Combining RNAscope, Immunohistochemistry (IHC) and Digital Image Analysis to Assess Podoplanin (PDPN) Protein and PDPN_mRNA Expression on Formalin-Fixed Paraffin-Embedded Normal Human Placenta Tissues.

The expression and function of podoplanin (PDPN) in the normal human placenta has been debated in placental evaluation. This study emphasizes the importance of a multimodal approach of PDPN expression in normal human placentas. A complete examination is performed using immunohistochemistry, RNAscope and automated Digital Image examination (DIA) interpretation. QuPath DIA-based analysis automatically generated the stromal and histological scores of PDPN expression for immunohistochemistry and RNAscope stains. The umbilical cord's isolated fibroblasts and luminal structures expressed PDPN protein and PDPN_mRNA. RNAscope detected PDPN_mRNA upregulation in syncytial placental knots trophoblastic cells, but immunohistochemistry did not certify this at the protein level. The study found a significant correlation between the IHC and RNAscope H-Score (p = 0.033) and Allred Score (p = 0.05). A successful multimodal strategy for PDPN assessment in human placentas confirmed PDPN expression heterogeneity in the full-term human normal placenta and umbilical cord at the protein and mRNA level. In placental syncytial knots trophoblastic cells, PDPN showed mRNA overexpression, suggesting a potential role in placenta maturation.

Impact of SARS-CoV-2 Pandemic on the Diagnosis of Cervical Cancer and Precursor Lesions-A Single-Center Retrospective Study.

Background and Objectives: Our aim was to perform a retrospective analysis of the volume of cervical screening tests, the number of patients treated with an excision method, and the incidence of invasive and non-invasive cervical during a pandemic and pre-pandemic period of 24 months. Materials and Methods: The study compared 404 patients who underwent cervical cone biopsy for cervical cancer. The study examined patients' specimens based on histopathological characteristics and categorized cervical lesions based on pap smear. Results: There was a statistically significant age difference between the two study periods. The mean difference was 32 years before the pandemic and 35 years during the pandemic (p-value > 0.05). The biggest patient loss ratio identified by age group was in the 50-59-year group, with a 14.53% loss in the pre-pandemic period and a 9.1% loss in the pandemic period. In the pandemic period, patients from rural areas presented in the clinical trial with a lower rate of 39.52% (83 patients) vs. 60.47% (127 patients) in urban areas. A higher percentage of patients experiencing cervicorrhagia as a clinical manifestation in the pandemic period vs. the pre-pandemic period, with an increase in more severe lesions in the pandemic period, had a statistical significance of 8% more newly diagnosed compared to the pre-pandemic period. Conclusions: The addressability of the patients during the COVID period was not affected in a drastic way in our study. We encountered a decrease in appointments in the age group of 50-59 years and a decrease in patients with rural residence. In our study, we found an increase in cervical bleeding as a reason for consultation in the pandemic period with a higher lesion degree, both on a pap smear and on a cervical biopsy.

Hyaluronic Acid/Bone Substitute Complex Implanted on Chick Embryo Chorioallantoic Membrane Induces Osteoblastic Differentiation and Angiogenesis, but not Inflammation.

Microscopic and molecular events related to alveolar ridge augmentation are less known because of the lack of experimental models and limited molecular markers used to evaluate this process. We propose here the chick embryo chorioallantoic membrane (CAM) as an in vivo model to study the interaction between CAM and bone substitutes (B) combined with hyaluronic acid (BH), saline solution (BHS and BS, respectively), or both, aiming to point out the microscopic and molecular events assessed by Runt-related transcription factor 2 (RUNX 2), osteonectin (SPARC), and Bone Morphogenic Protein 4 (BMP4). The BH complex induced osteoprogenitor and osteoblastic differentiation of CAM mesenchymal cells, certified by the RUNX2 +, BMP4 +, and SPARC + phenotypes capable of bone matrix synthesis and mineralization. A strong angiogenic response without inflammation was detected on microscopic specimens of the BH combination compared with an inflammatory induced angiogenesis for the BS and BHS combinations. A multilayered organization of the BH complex grafted on CAM was detected with a differential expression of RUNX2, BMP4, and SPARC. The BH complex induced CAM mesenchymal cells differentiation through osteoblastic lineage with a sustained angiogenic response not related with inflammation. Thus, bone granules resuspended in hyaluronic acid seem to be the best combination for a proper non-inflammatory response in alveolar ridge augmentation. The CAM model allows us to assess the early events of the bone substitutes⁻mesenchymal cells interaction related to osteoblastic differentiation, an important step in alveolar ridge augmentation.

