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.

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.

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.

Update on HPV Vaccination Policies and Practices in 17 Eastern European and Central Asian Countries and Territories.

BACKGROUND: In 2021, a survey was conducted as part of the regional program of the United Nations Population Fund (UNFPA) Regional Office for Eastern Europe and Central Asia (EECA) to assess the policies and practices relating to HPV vaccination and cervical cancer screening in the 17 countries and territories included in this region. Since then, very substantial progress has been made with HPV vaccination across the region so another survey was conducted establish the current situation. METHODS: A 10 question survey covering the policies, plans and practices for HPV vaccination was prepared. As cervical cancer prevention is a priority for the UNFPA, its offices in the 17 countries and territories included in this study are well placed to identify the people who can provide authoritative data for this survey. Working with the UNFPA offices, the questionnaires were sent to these national experts in May 2023, with data collected until 30 June 2023. All countries and territories returned completed questionnaires. RESULTS: In the period from 30 June 2021 to 30 June 2023, the number of countries and territories that have implemented or are implementing HPV vaccination programs has doubled. As of 30 June 2021, only 6 of 17 countries and territories had implemented national HPV vaccination programmes, and by 30 June 2023, another 6 could be added to this list. Of the 4 countries with sub-optimal vaccination coverage rates in 2021, none showed substantial improvement over the 2-year period. CONCLUSIONS: The implementation of HPV vaccination programs across the region is progressing very rapidly with ≈70% of the countries and territories implementing or having implemented national programs. However, greater attention needs to be given to ensuring that both the old and the new programs will achieve high coverage rates.

HPV Vaccination and Cervical Cancer Screening Policies and Practices in 18 Countries, Territories and Entities across Eastern Europe and Central Asia.

BACKGROUND: To assess readiness to achieve the WHO Global Strategy targets for HPV vaccination and cervical screening and to guide capacity building, the current status of these services in 18 Eastern European and Central Asian countries, territories and entities (CTEs) was evaluated. METHODS: In order to assess the current status of HPV vaccination and cervical cancer screening in these 18 CTEs, a 30 question survey tool was developed, covering: national policies, strategies and plans for cervical cancer prevention; status of cancer registration; status of HPV vaccination; and current practices for cervical cancer screening and treatment of precancerous lesions. As cervical cancer prevention comes within the mandate of the United Nations Fund for Population Development (UNFPA), the UNFPA offices in the 18 CTEs have regular contact with national experts who are directly involved in cervical cancer prevention actions and are well placed to provide the data required for this survey. Working through the UNFPA offices, the questionnaires were sent to these national experts in April 2021, with data collected from April to July 2021. All CTEs returned completed questionnaires. RESULTS: Only Armenia, Georgia, Moldova, North Macedonia, Turkmenistan and Uzbekistan have implemented national HPV vaccination programmes, with only the last 2 of these reaching the WHO target of 90% of girls fully vaccinated by age 15, while rates in the other 4 range from 8%-40%. Cervical screening is available in all CTEs but only Belarus and Turkmenistan have reached the WHO target of 70% of women screened once by age 35 and again by age 45, while rates elsewhere range from 2%-66%. Only Albania and Turkey follow the WHO recommendation to use a high-performance screening test, while the majority use cervical cytology as the main screening test and Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan use visual inspection. No CTEs currently operate systems to coordinate, monitor and quality assure (QA) the entire cervical screening process. CONCLUSIONS: Cervical cancer prevention services in this region are very limited. Achieving the WHO Global Strategy targets by 2030 will require substantial investments in capacity building by international development organisations.

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.

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.

VEGF, VEGF165b and EG-VEGF expression is specifically related with hormone profile in pituitary adenomas.

Vascular endothelial growth factor (VEGF), its inhibitory splice variant, VEGF165b and Endocrine Gland derived VEGF (EG-VEGF) have a controversial role in pituitary gland. We aim to study VEGF, VEGF165b and EG-VEGF expression in pituitary adenomas. A significant correlation was found between growth hormone (GH) and VEGF secretion (P=0.024). For prolactinomas, VEGF and prolactin expression, had a P-value of 0.02 for Kendall coefficient and a P-value of 0.043 for the Spearman coefficient. VEGF-mRNA amplification was detected in both tumor cells and folliculostellate cells. VEGF165b was positive in 16.66% of pituitary adenomas. EG-VEGF was significantly correlated with prolactin (P=0.025) and luteinizing hormone (P=0.028). Our data strongly support VEGF, VEGF165b and EG-VEGF as important players of pituitary adenomas tumorigenesis. Particular hormonal milieu heterogeneity, special vascular network with an unusual reactivity to tumor growth correlated with variability of VEGF, VEGF165b and EG-VEGF secretion may stratify pituitary adenomas in several molecular groups with a direct impact on therapy and prognosis.

The "game" of glial fibrillary acidic and S100 proteins in pituitary adenomas: two players or several?

INTRODUCTION: S100 protein and GFAP expression in pituitary adenomas tumour cells is not well known; few correlations with other prognostic or therapeutic factors have previously been reported in pituitary adenomas. We aim to elucidate their involvement in the pathogenesis of pituitary adenomas and to establish the correlation of their expression with different growth factors and growth factor receptors known to have a prognostic and/or therapeutic role. MATERIAL AND METHODS: Sixty-one cases of pituitary adenomas were immunohistochemically assessed for the expression of GFAP and S100 protein in both tumour cells and FS cells, in close relationship with hormone profile, and correlated with vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) expression, previously studied by our team. RESULTS: GFAP and S100 protein were expressed both in tumour cells and FS cells. Differences between morphology, distribution, and density of GFAP+ FS cells and S100+ FS cells were observed according to the hormone profile of pituitary adenomas. GFAP and S100 protein expression in tumour cells was significantly related to hormone profile of pituitary adenomas and also with VEGF and EGFR expression. CONCLUSIONS: GFAP and S100 protein expressions in tumour cells from pituitary adenomas are influenced by hormone profile. Our re-sults support the presence of two molecular subtypes of FS cells GFAP+/VEGF+/S100 respectively and another one that is GFAP-/S100+/EGFR+ simultaneously with the classical variant GFAP+/S100+. It is possible that S100+/EGFR+ pituitary adenomas represent a group of pituitary adenomas with an aggressive behaviour and a high ability of invasion and recurrence.

