattern skin flap in rats

To investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats. A random-pattern skin flap in size of 10.0 cm×2.5 cm was elevated on the dorsum of 72 Sprague Dawley rats. Then the 72 rats were randomly divided into 4 groups ( n=18) according to the therapy method. At immediate and within 4 days after operation, the rats were treated with normal saline injection in control group, normal saline injection combined with hyperbaric oxygen treatment in hyperbaric oxygen group, the natural hirudin injection in natural hirudin group, and the natural hirudin injection combined with hyperbaric oxygen treatment in combined group. The flap survival was observed after operation, and survival rate was evaluated at 6 days after operation. The skin samples were collected for histological analysis, microvessel density (MVD) measurement, and evaluation of tumor necrosis factor α (TNF-α) expression level by the immunohistochemical staining at 2 and 4 days after operation. Partial necrosis occurred in each group after operation, and the flap in combined group had the best survival. The survival rate of flap was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, and in combined group than in hyperbaric oxygen group and natural hirudin group ( P<0.05). There was no significant difference between hyperbaric oxygen group and natural hirudin group ( P>0.05). At 2 days, more microvascular structure was observed in hyperbaric oxygen group, natural hirudin group, and combined group in comparison with control group; while plenty of inflammatory cells infiltration in all groups. At 4 days, the hyperbaric oxygen group, natural hirudin group, and the combined group still showed more angiogenesis. Meanwhile, there was still infiltration of inflammatory cells in control group, inflammatory cells in the other groups were significantly reduced when compared with at 2 days. At 2 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group ( P<0.05); the expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group ( P<0.05). There was no significant difference in above indexes between hyperbaric oxygen group, natural hirudin group, and combined group ( P>0.05). At 4 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in natural hirudin group and combined group than in hyperbaric oxygen group ( P<0.05). The expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in combined group than in natural hirudin group and hyperbaric oxygen group ( P<0.05). Hyperbaric oxygen and natural hirudin therapy after random-pattern skin flap transplantation can improve the survival of flaps. Moreover, combined therapy is seen to exhibit significant synergistic effect. This effect maybe related to promotion of angiogenesis and the reduction of inflammation response.

Microvessel prediction in H&E Stained Pathology Images using fully convolutional neural networks

BackgroundPathological angiogenesis has been identified in many malignancies as a potential prognostic factor and target for therapy. In most cases, angiogenic analysis is based on the measurement of microvessel density (MVD) detected by immunostaining of CD31 or CD34. However, most retrievable public data is generally composed of Hematoxylin and Eosin (H&E)-stained pathology images, for which is difficult to get the corresponding immunohistochemistry images. The role of microvessels in H&E stained images has not been widely studied due to their complexity and heterogeneity. Furthermore, identifying microvessels manually for study is a labor-intensive task for pathologists, with high inter- and intra-observer variation. Therefore, it is important to develop automated microvessel-detection algorithms in H&E stained pathology images for clinical association analysis.ResultsIn this paper, we propose a microvessel prediction method using fully convolutional neural networks. The feasibility of our proposed algorithm is demonstrated through experimental results on H&E stained images. Furthermore, the identified microvessel features were significantly associated with the patient clinical outcomes.ConclusionsThis is the first study to develop an algorithm for automated microvessel detection in H&E stained pathology images.

Intraglandular transplantation of adipose‑derived stem cells combined with platelet‑rich fibrin extract for the treatment of irradiation‑induced salivary gland damage

The aim of the present study was to determine the effect of adipose-derived mesenchymal stem cells (ADSCs) combined with heterologous platelet-rich fibrin extract (PRFe) on irradiation-induced salivary gland (SG) damage. ADSCs were isolated from C3H mice, whereas PRFe was obtained from New Zealand rabbits. Twelve weeks post irradiation, the ADSCs or PRFe or their combination were transplanted into the submandibular glands of C3H mice with irradiation-induced damage. The salivary flow rate (SFR) was determined and histopathological analysis was performed at 12 weeks post transplantation. Immunofluorescence, microvessel density measurements and transmission electron microscopy were performed to assess α-amylase (AMY) production, apoptosis and microstructural changes in the cells. The administration of ADSCs combined with PRFe increased the SFR at 12 weeks post transplantation, whereas ADSCs alone or PRFe alone failed to do so. The ADSCs+PRFe-treated, irradiated SGs had fewer damaged and atrophied acinar cells, higher AMY levels and an increased microvessel density compared with the untreated irradiated SGs. Moreover, SG tissue from the ADSCs+PRFe group also showed decreased apoptotic and increased proliferative activity compared to that from the irradiated group. In conclusion, ADSCs or PRFe alone did not restore permanent, irradiation-induced damage of SG tissue when used alone, but when used together, they provided effective treatment outcomes.