Invasive ductal carcinoma of no special type and its corresponding lymph node metastasis: do they have the same immunophenotypic profile?

In the present study we compared the immunophenotypic subtypes of breast ductal invasive carcinomas with their ipsilateral, axillary lymph node metastasis. The ER (estrogen receptor), PR (progesterone receptor), Her2 (human epidermal growth factor receptor 2), and CK5 (cytokeratin 5) status and the proliferation marker Ki-67 were determined by immunohistochemistry on specimens from 43 women. All selected cases were diagnosed as invasive breast carcinomas, of no special type (NST), G2 grade of differentiation. The most frequently encountered subtype at both sites was luminal B. We determined that tumor profile evaluated by surrogate markers is not stable during the metastatic process. The total rate of shifted cases was 23.26% (10 cases), and the highest rate of shifting (6.98%) was encountered from luminal B/Ki-67 to luminal A subtype. In five cases, the subtype shifted to a poorer one according to prognosis. These data support the hypothesis that breast cancer is a heterogeneous disease, with substantial variability of cellular components within each category, a statement applicable to invasive breast carcinomas of NST type too. The receptor profile of this carcinoma, indicated by surrogate markers, is not stable throughout the metastatic process.

VEGF mRNA assessment in human pterygium: a new 'scope' for a future hope.

PURPOSE: The lack of powerful evidence to support the efficacy of anti-vascular endothelial growth factor (VEGF) therapy in human pterygium can be attributed to incomplete VEGF expression assessment by restrictive use of immunohistochemistry only and failure to use the molecular methods able to confirm immunohistochemical findings. By adding at least one more sensitive method to assess human pterygium VEGF expression, a more accurate selection of patients for bevacizumab therapy could be done and this would improve the efficacy of anti-VEGF therapy in human pterygium. METHODS: We assessed VEGF mRNA amplification on paraffin-embedded specimens by applying the RNAscope method for the first time in human pterygium, an in situ hybridization-based technique able to detect VEGF mRNA as a single gene copy on paraffin-embedded samples. RESULTS: Heterogeneous VEGF mRNA distribution and amplification inside the epithelial compartment of human pterygium were observed. Despite previous reports concerning the immunohistochemical expression of VEGF in the human pterygium fibrovascular compartment, no stromal components were characterized by VEGF mRNA amplification assessed by in situ hybridization in our study. A higher amplification score was observed in epithelium from recurrent pterygium, especially located in the basal and suprabasal epithelial cells. CONCLUSIONS: Based on our findings we consider that in situ hybridization assessment of VEGF for human pterygium specimens can be a useful tool for reconsidering the selection of pterygium patients to be enrolled in anti-VEGF therapy.

Evaluation of Vasculogenic Factors in the Developing Embryo at Weeks Five and Seven With a Special Focus on CD133 and TIE2 Markers.

