Cardiovascular Events and Androgen Receptor Signaling Inhibitors in Advanced Prostate Cancer: A Systematic Review and Meta-Analysis.

Importance: Cardiovascular (CV) events remain a substantial cause of mortality among men with advanced and metastatic prostate cancer (PCa). The introduction of novel androgen receptor signaling inhibitors (ARSI) has transformed the treatment landscape of PCa in recent years; however, their associated CV toxic effects remains unclear. Objective: To assess the incidence of CV events with addition of ARSI to standard of care (SOC) in locally advanced (M0) and metastatic (M1) PCa. Data Sources: Systematic searches of PubMed, Scopus, Web of Science, EMBASE, and ClinicalTrials.gov were performed from inception up to May 2023. Study Selection: Randomized clinical trials of ARSI agents (abiraterone, apalutamide, darolutamide, enzalutamide) that reported CV events among individuals with M0 and M1, hormone-sensitive prostate cancer (HSPC) and castration-resistant prostate cancer (CRPC). Data Extraction and Synthesis: A systematic review was performed in accordance with PRISMA guidance. Two authors screened and independently evaluated studies eligible for inclusion. Data extraction and bias assessment was subsequently performed. Main Outcomes and Measures: A random-effects meta-analysis was performed to estimate risk ratios for the incidence of all grade and grade 3 or higher CV events (primary outcomes), in addition to hypertension, acute coronary syndrome (ACS), cardiac dysrhythmia, CV death, cerebrovascular event, and venous thromboembolism (secondary outcomes). Sources of heterogeneity were explored using meta-regression. Results: There were 24 studies (n = 22 166 patients; median age range, 63-77 years; median follow-up time range, 3.9-96 months) eligible for inclusion. ARSI therapy was associated with increased risk of all grade CV event (risk ratio [RR], 1.75; 95% CI, 1.50-2.04; P < .001) and grade 3 or higher CV events (RR, 2.10; 95%, 1.72-2.55; P < .001). ARSI therapy also was associated with increased risk for grade 3 or higher events for hypertension (RR, 2.25; 95% CI, 1.74-2.90; P < .001), ACS (RR, 1.93; 95% CI, 1.43-1.60; P < .01), cardiac dysrhythmia (RR, 1.64; 95% CI, 1.23-2.17; P < .001), cerebrovascular events (RR, 1.86; 95% CI, 1.34-2.59; P < .001) and for CV-related death (RR, 2.02; 95% CI, 1.32-3.10; P = .001). Subgroup analysis demonstrated increased risk of all CV events across the disease spectrum (M0 HSPC: RR, 2.26; 95% CI, 1.36-3.75; P = .002; M1 HSPC: RR, 1.85; 95% CI, 1.47-2.31; P < .001; M0 CRPC: RR, 1.79; 95% CI, 1.13-2.81; P = .01; M1 CRPC: RR, 1.46; 95% CI, 1.16-1.83; P = .001). Conclusions and Relevance: This systematic review and meta-analysis found that the addition of ARSIs to traditional ADT was associated with increased risk of CV events across the prostate cancer disease spectrum. These results suggest that patients with prostate cancer should be advised about and monitored for the potential of increased risk of CV events with initiation of ARSI therapy alongside conventional hormonal therapy.

Classification of formalin-fixed bladder cancer cells with laser tweezer Raman spectroscopy.

Bladder cancer is a common cancer that is relatively hard to detect at an early stage because of its non-obvious symptoms. It is known that bladder cells can be found in urine samples which potentially could be used for early detection of bladder cancer. Raman spectroscopy is a powerful non-invasive tool for accessing biochemical information of cells. Combined with laser tweezers, to allow isolation of single cells, Raman spectroscopy has been used to characterise a number of bladder cells that might be found in a urine sample. Using principal component-canonical variates analysis (PC-CVA) and k-fold validation, the results shows that the invasive bladder cancer cells can be identified with accuracy greater than 87%. This demonstrates the potential of developing an early detection method that identifies the invasive bladder cancer cells in urine samples.

Non-canonical EphA2 activation underpins PTEN-mediated metastatic migration and poor clinical outcome in prostate cancer.

BACKGROUND: The key process of mesenchymal to amoeboid transition (MAT), which enables prostate cancer (PCa) transendothelial migration and subsequent development of metastases in red bone marrow stroma, is driven by phosphorylation of EphA2S897 by pAkt, which is induced by the omega-6 polyunsaturated fatty acid arachidonic acid. Here we investigate the influence of EphA2 signalling in PCa progression and long-term survival. METHODS: The mechanisms underpinning metastatic biopotential of altered EphA2 signalling in relation to PTEN status were assessed in vitro using canonical (EphA2D739N) and non-canonical (EphA2S897G) PC3-M mutants, interrogation of publicly available PTEN-stratified databases and clinical validation using a PCa TMA (n = 177) with long-term follow-up data. Spatial heterogeneity of EphA2 was assessed using a radical prostatectomy cohort (n = 67). RESULTS: Non-canonical EphA2 signalling via pEphA2S897 is required for PCa transendothelial invasion of bone marrow endothelium. High expression of EphA2 or pEphA2S897 in a PTENlow background is associated with poor overall survival. Expression of EphA2, pEphA2S897 and the associated MAT marker pMLC2 are spatially regulated with the highest levels found within lesion areas within 500 µm of the prostate margin. CONCLUSION: EphA2 MAT-related signalling confers transendothelial invasion. This is associated with a substantially worse prognosis in PTEN-deficient PCa.

