Lipid imaging mass spectrometry: Towards a new molecular histology.

Lipid research is attracting greater attention, as these molecules are key components to understand cell metabolism and the connection between genotype and phenotype. The study of lipids has also been fueled by the development of new and powerful technologies, able to identify an increasing number of species in a single run and at decreasing concentrations. One of such key developments has been the image techniques that enable the visualization of lipid distribution over a tissue with cell resolution. Thanks to the spatial information reported by such techniques, it is possible to associate a lipidome trait to individual cells, in fixed metabolic stages, which greatly facilitates understanding the metabolic changes associated to diverse pathological conditions, such as cancer. Furthermore, the image of lipids is becoming a kind of new molecular histology that has great chances to make an impact in the diagnostic units of the hospitals. Here, we examine the current state of the technology and analyze what the next steps to bring it into the diagnosis units should be. To illustrate the potential and challenges of this technology, we present a case study on clear cell renal cell carcinoma, a good model for analyzing malignant tumors due to their significant cellular and molecular heterogeneity.

A functional role for glycosylated B7-H5/VISTA immune checkpoint protein in metastatic clear cell renal cell carcinoma.

Increased expression of the B7 family of immune checkpoint proteins hinders tumor elimination by the immune system. Expression levels of the B7-H5 protein were found to be upregulated in clear cell renal cell carcinomas (ccRCC). We here report the molecular, functional, and clinical characterization of B7-H5 from renal cancer cells and metastatic ccRCC tumors. B7-H5 was highly glycosylated and mainly expressed in the cell membrane. Mutagenic studies on B7-H5 identified the residues targeted by N-glycosylation and revealed an impact of B7-H5 glycosylation on protein expression levels and localization. B7-H5 knockdown decreased the cell proliferation and viability of renal cancer cells. We analyzed B7-H5 expression on tumor cells and tumor-infiltrated leukocytes (TILs) in samples from metastatic ccRCC patients and found that B7-H5 expression on TILs correlated with syncronous metastases and poor outcomes. These results provide insights into the molecular properties and clinical impact of B7-H5 and support B7-H5 as a new immunotherapeutic target in metastatic ccRCC.

Predicting Survival of Metastatic Clear Cell Renal Cell Cancer Treated with VEGFR-TKI-Based Sequential Therapy.

(1) Objective: To develop a clinically useful nomogram that may provide a more individualized and accurate estimation of cancer-specific survival (CSS) for patients with clear-cell (CC) metastatic renal cell carcinoma (mRCC) treated with nephrectomy and vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGFR-TKI)-based sequential therapy. (2) Methods: A prospectively maintained database of 145 patients with mRCC treated between 2008 and 2018 was analyzed to predict the CSS of patients receiving sunitinib and second- and third-line therapies according to current standards of practice. A nomogram based on four independent clinical predictors (Eastern Cooperative Oncology Group status, International Metastatic RCC Database Consortium score, the Morphology, Attenuation, Size and Structure criteria and Response Evaluation Criteria in Solid Tumors response criteria) was calculated. The corresponding 1- to 10-year CSS probabilities were then determined from the nomogram. (3) Results: The median age was 60 years (95% CI 57.9-61.4). The disease was metastatic at diagnosis in 59 (40.7%), and 86 (59.3%) developed metastasis during follow-up. Patients were followed for a median 48 (IQR 72; 95% CI 56-75.7) months after first-line VEGFR-TKI initiation. The concordance probability estimator value for the nomogram is 0.778 ± 0.02 (mean ± SE). (4) Conclusions: A nomogram to predict CSS in patients with CC mRCC that incorporates patient status, clinical risk classification and response criteria to first-line VEGFR-TKI at 3 months is presented. This new tool may be useful to clinicians assessing the risk and prognosis of patients with mRCC.

Identification of miRNAs and Their Target Genes Associated with Sunitinib Resistance in Clear Cell Renal Cell Carcinoma Patients.