Tumor angiogenesis and bone metastasis - Correlation in invasive breast carcinoma

PurposeTo test the hypotheses that pathological biomarkers including CD34 positive endothelial cell and microvessel density (MVD) of the primary breast tumor can be used to predict the probability of occurrence for bone metastases and provide information for appropriate therapeutic strategies at an early stage. MethodsThree groups of CD34 immunohistochemical stained slices (n=60) were acquired from surgical specimens of sixty patients including non-metastasis (group 1), only lymph node metastasis (group 2), and bone metastasis (group 3). MVD was measured by TissueGnostics system. ResultsThe MVD measurement results were 57.14±23.00 in group 1, 86.44±21.13 in group 2, and 126.85±47.89 in group 3. There were statistical differences between group 1 and group 2 (P=0.0002), between group 2 and group 3 (P=0.0014) and between group 1 and group 3 (P<0.0001). The strong correlations were found between CD34 positive cell measurement and its percentage (group 1, r=0.74, P=0.0002; group 2, r=0.62, P=0.0034; group 3, r=0.84, P<0.0001), and between CD34 positive endothelial cell measurement and MVD (r=0.61, P<0.0001). ConclusionsQuantitative CD34 positive endothelial cell and MVD measurements of the primary breast tumor have a strong correlation with the occurrence rate of bone metastases, which predicts the probability of occurrence for bone metastases at an early stage.

Effect of Treatment Sequencing on Tumor Response to Combined Treatment With Ultrasound-Driven Microbubbles and Radiation Therapy

Ultrasound-driven microbubbles (USMB) have synergistic effects with radiotherapy (RT). Sequence and timing of RT and vascular-disrupting agents have a large effect on cytotoxicity. We investigated outcomes based on USMB and RT sequencing in a preclinical prostate cancer model. PC-3 prostate cancer xenografts were treated with ultrasound-driven lipid microspheres and 8 Gy of external-beam RT. Sequence 1 (S1) consisted of USMB treatment preceding RT by 3, 6, 12, and 24 hours; Sequence 2 (S2) delivered USMB after RT at the same times. 5 tumors were treated at baseline (no treatment) and at each time point. Tumor effect was assessed via CD31 staining, TUNEL and H&E staining, and Carbonic Anhydrase 9 (CA9) staining to measure microvessel density, cell death and hypoxia, respectively. For the tumors treated using S1, microvessel density (MVD) was 18.8±4.8 counts per field (cpf) at baseline and 9.8±2.3, 9.1±2.5, 11.0±3.9 and 12.8±3.3 cpf when USMB preceded RT by 3, 6, 12 and 24 hours, respectively. Using S2, MVD was 18.0±1.8 cpf at baseline and 12.2±0.7, 10.7±1.1, 12.1±1.9 and 14.2±1.8 cpf, respectively, when RT preceded USMB by the same times. Each MVD measurement was significantly lower than baseline (p<0.05) except for S2 separated by 24 hours (S2-24). The MVD using S1 was larger than S2 at all time points, but none of these differences were statistically significant. Percent cell death (CD) using S1 was 19.2%±4.4% at baseline and 28.0%±6.4%, 35.6%±10.4%, 30.8%±9.0% and 26.4%±3.4% when USMB preceded RT by 3, 6, 12 and 24 hours, respectively. Using S2, CD was 14.8%±4.4% at baseline and 24.2%±7.0%, 27.8%±5.2%, 24.8%±6.1% and 22.4%±5.2%, respectively, at the same times. Only the S1-6 measurement differed significantly from baseline (p<0.01). There was more cell death with S1 than S2 at all time points but these differences were not statistically significant (p>0.05). By both MVD and CD, maximum effect occurred using S1-6. Among S2 measurements, maximum effect was using S2-6. However, despite the clear trend of increasing effect with longer separation time up to 6 hours followed by a decrease with longer separation, the measurements at 3, 6, 12 and 24 hours were not significantly different from each other using either S1 or S2 (p>0.05). The only hypoxia measurement significantly different from baseline was S1-6 (p<0.01). The lowest S2 measurement was S2-6, but it did not differ significantly from baseline (p>0.05). Less hypoxia was measured using S1 than S2 at all time points but these differences were not statistically significant (p>0.05). A trend was seen toward S1 yielding greater tumor effect than S2 and toward maximum tumor effect with treatment separation of 6 hours. However, our sample was not large enough to establish statistical significance. Testing of a larger sample should confirm this finding after which further testing is warranted to develop clinical protocols for combining USMB and RT.