Background Human embryo vasculogenesis (blood vessel development starting from endothelial precursors) includes the ability of mesenchymal cells and pluripotent stem cells to differentiate into endothelial cells. Quantification of endothelial progenitor cells is difficult to assess during the early steps of human embryo development due to several factors, especially due to the paucity of human embryo tissue which is usually discarded after early-stage pregnancy abortive methods. CD133 (Promimin-1) is a general marker of progenitor cells, but combined with other endothelial markers such as CD34, it may identify endothelial progenitor cells during embryonic development. CD34 immunohistochemistry was previously performed by our team to identify human embryo capillaries and comparatively assess microvessel density between different human embryonic tissues. TIE2 is an angiopoietin receptor strongly involved in the newly formed blood vessel maturation due to its expression in some mesenchymal precursors for future pericytes. CD34 assesses the presence of endothelial cells but its single use does not evaluate the endothelial progenitor state as CD133 may do nor vessel maturation as TIE2 may do. Data about the dynamics of CD133/TIE2 expression in the early stages of human embryo development are scarce. Hence, in this study, we aimed to comparatively assess the dynamic of CD133+ endothelial precursors and TIE2 expression on five and seven-week-old human embryonic tissues with a special emphasis on their expression on embryonic vascular beds. Methodology CD133 and TIE2 immunohistochemistry was performed on five and seven-week-old human embryonic tissues followed by their quantification using the Qu Path digital image analysis (DIA) automated method. Results CD133 and TIE2 showed divergent patterns of expression during the initial phases of human embryonic development, specifically in the vascular endothelium of tiny capillaries. The expression of CD133 in endothelial cells lining the perfused lumen gradually decreased from five to seven-week-old embryos. It remained expressed with greater intensity in cells located at the tip of the vascular bud that emerged into pre-existing capillaries. TIE2 was much more specific than CD133, being restricted to the level of the vascular endothelium; therefore, it was easier to quantify using digital image analysis. The endothelium of the embryonic aorta was an exception to the divergent expression, as CD133 and TIE2 were consistently co-expressed in the seven-week-old embryo. The Qu Path DIA assessment increased the accuracy of CD133 and TIE2 evaluation, being the first time they were quantified by using automated software and not manually. Conclusions High heterogeneity of CD133 and TIE2 was observed between five and seven-week-old embryonic tissues as well as between different embryonic regions from the same gestational age. The unique finding of CD133/TIE2 co-expression persistence inside aortic endothelium needs further studies to elucidate the role of this co-expression.

Exploring vasculogenesis in the normal human kidney and clear cell renal cell carcinoma: insights from development to tumor progression and biomarkers for therapy response.

Vasculogenesis, which refers to the development of blood vessels from precursor cells, is a process that occurs predominantly during early embryonic life. It plays a crucial role in the establishment of the primitive vascular network. Vasculogenesis diminishes throughout the fetal vascular remodeling process, giving way to angiogenesis, which becomes the predominant mechanism after birth. At first, the development of the kidney's blood vessels depends on vasculogenesis, and then both vasculogenesis and angiogenesis happen simultaneously. Both processes are necessary for the normal development of the renal vasculature. Although the kidneys are highly vascularized, our understanding of normal kidney vasculogenesis is still incomplete. This lack of knowledge may explain the limited data available on the role of vasculogenesis in the progression and spread of renal cancers. In other types of cancer, researchers have well documented the phenomenon of tumor vasculogenesis. However, there is currently limited and fragmented information about the occurrence of clear-cell renal cell carcinomas (cc-RCC). In this article, we provide a comprehensive review of the current understanding of normal kidney vasculogenesis and vasculogenic pathways in clear cell renal cell carcinoma (cc-RCC). We specifically focus on cellular precursors, growth factors, and the influence of the normal and tumor environments on these processes. It will carefully look at how tumor vasculogenesis might affect the growth and metastasis of clear cell renal cell carcinoma (cc-RCC), as well as how it might affect the effectiveness of drugs and the development of therapy resistance.

Heterogeneity of Cervical Cancer-Associated Tertiary Lymphoid Structures (TLSs) and Their Specific Interrelation With Clinicopathological Parameters.