Sunitinib has greatly improved the survival of clear cell renal cell carcinoma (ccRCC) patients in recent years. However, 20-30% of treated patients do not respond. To identify miRNAs and genes associated with a response, comparisons were made between biopsies from responder and non-responder ccRCC patients. Using integrated transcriptomic analyses, we identified 37 miRNAs and 60 respective target genes, which were significantly associated with the NF-kappa B, PI3K-Akt and MAPK pathways. We validated expression of the miRNAs (miR-223, miR-155, miR-200b, miR-130b) and target genes (FLT1, PRDM1 and SAV1) in 35 ccRCC patients. High levels of miR-223 and low levels of FLT1, SAV1 and PRDM1 were associated with worse overall survival (OS), and combined miR-223 + SAV1 levels distinguished responders from non-responders (AUC = 0.92). Using immunohistochemical staining of 170 ccRCC patients, VEGFR1 (FLT1) expression was associated with treatment response, histological grade and RECIST (Response Evaluation Criteria in Solid Tumors) score, whereas SAV1 and BLIMP1 (PRDM1) were associated with metachronous metastatic disease. Using in situ hybridisation (ISH) to detect miR-155 we observed higher tumoural cell expression in non-responders, and non-tumoural cell expression with increased histological grade. In summary, our preliminary analysis using integrated miRNA-target gene analyses identified several novel biomarkers in ccRCC patients that surely warrant further investigation.

Revisiting Pulmonary Sclerosing Pneumocytoma.

Pulmonary sclerosing pneumocytoma (PSP) is a quite rare tumor outside Eastern countries. This rarity, together with a wide histological appearance, makes its correct identification a diagnostic challenge for pathologists under the microscope. Historically, PSP was considered a vascular-derived neoplasm (sclerosing hemangioma), but its immunohistochemical profile clearly supports its epithelial origin. No specific molecular fingerprint has been detected so far. This short narrative revisits the clinical, histological, immunohistochemical, and molecular aspects of this tumor, paying special attention to some controversial points still not well clarified, i.e., clinical aggressiveness and metastatic spread, multifocality, the supposed development of sarcomatoid change in a subset of cases, and tumor associations with lung adenocarcinoma and/or well-differentiated neuroendocrine hyperplasia/tumors. The specific diagnostic difficulties on fine-needle aspiration cytology/biopsy and perioperative frozen sections are also highlighted. Finally, a teaching case of tumor concurrence of lung adenocarcinoma, neuroendocrine lesions, and PSP, paradigmatic of tumor association in this context, is also presented.

Advances, recognition, and interpretation of molecular heterogeneity among conventional and subtype histology of urothelial carcinoma (UC): a survey among urologic pathologists and comprehensive review of the literature.

AIMS: Urothelial carcinoma (UC) demonstrates significant molecular and histologic heterogeneity. The WHO 2022 classification has hinted at adding molecular signatures to the morphologic diagnosis. As morphology and associated molecular repertoire may potentially translate to choices of and response to therapy and relapse rate, broader acceptability of recognizing these key features among uropathologists is needed. This prompted an international survey to ascertain the practice patterns in classical/subtype UC among uropathologists across the globe. METHODS AND RESULTS: A survey instrument was shared among 98 uropathologists using SurveyMonkey software. Anonymized respondent data were analysed. The response rate was 85%. A majority were in concordance with the profiles of luminal (93%) and basal (82%) types. Opinion on the FGFR3 testing platform was variable. While 95% concurred that TERT promoter mutation is the key driver in UC, 72% had the opinion that APOBEC mutagenesis is the main signature in muscle invasive bladder cancer (MIBC). Uropathologists have divergent opinions on MIBC and ERCC2 mutations. Among the participants, 94% would quantify aggressive micropapillary and sarcomatoid histology, while 88% would reevaluate another transurethral resection of the bladder tumour specimen in nonmuscle invasive tumour with micropapillary, small cell, or sarcomatoid histology. A leading number agreed to specific molecular signatures of micropapillary (93%), plasmacytoid (97%), and small cell (86%) subtypes. Ninety-six percent of participants agreed that a small-cell component portends a more aggressive course and should be treated with neoadjuvant chemotherapy and 63% would perform HER2/neu testing only on oncologist's request in advanced tumours. Ninety percent agreed that microsatellite instability testing, although not a standard protocol, should be considered in young patients with upper tract UC. Eighty-six percent agreed that UC with high tumour mutational burden would be a better candidate for immunotherapy. CONCLUSION: In the era of precision medicine, enhanced understanding of molecular heterogeneity of UC will contribute to better therapeutic options, novel biomarker discovery, innovative management protocols, and outcomes. Our survey provides a broad perspective of pathologists' perceptions and experience regarding incorporation of histomolecular approaches to "personalize" therapy. Due to variable clinical adoption, there is a need for additional data using uniform study criteria. This will drive generation of best practice guidelines in this area for widespread and consistent clinical utility.