PD-L1 expression correlates with VEGF and microvessel density in patients with uniformly treated classical Hodgkin lymphoma.

Recent studies have reported the associations between programmed death-ligand 1 (PD-L1) or PD-L2/PD-1 pathways and pro-angiogenic genes including hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) in several malignancies. However, no study has examined the relationship or prognostic implication of PD-L1, PD-L2, PD-1, VEGF expression, and microvessel density (MVD) in classical Hodgkin lymphoma (cHL) patients. Diagnostic tissues from 109 patients with doxorubicin, bleomycin, vinblastine, and dacarbazine-treated cHL were evaluated retrospectively by immunohistochemical analysis for PD-L1, PD-L2, PD-1, VEGF expression, and for CD31 expression as a measure of MVD. There was a positive correlation between PD-L1 and VEGF expression (P=0.008) and additionally between PD-L2 and VEGF expression (P=0.001). The mean MVD in tumors positive for both PD-L1 and VEGF was significantly (P=0.022) higher than the mean MVD in tumors negative for both markers. High PD-1 expression group had lower (P=0.019) 5-year overall survival rate than low PD-1 expression group. Multivariate analysis revealed that PD-1 was an independent prognostic factor for cHL with significance (P=0.026). However, PD-L1, PD-L2, and VEGF expression had no prognostic impact. Our data confirmed the positive correlations between PD-L1, VEGF, or MVD. Our findings provided evidence supporting new therapeutic approaches including combinations of anti-PD-L1/PD-1 and anti-VEGF therapy in addition to the current standard regimen for cHL.

Epstein-Barr virus positivity is associated with angiogenesis in, and poorer survival of, patients receiving standard treatment for classical Hodgkin's lymphoma.

Epstein-Barr virus (EBV) is a significant contributor to the development of classical Hodgkin's lymphoma (cHL). Recent studies have documented associations between angiogenesis and EBV-associated malignancies. No study has yet examined the associations among, and prognostic implications of, EBV infection, vascular endothelial growth factor (VEGF) expression, and microvessel density (MVD) in cHL patients. Diagnostic tissues from 135 cHL patients treated with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) were retrospectively evaluated by in situ hybridization of EBV-encoded small RNA (EBER) and immunohistochemical staining for VEGF and CD31 (a measure of MVD). EBER and VEGF expression were positively correlated (P=0.038). The mean MVD value of EBER-positive tumors was significantly higher than that of EBER-negative tumors (P=0.034). The mean MVD of tumors positive for both EBER and VEGF was significantly higher than that of tumors negative for both markers (P=0.008). EBER-positive patients had a lower 5-year overall survival (OS) rate than EBER-negative patients (P=0.046). A high MVD was also associated with a poorer OS (P=0.01); multivariate analysis showed that this was a significant and independent prognostic factor (P=0.026). We found positive correlations between EBER and VEGF levels, and the MVD, indicating that EBV plays an important role in tumor angiogenesis. Targeting of both angiogenesis and EBV may be important when treating cHL patients who are EBER-positive and/or have a high MVD.

Development and Validation of a Histological Method to Measure Microvessel Density in Whole-Slide Images of Cancer Tissue.

Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.