OBJECTIVE: The study investigates morphological variants of tertiary lymphoid structures (TLSs) in relation to cervical cancer development, from intraepithelial neoplastic lesions to invasive carcinomas with locoregional lymph node metastases. MATERIALS AND METHODS: This retrospective analysis comprised 100 cervical cancer cases who had had total hysterectomy with lymphadenectomy in the Obstetrics and Gynecology Clinic of the Municipal Emergency Clinical Hospital of Timisoara, Romania, from 2020 to 2023. Bilateral ilio obturator lymphadenectomy and total hysterectomy were used to acquire biopsy samples. The presence of germinal centers, other stromal structures, TLS density, topography relative to the tumor lesion, and malignant cell islets are used to evaluate and classify TLS. RESULTS: We first globally evaluated the total number of TLSs (TLS.T). We observed topographically two places in the cervical stroma: TLS immediately peritumorally positioned and TLS away from tumor lesions. Invasive carcinomas have bigger superficial TLSs with a well-defined germinal center. As they approached the tumor, TLSs increased in size and density. We also detected a special type of TLS associated with nerve fibers, which we named tertiary lymphoid structures associated with nerves (TLS.N). The total number of TLSs did not correlate with age, but 85.71% of patients presenting TLS.N were aged between 59 and 72 years old. Our findings showed a strong correlation between age (postmenopausal, p = 0.005) and TLS-N presence. Similarly, TLS parameters evolved with tumor differentiation. Only in the TLS.N group did the tumoral grading (G) 3 correlate with TLS (p = 0.041), while TLS.T did not correlate with G. All TLS.N. patients, except one, had lymphovascular invasion and massive histiocytosis. On the first point, TLS.N correlated with lymphovascular invasion (p = 0.032). CONCLUSION: Tertiary lymphoid structures associated with nerves have not been previously reported in cervical cancer, and their effects on prognosis and aggression are unknown. There was a substantial association between TLSs.N presence and age over 60, suggesting it is exclusive to menopausal women. They were also substantially connected with lymphovascular invasion and G3, suggesting they may be a poor cervical cancer prognostic factor.

Clinical Outcomes and Molecular Predictors of Pembrolizumab (Keytruda) as a PD-1 Immune Checkpoint Inhibitor in Advanced and Metastatic Cervical Cancer: A Systematic Review and Meta-Analysis.

This systematic review evaluates the clinical outcomes and molecular predictors of response to pembrolizumab in patients with advanced and metastatic cervical cancer. We adhered to the PRISMA guidelines for systematic reviews, conducting a database search in PubMed, Scopus, and Embase. The eligibility criteria centered on clinical outcomes, including the overall survival (OS), progression-free survival (PFS), and immune-related biomarkers post-pembrolizumab therapy. We included both prospective and retrospective studies that detailed clinical outcomes and molecular characteristics predictive of therapeutic response. Our search yielded six studies involving 846 patients treated with pembrolizumab from 2017 to 2022. The meta-analysis of these studies showed that pembrolizumab, used as monotherapy or in combination with chemotherapy, extended the OS by a weighted median of 10.35 months and the PFS by 8.50 months. The treatment demonstrated a pooled objective response rate (ORR) of 22.39%, although the I2 test result of 67.49% showed a high heterogeneity among the studies. Notably, patients with high PD-L1 expression (CPS ≥ 10) experienced improved outcomes in terms of the PFS and OS. The most common complications were fatigue, diarrhea, and immune-related adverse events. Pembrolizumab significantly enhances clinical outcomes in metastatic cervical cancer, particularly among patients with high PD-L1 expression. The drug maintains a good safety profile, reinforcing its treatment potential for patients with advanced and metastatic cervical cancer. Future studies should explore long-term effects and strategies to integrate pembrolizumab optimally into current treatment regimens, aiming to maximize patient benefits and effectively manage side effects.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

BACKGROUND/AIM: Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MATERIALS AND METHODS: MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. RESULTS: VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. CONCLUSION: Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Artificial Intelligence (AI) Based Analysis of In Vivo Polymers and Collagen Scaffolds Inducing Vascularization.