B7-H3: a robust target for immunotherapy in prostate cancer.

B7-H3, an immune checkpoint glycoprotein, facilitates immune evasion and the promotion of tumors and is highly expressed on the surface of prostate cancer (PCa) cells, which makes it a feasible and robust candidate for immunotherapies against advanced prostate cancer. Here, we summarize and discuss recent findings on the suitability of targeting B7-H3 in PCa treatment.

Systemic cellular migration: The forces driving the directed locomotion movement of cells.

Directional motility is an essential property of cells. Despite its enormous relevance in many fundamental physiological and pathological processes, how cells control their locomotion movements remains an unresolved question. Here, we have addressed the systemic processes driving the directed locomotion of cells. Specifically, we have performed an exhaustive study analyzing the trajectories of 700 individual cells belonging to three different species (Amoeba proteus, Metamoeba leningradensis, and Amoeba borokensis) in four different scenarios: in absence of stimuli, under an electric field (galvanotaxis), in a chemotactic gradient (chemotaxis), and under simultaneous galvanotactic and chemotactic stimuli. All movements were analyzed using advanced quantitative tools. The results show that the trajectories are mainly characterized by coherent integrative responses that operate at the global cellular scale. These systemic migratory movements depend on the cooperative nonlinear interaction of most, if not all, molecular components of cells.

Non-coding RNA and gene expression analyses of papillary renal neoplasm with reverse polarity (PRNRP) reveal distinct pathological mechanisms from other renal neoplasms.

Papillary renal neoplasm with reversed polarity (PRNRP) is a recently described rare renal neoplasm. Traditionally, it was considered a variant of papillary renal cell carcinoma (PRCC). However, several studies reported significant differences between PRNRP and PRCC in terms of clinical, morphological, immunohistochemical and molecular features. Nonetheless, PRNRP remains a poorly understood entity. We used microarray analysis to elucidate the non-coding RNA (ncRNA) and gene expression profiles of 10 PRNRP cases and compared them with other renal neoplasms. Unsupervised cluster analysis showed that PRNRP had distinct expression profiles from either clear cell renal cell carcinoma (ccRCC) or PRCC cases at the level of ncRNA but were less distinct at the level of gene expression. An integrated omic approach determined miRNA:gene interactions that distinguished PRNRP from PRCC and we validated 10 differentially expressed miRNAs and six genes by quantitative RT-PCR. We found that levels of the miRNAs, miR-148a, miR-375 and miR-429, were up-regulated in PRNRP cases compared to ccRCC and PRCC. miRNA target genes, including KRAS and VEGFA oncogenes, and CXCL8, which regulates VEGFA, were also differentially expressed between renal neoplasms. Gene set enrichment analysis (GSEA) determined different activation of metabolic pathways between PRNRP and PRCC cases. Overall, this study is by far the largest molecular study of PRNRP cases and the first to investigate either ncRNA expression or their gene expression by microarray assays.

Clear Cell Renal Cell Carcinoma: A Test Bench for Investigating Tumor Complexity.

Clear cell renal cell carcinoma (CCRCC), by far the most common renal cancer subtype, is an aggressive tumor variant, serving in recent years as a prolific test bench in cancer research [...].

The Expression of Alamandine Receptor MrgD in Clear Cell Renal Cell Carcinoma Is Associated with a Worse Prognosis and Unfavorable Response to Antiangiogenic Therapy.

Renal cell carcinoma (RCC) ranks among the most prevalent malignancies in Western countries, marked by its notable heterogeneity, which contributes to an unpredictable clinical trajectory. The insufficiency of dependable biomarkers adds complexity to assessing this tumor progression. Imbalances of several components of the intrarenal renin-angiotensin system (iRAS) significantly impact patient prognoses and responses to first-line immunotherapies. In this study, we analyzed the immunohistochemical expression of the Mas-related G-protein-coupled receptor D (MrgD), which recognizes the novel RAS peptide alamandine (ALA), in a series of 87 clear cell renal cell (CCRCCs), 19 papillary (PRCC), 7 chromophobe (ChRCC) renal cell carcinomas, and 11 renal oncocytomas (RO). MrgD was expressed in all the renal tumor subtypes, with a higher mean staining intensity in the PRCCs, ChRCCs, and ROs. A high expression of MrgD at the tumor center and at the infiltrative front of CCRCC tissues was significantly associated with a high histological grade, large tumor diameter, local invasion, and locoregional node and distant metastasis. Patients with worse 5-year cancer-specific survival and a poorer response to antiangiogenic tyrosine-kinase inhibitors (TKIs) showed higher MrgD expression at the center of their primary tumors. These findings suggest a possible role of MrgD in renal carcinogenetic processes. Further studies are necessary to unveil its potential as a novel biomarker for CCRCC prognosis and response to frontline therapies.