Effects of prolonged hyperoxia on pulmonary microvascular development and expression of Ehprin-B2 in neonatal mouse lungs

Objective To investigate the effects of long-term oxygen exposure on the pulmonary microvascular development and the expression of Ephrin-B2 of lungt issue in neonatal mice. Methods Forty-eight 2-day-old Kunming mice were randomly divided into hyperoxia group and air group, with 24 mice in each group. Mice in hyperoxia group were exposed to 70% oxygen to establish a model of bronchopulmonary dysplasia (BPD). Six mice from each group were sacrificed at 3, 7, 14 and 21 days of age, and lung tissue was collected for further test. The lung sections were stained with hematoxylin and eosin for histological evaluation, radial alveolar counts (RAC) and microvessel density (MVD) measurement by CD34 immunohistochemistry. Location and expression of Ephrin-B2 in lung tissue were measured by immunohistochemistry. Ephrin-B2 mRNA and protein levels were detected by fluorescent quantitative reverse transcriptase-polymerase chain reaction and Western blot, respectively. Two independent samples t-test was used for statistical analysis. Results (1) Pathological changes: The pathology of lung tissue in hyperoxia group showed typical BPD-like changes with advancing postnatal age, presenting mainly with simplified alveolar development and decreased microvessel number. Compared with the air group, RAC and MVD were significantly decreased in 14-day-old mice (6.067±0.432 vs 6.950±0.243, t=4.365, P<0.05; 4.133±0.476 vs 4.867±0.472, t=2.680, P<0.01) and 21-day-old mice in the hyperoxia group (8.050±0.362 vs 9.817±0.487, t=7.127, P<0.05; 4.333±0.532 vs 6.017±0.937, t=3.828, P<0.01). (2) Location and expression of Ephrin-B2: Ephrin-B2 was mainly expressed in alveolar epithelial cells, and weakly expressed in alveolar septum. Compared with the air group, the average optical density of Ephrin-B2 was significantly decreased in 7-day-old (0.146±0.013 vs 0.153±0.009), 14-day-old (0.140±0.007 vs 0.161±0.006) and 21-day-old mice in the hyperoxia group (0.138±0.008 vs 0.166±0.009)(t=-2.049, -9.442 and -10.087, all P<0.05). (3) Ephrin-B2 mRNA and protein levels: The Ephrin-B2 mRNA levels in 14-day-old (0.65±0.14 vs 1.05±0.16, t=4.609, P<0.01) and 21-day-old mice (0.57±0.09 vs 1.13±0.18, t=6.816, P<0.01) were significantly lower in hyperoxia group than in the air group. The Ephrin-B2 protein levels were also significantly lower in hyperoxia group than in the air group in 21-day-old mice (0.13±0.03 vs 0.29±0.08, t=4.587, P=0.000). Conclusions Oxygen-induced BPD model mice have simplified alveolar development, reduced MVD and decreased expression of Ephrin-B2 in lung tissue, which may play an important role in the pathogenesis of BPD. Key words: Bronchopulmonary dysplasia; Microvessels; Ephrin-B2; Disease models, animal

Inhibitory effect of endostatin gene therapy combined with phosphorus-32 colloid on tumour growth in Wistar rats.

Eighty healthy male Wistar rats, aged 5 weeks, weighing 100–120 g, were utilized for establishing tumour-bearing models by immediate Walker-256 cancerous ascites injection and randomly divided to four groups (n=20) treated with 0.2 ml solution containing saline, 32P-colloid (0.3 mCi), endostatin gene (20 μg), endostatin gene combined with colloid 32P. The effect of endostatin combined with a small dose of 32P-colloidal on tumour growth in vivo was evaluated and the potential mechanism underlying the combined therapy was explored. We found that 32P-colloid combined with endostatin exhibited higher inhibitory effect upon tumour growth compared with application of 32P-colloid or endostatin alone, although three therapies all significantly inhibited tumour growth compared with saline control group. The higher inhibitory effect of 32P-colloid combined with endostatin upon tumour growth might be attributed to a synergistic effect of inhibiting angiogenesis by endostatin and inducing apoptosis by 32P-colloid, as demonstrated by microvessel density (MVD) and apoptotic index (AI) measurement. Combined therapy of 32P-colloid and endostatin probably serves as a novel and efficacious therapy of tumour growth.