High Ki-67 expression is associated with prolactin secreting pituitary adenomas.

Pituitary adenomas represent the third most common primary intracranial tumor in neurosurgical practice. To understand the biological behaviour of the pituitary adenomas previous studies have determined the tumor proliferation rate using monoclonal antibodies targeted against the Ki-67 antigen. The aim of this study was to correlate the Ki-67 index with hormonal profiles of pituitary adenomas. The study included 50 pituitary adenomas. For histopathologic evaluation, the sections were stained with routine hematoxylin and eosin method. Additional paraffin sections from each tumor were immunostained using primary antibodies against the following pituitary hormones: somatotropin (STH), prolactin (PRL), adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). To detect the expression of Ki-67 we used a mouse anti-human monoclonal antibody (clone K2). The percentage of Ki-67 positive nuclei (Ki-67 labeling index) was assessed by counting approximately 1000 nuclei of the tumor cells at ×400 magnification. Out of the 50 tumor samples, 31 (62%) pituitary adenomas showed proliferative activity, and the proliferation rate was variable in this group. The overall mean Ki-67 labeling index was 1.59 ± 1.47, ranging from 0.3% to 6.6%. In 5 cases, the Ki-67 index was >3%, all of them being prolactinomas. The Ki-67 index was higher in PRL-secreting adenomas (mean ± SD was 3.37 ± 1.80, range 0.9 - 6.6%). Our study provides the evidence that a higher Ki-67 value is associated with pituitary adenomas that secrete PRL (prolactinomas and mixed STH/PRL-secreting adenomas).

Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 in Cancer Development and Tumor Angiogenesis.

A lot of data suggests endocrine gland-derived vascular endothelial growth factor (EG-VEGF) to be restricted to endocrine glands and to some endocrine-dependent organs. Many evidences show that EG-VEGF stimulates angiogenesis and cell proliferation, although it is not a member of the VEGF family. At the time, a lot of data regarding the role of this growth factor in normal development are available. However, controversial results have been published in the case of pathological conditions and particularly in malignant tumors. Thus, our present paper has been focused on the role of EG-VEGF in normal tissues and various malignant tumors and their angiogenic processes.

Geographic-Related Differences of Pituitary Adenomas Hormone Profile: Analysis of Two Groups Coming from Southeastern and Eastern Europe.

We compared the immunoprofile of pituitary adenomas from Romania and Moldova. One hundred and eighty cases coming from Romania (94 cases, group 1) and Moldova (86 cases, group 2) were assessed by immunohistochemistry regarding all six basic hormones expressed in pituitary adenomas. Specific differences and similarities were found and stated for both groups. In group 1, 70% of cases were pituitary adenomas positive for one hormone, 13% were plurihormonal, while 17% were negative. In group 2, 50,3% of the cases expressed only one hormone and 12,5% were negative for all hormones. The highest difference was observed for plurihormonal adenomas, found in about 37,2% of cases for group 2 (2.86 times higher for group 2 compared with group 1). A higher incidence of GH-secreting adenomas characterized group "1," while group "2" had the highest percent of LH-secreting adenomas, 55% of cases being positive. Triple association was noticed in 4.25% of cases of group 1 and in 8,13% out of total cases, from group 2. Four-hormone association was found only in group 2, noticed in 15,56% of the cases. The present paper highlights strong evidences of a particular and different immunoprofile of pituitary adenomas coming from Romania and Moldova.

Crosstalk between tumor blood vessels heterogeneity and hormonal profile of pituitary adenomas: evidence and controversies.

AIM: Pituitary adenomas are intracranial tumors with controversial histopathology and heterogeneous clinical behaviour. Angiogenesis and tumor blood vessels' role in pathogenesis, remain one of the great pituitary tumor mysteries. No connection between tumor vessel heterogeneity, hormonal profile and biological behaviour has been reported. We aimed to study pituitary adenomas blood vessels concerning their immature, intermediate or mature phenotype and microvessel density, correlated with immunohistochemical hormonal profile and hormone values in serum and cerebrospinal fluid. MATERIALS AND METHODS: We classified pituitary adenomas according to hormone profile and we applied a double immunostaining highlighting both endothelial and perivascular cells for a more accurate assessment of blood vessel types. RESULTS: Overall microvessel density was found to be highest in growth hormone-secreting adenomas (48.51 ± 12.15) and lowest in prolactinomas (29.15 ± 18.78). When we differentially counted tumor blood vessels we observed a predominance of immature and intermediate blood vessels compared to mature ones. A significant correlation was found between immature tumor blood vessels and tissue prolactin expression, as assessed by immunhistochemistry (p=0.044). A partial correlation was found between serum (p=0.036) and cerebrospinal prolactin values (p=0.006) with immature and intermediate blood vessels. Also, a partial correlation has been reported only between mature blood vessels and cerebrospinal fluid prolactin values (p=0.008). No correlation was obtained for other types of pituitary adenomas. CONCLUSION: Our results suggest a strong involvement of prolactin with a dual role in pituitary adenomas vasculature remodelling by acting both on endothelial and perivascular cells, a finding that could partially explain discrepancies between clinical diagnosis and hormonal profile.