Fatty-Acid Uptake in Prostate Cancer Cells Using Dynamic Microfluidic Raman Technology.

It is known that intake of dietary fatty acid (FA) is strongly correlated with prostate cancer progression but is highly dependent on the type of FAs. High levels of palmitic acid (PA) or arachidonic acid (AA) can stimulate the progression of cancer. In this study, a unique experimental set-up consisting of a Raman microscope, coupled with a commercial shear-flow microfluidic system is used to monitor fatty acid uptake by prostate cancer (PC-3) cells in real-time at the single cell level. Uptake of deuterated PA, deuterated AA, and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were monitored using this new system, while complementary flow cytometry experiments using Nile red staining, were also conducted for the validation of the cellular lipid uptake. Using this novel experimental system, we show that DHA and EPA have inhibitory effects on the uptake of PA and AA by PC-3 cells.

Microenvironmental IL1ß promotes breast cancer metastatic colonisation in the bone via activation of Wnt signalling.

Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1ß stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1ß-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis.

Primary Mutational Landscape Linked with Pre-Docetaxel Lactate Dehydrogenase Levels Predicts Docetaxel Response in Metastatic Castrate-Resistant Prostate Cancer.

BACKGROUND: Docetaxel chemotherapy is a standard of care for metastatic castrate-resistant prostate cancer (mCRPC): 40-50% of patients achieve a biochemical response. However, there is a lack of response predictive biomarkers. OBJECTIVE: To assess lactate dehydrogenase (LDH) as a docetaxel response biomarker in mCRPC and to examine the association of LDH with genomic alterations in primary diagnostic biopsies. DESIGN, SETTING, AND PARTICIPANTS: Clinical and associated primary tumour-targeted next-generation sequencing data from matched training (n=150) and test (n=120) cohorts of progressive mCRPC patients receiving docetaxel therapy were analysed. Data were correlated with large-scale prostate cancer genomic datasets. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Prostate-specific antigen (PSA) response, radiographic response, biochemical progression-free survival (PFS), overall survival (OS), genomic analysis of primary biopsies, and genomic datasets (Memorial Sloan Kettering Cancer Center [MSKCC] and SU2C/PCF). RESULTS AND LIMITATIONS: Serum LDH ≥450U/l is a reliable prognostic biomarker (area under the curve: 0.757 [standard deviation 0.054, 95% confidence interval [CI] 0.650-0.864, p<0.001]) in progressive mCRPC, predicting PFS at 3 mo. Patients with LDH ≥450U/l were poorer PSA responders, with shorter PFS (213 vs 372 d, hazard ratio [HR] 1.876, 95% CI 1.289-2.7300) and OS (362 vs 563 d, HR 1.630, 95% CI 1.127-2.357). High LDH is an independent surrogate marker for survival following docetaxel and predicts a poor radiological response (p=0.043). Of the 14 patients with LDH ≥450U/l available for next-generation sequencing, nine (64.3%) were more likely to have DNA repair gene mutation(s) (BRCA1/2, ATM, CHEK2, Fanconi anaemia gene) in their primary biopsy. Cross correlation with MSKCC and SU2C/PCF databases revealed a positive correlation between LDHA, PARP1 (r=0.667, p<0.01), and other DNA repair genes. CONCLUSIONS: Genomic abnormalities of LDHA and DNA repair in primary biopsies link to high pretreatment LDH and poor response to docetaxel in mCRPC. PATIENT SUMMARY: The presence of mutations of the lactate dehydrogenase and DNA repair pathways are associated with aggressive prostate cancer and poor response to chemotherapy later in the disease.

High-throughput quantum cascade laser (QCL) spectral histopathology: a practical approach towards clinical translation.

Infrared microscopy has become one of the key techniques in the biomedical research field for interrogating tissue. In partnership with multivariate analysis and machine learning techniques, it has become widely accepted as a method that can distinguish between normal and cancerous tissue with both high sensitivity and high specificity. While spectral histopathology (SHP) is highly promising for improved clinical diagnosis, several practical barriers currently exist, which need to be addressed before successful implementation in the clinic. Sample throughput and speed of acquisition are key barriers and have been driven by the high volume of samples awaiting histopathological examination. FTIR chemical imaging utilising FPA technology is currently state-of-the-art for infrared chemical imaging, and recent advances in its technology have dramatically reduced acquisition times. Despite this, infrared microscopy measurements on a tissue microarray (TMA), often encompassing several million spectra, takes several hours to acquire. The problem lies with the vast quantities of data that FTIR collects; each pixel in a chemical image is derived from a full infrared spectrum, itself composed of thousands of individual data points. Furthermore, data management is quickly becoming a barrier to clinical translation and poses the question of how to store these incessantly growing data sets. Recently, doubts have been raised as to whether the full spectral range is actually required for accurate disease diagnosis using SHP. These studies suggest that once spectral biomarkers have been predetermined it may be possible to diagnose disease based on a limited number of discrete spectral features. In this current study, we explore the possibility of utilising discrete frequency chemical imaging for acquiring high-throughput, high-resolution chemical images. Utilising a quantum cascade laser imaging microscope with discrete frequency collection at key diagnostic wavelengths, we demonstrate that we can diagnose prostate cancer with high sensitivity and specificity. Finally we extend the study to a large patient dataset utilising tissue microarrays, and show that high sensitivity and specificity can be achieved using high-throughput, rapid data collection, thereby paving the way for practical implementation in the clinic.