BACKGROUND/AIM: Biomaterials are essential in modern medicine, both for patients and research. Their ability to acquire and maintain functional vascularization is currently debated. The aim of this study was to evaluate the vascularization induced by two collagen-based scaffolds (with 2D and 3D structures) and one non-collagen scaffold implanted on the chick embryo chorioallantoic membrane (CAM). MATERIALS AND METHODS: Classical stereomicroscopic image vascular assessment was enhanced with the IKOSA software by using two applications: the CAM assay and the Network Formation Assay, evaluating the vessel branching potential, vascular area, as well as tube length and thickness. RESULTS: Both collagen-based scaffolds induced non-inflammatory angiogenesis, but the non-collagen scaffold induced a massive inflammation followed by inflammatory-related angiogenesis. Vessels branching points/Region of Interest (Px^2) and Vessel branching points/Vessel total area (Px^2), increased exponentially until day 5 of the experiment certifying a sustained and continuous angiogenic process induced by 3D collagen scaffolds. CONCLUSION: Collagen-based scaffolds may be more suitable for neovascularization compared to non-collagen scaffolds. The present study demonstrates the potential of the CAM model in combination with AI-based software for the evaluation of vascularization in biomaterials. This approach could help to reduce and replace animal experimentation in the pre-screening of biomaterials.

Glycosaminoglycans Modulate the Angiogenic Ability of Type I Collagen-Based Scaffolds by Acting on Vascular Network Remodeling and Maturation.

Type I collagen, prevalent in the extracellular matrix, is biocompatible and crucial for tissue engineering and wound healing, including angiogenesis and vascular maturation/stabilization as required processes of newly formed tissue constructs or regeneration. Sometimes, improper vascularization causes unexpected outcomes. Vascularization failure may be caused by extracellular matrix collagen and non-collagen components heterogeneously. This study compares the angiogenic potential of collagen type I-based scaffolds and collagen type I/glycosaminoglycans scaffolds by using the chick embryo chorioallantoic membrane (CAM) model and IKOSA digital image analysis. Two clinically used biomaterials, Xenoderm (containing type I collagen derived from decellularized porcine extracellular matrix) and a dual-layer collagen sponge (DLC, with a biphasic composition of type I collagen combined with glycosaminoglycans) were tested for their ability to induce new vascular network formation. The AI-based IKOSA app enhanced the research by calculating from stereomicroscopic images angiogenic parameters such as total vascular area, branching sites, vessel length, and vascular thickness. The study confirmed that Xenoderm caused a fast angiogenic response and substantial vascular growth, but was unable to mature the vascular network. DLC scaffold, in turn, produced a slower angiogenic response, but a more steady and organic vascular maturation and stabilization. This research can improve collagen-based knowledge by better assessing angiogenesis processes. DLC may be preferable to Xenoderm or other materials for functional neovascularization, according to the findings.

DNA damage in human pterygium: one-shot multiple targets.

PURPOSE: Little is known about DNA damage in human pterygium, and no data about DNA damage involvement as a potential angiogenic factor are available. We studied, with immunohistochemistry, the presence and localization of thymine dimers in the epithelial and stromal components of the human primary pterygium and its recurrences with a special emphasis on the vascular network and its interactions with the p53 tumor suppressor gene protein. METHODS: Thirty-five primary human pterygium, three recurrences, and three normal bulbar conjunctiva were included in the present study. Formalin-fixed, paraffin-embedded tissues were submitted for immunohistochemical analysis with antithymine dimers and p53 antibodies. Thymine dimer and p53 nuclear staining was assessed in the epithelial and stromal components of pterygial tissues and normal counterparts. RESULTS: Thymine dimers were present in the epithelial and stromal components of human pterygium and its recurrences. The thymine dimers were detected in the epithelial component of the human pterygium with a higher density and intensity in the basal layer of the epithelium. Small blood vessels' endothelial cells showed positive reaction for antithymine dimer antibodies together with isolated positive expression found in the nuclei of perivascular cells. For the recurrent pterygium, dimer expression was found only in the subepithelial fibrovascular layer components and in scattered cells from the basal layer of the epithelium. P53 expression was positive in 38.5% of the cases in the epithelial compartment, and in two cases, scattered p53 positive endothelial, fibroblast-like, and perivascular cells were detected in the fibrovascular compartment. CONCLUSIONS: Thymine dimers in human pterygium and its recurrences suggest that DNA damage is involved not only in pterygium epithelial and fibrous proliferation but also in angiogenesis and lymphangiogenesis from this ocular lesion in a still incomplete elucidated pathogenic mechanism.