Palisading adenocarcinoma. Case report on a newly recognized tumor in the salivary glands.

A palisading adenocarcinoma arising in the left submandibular salivary gland in a 65-year old woman is presented here. This tumor has been identified only very recently and its recognition as a true entity in the list of salivary gland tumors is still pending. Its distinct clinical-pathological context includes a predilection for women, sublingual or submandibular gland involvement, low-grade cytology with pseudo-neuroendocrine features, and sharp immunohistochemical expression.

Convergent insights into intratumor heterogeneity.

Mathematics, conventional histology, and genomics converge to confirm that highly aggressive clear cell renal cell carcinomas (CCRCCs) display low levels of intratumor heterogeneity (ITH). We hypothesize that therapeutic strategies aimed at maintaining high ITH levels would be advisable to slow down cancer evolution and to improve survival.

Early Evolution in Cancer: A Mathematical Support for Pathological and Genomic Evidence in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (CCRCC) is an aggressive form of cancer and a paradigmatic example of intratumor heterogeneity (ITH). The hawk-dove game is a mathematical tool designed to analyze competition in biological systems. Using this game, the study reported here analyzes the early phase of CCRCC development, comparing clonal fitness in homogeneous (linear evolutionary) and highly heterogeneous (branching evolutionary) models. Fitness in the analysis is a measure of tumor aggressiveness. The results show that the fittest clone in a heterogeneous environment is fitter than the clone in a homogeneous context in the early phases of tumor evolution. Early and late periods of tumor evolution in CCRCC are also compared. The study shows the convergence of mathematical, histological, and genomics studies with respect to clonal aggressiveness in different periods of the natural history of CCRCC. Such convergence highlights the importance of multidisciplinary approaches for obtaining a better understanding of the intricacies of cancer.

Benign Mesothelial Proliferations of the Tunica Vaginalis Testis.

The correct diagnosis of mesothelial proliferations is a classic problem for pathologists, and one which has important clinical implications. A significant number of such cases appear associated with recurrent hydrocele, as an irritative/reactive response to this condition. The morphological spectrum of mesothelial lesions in this topography is broad, and a set of benign conditions may appear, sometimes with florid gross features and cytologic pseudo-atypia. Here, we present two different examples in which malignancy was initially considered in the differential diagnosis.

Interpretability of radiomics models is improved when using feature group selection strategies for predicting molecular and clinical targets in clear-cell renal cell carcinoma: insights from the TRACERx Renal study.

BACKGROUND: The aim of this work is to evaluate the performance of radiomics predictions for a range of molecular, genomic and clinical targets in patients with clear cell renal cell carcinoma (ccRCC) and demonstrate the impact of novel feature selection strategies and sub-segmentations on model interpretability. METHODS: Contrast-enhanced CT scans from the first 101 patients recruited to the TRACERx Renal Cancer study (NCT03226886) were used to derive radiomics classification models to predict 20 molecular, histopathology and clinical target variables. Manual 3D segmentation was used in conjunction with automatic sub-segmentation to generate radiomics features from the core, rim, high and low enhancing sub-regions, and the whole tumour. Comparisons were made between two classification model pipelines: a Conventional pipeline reflecting common radiomics practice, and a Proposed pipeline including two novel feature selection steps designed to improve model interpretability. For both pipelines nested cross-validation was used to estimate prediction performance and tune model hyper-parameters, and permutation testing was used to evaluate the statistical significance of the estimated performance measures. Further model robustness assessments were conducted by evaluating model variability across the cross-validation folds. RESULTS: Classification performance was significant (p < 0.05, H0:AUROC = 0.5) for 11 of 20 targets using either pipeline and for these targets the AUROCs were within ± 0.05 for the two pipelines, except for one target where the Proposed pipeline performance increased by > 0.1. Five of these targets (necrosis on histology, presence of renal vein invasion, overall histological stage, linear evolutionary subtype and loss of 9p21.3 somatic alteration marker) had AUROC > 0.8. Models derived using the Proposed pipeline contained fewer feature groups than the Conventional pipeline, leading to more straightforward model interpretations without loss of performance. Sub-segmentations lead to improved performance and/or improved interpretability when predicting the presence of sarcomatoid differentiation and tumour stage. CONCLUSIONS: Use of the Proposed pipeline, which includes the novel feature selection methods, leads to more interpretable models without compromising prediction performance. TRIAL REGISTRATION: NCT03226886 (TRACERx Renal).