Reassessing Breast Cancer-Associated Fibroblasts (CAFs) Interactions with Other Stromal Components and Clinico-Pathologic Parameters by Using Immunohistochemistry and Digital Image Analysis (DIA).

BACKGROUND: Breast cancer (BC) stroma has CD34- and αSMA-positive cancer-associated fibroblasts (CAFs) differently distributed. During malignant transformation, CD34-positive fibroblasts decrease while αSMA-positive CAFs increase. The prevalence of αSMA-positive CAFs in BC stroma makes microscopic examination difficult without digital image analysis processing (DIA). DIA was used to compare CD34- and αSMA-positive CAFs among breast cancer molecular subgroups. DIA-derived data were linked to age, survival, tumor stroma vessels, tertiary lymphoid structures (TLS), invasion, and recurrence. METHODS: Double immunostaining for CD34 and αSMA showed different CAF distribution patterns in normal and BC tissues. Single CD34 immunohistochemistry on supplemental slides quantified tumor stroma CD34_CAFs. Digital image analysis (DIA) data on CAF density, intensity, stromal score, and H-score were correlated with clinico-pathologic factors. RESULTS: CD34/αSMA CAF proportion was significantly related to age in Luminal A (LA), Luminal B (LB), and HER2 subtypes. CD34_CAF influence on survival, invasion, and recurrence of LA, LB-HER2, and TNBC subtypes was found to be significant. The CD34/αSMA-expressing CAFs exhibited a heterogeneous impact on stromal vasculature and TLS. CONCLUSION: BC stromal CD34_CAFs/αSMA_CAFs have an impact on survival, invasion, and recurrence differently between BC molecular subtypes. The tumor stroma DIA assessment may have predictive potential to prognosis and long-term follow-up of patients with breast cancer.

Tertiary Lymphoid Structures (TLSs) and Stromal Blood Vessels Have Significant and Heterogeneous Impact on Recurrence, Lymphovascular and Perineural Invasion amongst Breast Cancer Molecular Subtypes.

BACKGROUND: Tertiary lymphoid structures (TLSs) mediate local antitumor immunity, and interest in them significantly increased since cancer immunotherapy was implemented. We examined TLS- tumor stromal blood vessel interplay for each breast cancer (BC) molecular subtype related to recurrence, lymphovascular invasion (LVI), and perineural invasion (PnI). METHODS: TLSs were quantified on hematoxylin and eosin stain specimens followed by CD34/smooth muscle actin (SMA) double immunostaining for stromal blood vessel maturation assessment. Statistical analysis linked microscopy to recurrence, LVI, and PnI. RESULTS: TLS negative (TLS-) subgroups in each BC molecular subtype (except to Luminal A) have higher LVI, PnI, and recurrence. A significant rise in LVI and PnI were observed for the HER2+/TLS- subgroup (p < 0.001). The triple negative breast cancer (TNBC)/TLS- subgroup had the highest recurrence and invasion risk which was also significantly related to tumor grade. PnI but not LVI significantly influenced recurrence in the TNBC/TLS+ subgroup (p < 0.001). TLS-stromal blood vessel interrelation was different amongst BC molecular subtypes. CONCLUSION: BC invasion and recurrence are strongly influenced by TLS presence and stromal blood vessels, especially for HER2 and TNBC BC molecular subtypes.