Novel anti-PTEN C2 domain monoclonal antibodies to analyse the expression and function of PTEN isoform variants.

PTEN is a major tumor suppressor gene frequently mutated in human tumors, and germline PTEN gene mutations are the molecular diagnostic of PTEN Hamartoma Tumor Syndrome (PHTS), a heterogeneous disorder that manifests with multiple hamartomas, cancer predisposition, and neurodevelopmental alterations. A diversity of translational and splicing PTEN isoforms exist, as well as PTEN C-terminal truncated variants generated by disease-associated nonsense mutations. However, most of the available anti-PTEN monoclonal antibodies (mAb) recognize epitopes at the PTEN C-terminal tail, which may introduce a bias in the analysis of the expression of PTEN isoforms and variants. We here describe the generation and precise characterization of anti-PTEN mAb recognizing the PTEN C2-domain, and their use to monitor the expression and function of PTEN isoforms and PTEN missense and nonsense mutations associated to disease. These anti-PTEN C2 domain mAb are suitable to study the pathogenicity of PTEN C-terminal truncations that retain stability and function but have lost the PTEN C-terminal epitopes. The use of well-defined anti-PTEN mAb recognizing distinct PTEN regions, as the ones here described, will help to understand the deleterious effects of specific PTEN mutations in human disease.

Deep learning approach for accurate prostate cancer identification and stratification using combined immunostaining of cytokeratin, p63, and racemase.

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer in men worldwide, affecting around 1.4 million individuals. Current PCa diagnosis relies on histological analysis of prostate biopsy samples, an activity that is both time-consuming and prone to observer bias. Previous studies have demonstrated that immunostaining of cytokeratin, p63, and racemase can significantly improve the sensitivity and the specificity of PCa detection compared to traditional H&E staining. METHODS: This study introduces a novel approach that combines diagnosis-specific immunohistochemical (IHC) staining and deep learning techniques to provide reliable stratification of prostate glands. Our approach leverages a customized segmentation network, called K-PPM, that incorporates adaptive kernels and multiscale feature integration to enhance the functional information of IHC. To address the high class-imbalance problem in the dataset, we propose a weighted adaptive patch-extraction and specific-class kernel update. RESULTS: Our system achieved noteworthy results, with a mean Dice Score Coefficient of 90.36% and a mean absolute error of 1.64 % in specific-class gland quantification on whole slides. These findings demonstrate the potential of our system as a valuable support tool for pathologists, reducing workload and decreasing diagnostic inter-observer variability. CONCLUSIONS: Our study presents innovative approaches that have broad applicability to other digital pathology areas beyond PCa diagnosis. As a fully automated system, this model can serve as a framework for improving the histological and IHC diagnosis of other types of cancer.

Fibroblast Activation Protein-α (FAP) Identifies Stromal Invasion in Colorectal Neoplasia.

The increasing detection of colorectal adenomas and early adenocarcinomas (ADCs) in the context of nationwide screening programs has led to a significant increase in the incidence of inconclusive diagnoses in which histopathologic analysis of endoscopic biopsies does not allow pathologists to provide a reliable diagnosis of stromal invasion. The objective of this study was to analyze the discriminative capacity of the immunohistochemical expression of fibroblast activation protein-α (FAP) in distinguishing colorectal adenomas with low-grade dysplasia (LGD) and high-grade dysplasia (HGD) from invasive intestinal-type ADCs. The study analyzed the first endoscopic biopsies from a series of patients classified as inconclusive or conclusive for stromal invasion based on the pathologic report. In total, 30 ADCs, 52 HGDs, and 15 LGDs were included in the study. FAP expression was detected in 23/30 ADCs and was negative in all adenomas with either LGD or HGD features (100% specificity and 76.7% sensitivity, area under the curve=0.883, CI=0.79-0.98). Considering these findings, we conclude that FAP is a potentially useful tool for helping pathologists identify invasive lesions in colorectal endoscopic biopsies, avoiding unnecessary biopsy repetitions.