Age, Sex, Metabolic and Pharmacologic Factors May Predict Nonresponse Status to Rheumatoid Arthritis Therapies.

BACKGROUND/AIM: A real challenge for patients with rheumatoid arthritis (RA) and rheumatologists is primary nonresponse status (PNRS) or secondary nonresponse status (SNRS) to various therapies. Despite their detrimental influence on patient life quality, PNRS and SNRS have no accurate definition and no early predictive criteria for their development exist. Patients with RA under 40 years of age are rare, hence PNRS and SNRS data for this age group are scarce. This study examined the PNRS and SNRS according to sex, age, BMI, therapy type, and duration. PATIENTS AND METHODS: Retrospectively, 115 patients with RA having PNRS and/or SNRS were stratified by age (22-39, 40-59, and 60-81). The association between body mass index (BMI), proinflammatory cytokines inhibitors, JAK inhibitors, and TNF-alpha inhibitors, sex, age, and PNRS and SNRS was examined. RESULTS: All three proinflammatory cytokine inhibitors (rituximab, tocilizumab, and abatacept) were associated with PNRS and SNRS in women with a high BMI aged 40-59 years. Abatacept-related PNRS and SNRS was significant in women with normal BMI aged 60-81 years. Adalimumab, infliximab, and golimumab affected SNRS differently in women with normal BMI aged 22-39 years and women with high BMI aged 60-81 years. Etanercept and infliximab were associated with SNRS status in men with high-BMI aged 40-59 years. CONCLUSION: PNRS and SNRS development in patients with RA is significantly influenced by age, sex, and BMI, but most importantly is closely and differentially related to therapy type and duration.

Patterns of Tumor Vasculogenesis on a Chick Embryo Chorioallantoic Membrane In Vivo Model of Breast Cancer.

BACKGROUND/AIM: Understanding tumor vasculogenesis is a cornerstone for the inhibition of tumor progression. This study aimed to generate an in vivo breast cancer environment to analyze the patterns of tumor vasculogenesis. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSC) and breast cancer MCF-7 cells (MCF-7) were seeded onto a chorioallantoic membrane (CAM) and, after a 7-day incubation, we performed a morphological and immunohistochemical analysis of CAM. RESULTS: hMSC and MCF-7 activated vasculogenesis and hematopoiesis on CAM. They stimulated the development of cord/capillary-like structures (CLS), formed by endothelial-like cells and hematopoietic cells. CLS presented a polygonal pattern, evolving towards a clearly visible plexus. Immunohistochemically, CLS were CD105+/AC133+/Oct3/4+, and the intensity was weak-moderate in the endothelial-like cells (inconstant) and weak in the hematopoietic cells. CONCLUSION: Tumor and embryonic vasculogenesis share a common paradigm, while CD105, AC133, and Oct3/4 were found to play a role in establishing the vasculogenic and hematopoietic stage.

Heterogeneity of Platelet Derived Growth Factor Pathway Gene Expression Profile Defines Three Distinct Subgroups of Renal Cell Carcinomas.

BACKGROUND/AIM: We previously described four different vascular patterns (reticular, diffuse, fasciculate, and trabecular) in renal cell carcinoma (RCC) suggesting an early and heterogeneous acquisition of perivascular cells most probably due to a particular PDGF pathway gene expression profile. The aim of the study was to study PDGF pathway gene expression profiles, separately for each vascular pattern. MATERIALS AND METHODS: TaqMan assay for the PDGF pathway was performed on twelve cases of ccRCC previously evaluated by histopathology, immunohistochemistry, and RNAscope. Gene expression profile was correlated with grade, invasion, vascular patterns, and VEGF. RESULTS: PIK3C3 and SLC9A3 genes were overexpressed in all vascular patterns, but they were significantly correlated with high VEGF mRNA in the reticular and diffuse pattern. STAT1, JAK2, SHC2, SRF and CHUK (IKK) were exclusively overexpressed in cases with diffuse vascular pattern. SLC9A3, CHUK and STAT3 were overexpressed in G2 tumors. CONCLUSION: Three ccRCC subgroups were defined: 1) PIK3C3 (VSP34)/SLC9A3 which may be proper for anti PIK3C3 inhibitors; 2) VEGFhigh subgroup where association of anti VEGF may be a benefit and 3) JAK2/STAT1 subgroup, potentially being eligible for anti JAK/STAT therapy associated with IKK inhibitors.

VEGF Pathway Gene Expression Profile of Proliferating versus Involuting Infantile Hemangiomas: Preliminary Evidence and Review of the Literature.

Background. Infantile hemangiomas may have unexpected behavior. Initial regression (spontaneously or drug-induced) may be followed by unexplained recurrences. At this moment, there are no well-established criteria to predict infantile hemangioma reccurrences. Methods. We compared the VEGF pathway gene expression profile for one case of involuting infantile hemangioma versus one case of recurrent proliferative infantile hemangioma using TaqMan Array. Results. We found ten genes upregulated for both involuting and recurrent proliferative hemangiomas: ACTB, KRAS, MAP2K1, HRAS, NOS3, BAD, HSPB1, HPRT1, GUSB, and CASP9. Thirteen genes were downregulated for both involuting and proliferative hemangiomas: FIGF, ACTG1, GRB2, MAPKAPK2, ACTG2, MAP2K2, MAPK3, HSP90AA1, MAP2K6, NRAS, ACTA1, KDR, and MAPK1. Three genes showed divergent expression between proliferating and involuting hemangiomas. Proliferating hemangioma had MAPK14 and AKT1 gene upregulation and ACTA2 downregulation. Involuting infantile hemangioma was characterized by ACTA2 upregulation and AKT1 and MAPK14 downregulation. Conclusions. Three genes, AKT1, p38/MAPK14, and ACTA2, were found to have divergent expression in proliferating and involuting infantile hemangiomas. Excepting AKT1, which was mentioned in the last ISSVA classification (strictly related to Proteus Syndrome), none of the other genes were reported. An accurate gene expression profile mapping of infantile hemangiomas together with a gene expression-based hemangioma classification is stringently needed.

The Mutually Mediated Chloride Intracellular Channel Protein 1 (CLIC1) Relationship between Malignant Cells and Tumor Blood Vessel Endothelium Exhibits a Significant Impact on Tumor Angiogenesis, Progression, and Metastasis in Clear Cell Renal Cell Carcinoma (ccRCC).

Background: Overexpression of chloride intracellular channel protein 1 (CLIC1) in tumor cells has been confirmed, but it has received less attention in the tumor blood vessel endothelium. Aim: The assessment of CLIC1 expression in ccRCC tumor blood vessels and its relationship with TNM parameters and tumor cell CLIC1 expression. Methods: CLIC1 immunostaining in ccRCC was evaluated in 50 cases in both malignant cells and tumor blood vessels (CLIC1 microvessel density-CLIC1-MVD) and was correlated with TNM staging parameters. Results: CLIC1-MVD was observed in approximately 65% of cases, and CLIC1 co-localization in both tumor and endothelial cells was observed in 59% of cases. ccRCC was classified into four groups (Classes 0−3) based on the percentage of positive tumor cells, with each group including sub-groups defined by CLIC1 expression in the endothelium. Class 3 (60−100% positive tumor cells) had the highest CLIC1-MVD, with an impact on T and M parameters (p value = 0.007 for T, and p value = 0.006 for M). For cases with CLIC1 intracellular translocation, there was a strong correlation between CLIC1-MVD and M (p value < 0.001). Conclusions: Co-expression of ccRCC tumor and endothelial cells promotes tumor progression and metastasis and should be investigated further as a potential therapeutic target for ccRCC and other human malignancies.