Stroma-induced Jagged1 expression drives PC3 prostate cancer cell migration; disparate effects of RIP-generated proteolytic fragments on cell behaviour and Notch signaling.

The Notch ligand Jagged1 is subject to regulated intramembrane proteolysis (RIP) which yields a soluble ectodomain (sJag) and a soluble Jagged1 intracellular domain (JICD). The full-length Jagged1 protein enhances prostate cancer (PCa) cell proliferation and is highly expressed in metastatic cells. However, little is known regarding the mechanisms by which Jagged1 or its RIP-generated fragments might promote PCa bone metastasis. In the current study we show that bone marrow stroma (BMS) induces Jagged1 expression in bone metastatic prostate cancer PC3 cells and that this enhanced expression is mechanistically linked to the promotion of cell migration. We also show that RIP-generated Jagged1 fragments exert disparate effects on PC3 cell behaviour and Notch signaling. In conclusion, the expression of both the full-length ligand and its RIP-generated fragments must be considered in tandem when attempting to regulate Jagged1 as a possible PCa therapy.

SR-FTIR spectroscopy of renal epithelial carcinoma side population cells displaying stem cell-like characteristics.

It is hypothesized that cells with stem cell-like properties may be influential in carcinogenesis, possessing the ability to self-renew, produce differentiated daughter cells and resist environmental or therapeutic injury. This has led to a surge in interest in identifying and characterizing the tumour initiating or cancer stem cell (CSC) with the aim of discovering novel diagnostic and prognostic markers and of understanding the basic biology with the ultimate aim of generating new therapeutic approaches and biomarkers. However, a major hurdle to this process has been the lack of a truly specific cancer stem cell biomarker allied to the rarity of these cells. This has led to problems in characterising these CSCs by traditional '-omic' techniques. Using a renal carcinoma cell line model, we show that synchrotron radiation-Fourier transform infrared (SR-FTIR) spectroscopy is a suitable tool to measure discrete differences in the biochemistry of small numbers of single-cells. Using the chemometric techniques of Principal Component and Linear Discriminant Analysis (PCA and LDA) for multivariate reduction, biochemical differences between the cells from different sub-populations were evaluated. Results found lipid and phosphodiester vibrations to be particularly good discriminating markers in the spectra of these stem-like cells, relative to the more differentiated, proliferating cells that make up the majority of the cell population.

Classification of fixed urological cells using Raman tweezers.

In this paper we report on preliminary investigations into using Raman tweezers to classify urological cell lines. This builds on earlier work within the group, whereby Raman tweezer methodologies were developed, and the application of this technique to differentiate between live prostate cancer (CaP) and bladder cells lines (PC-3 and MGH-U1 respectively) was demonstrated.In this present study we analysed chemically fixed cells using two different fixative methods; SurePath (a commercial available liquid based cytology media) and 4% v/v formalin/PBS fixatives. The study has been expanded from our previous live cell study to include the androgen sensitive CaP cell line LNCaP, primary benign prostate hyperplasia (BPH) cells as well as primary urethral cells. Raman light from the cells was collected using a 514.5 nm Ar-ion laser excitation source in back-scattering configuration mode.Principal component-linear discriminate analysis (PC-LDA) models of resulting cell spectra were generated and these were validated using a blind comparison. Sensitivities and specificities of > 72% and 90% respectively, for SurePath fixed cells, and > 93% and 98% respectively for 4% v/v formalin/PBS fixed cells was achieved. The higher prediction results for the formalin fixed cells can be attributed to a better signal-to-noise ratio for spectra obtained from these cells.Following on from this work, urological cell lines were exposed to urine for up to 12 hours to determine the effect of urine on the ability to classify these cells. Results indicate that urine has no detrimental effect on prediction results.

Molecular mechanisms of metastasis in prostate cancer.

Prostate cancer (PCa) preferentially metastasizes to the bone marrow stroma of the axial skeleton. This activity is the principal cause of PCa morbidity and mortality. The exact mechanism of PCa metastasis is currently unknown, although considerable progress has been made in determining the key players in this process. In this review, we present the current understanding of the molecular processes driving PCa metastasis to the bone.

Measurement of elastic properties of prostate cancer cells using AFM.

This communication reports that three prostate cancer cells of differing metastatic potential were discriminated based on their Young's moduli (LNCaP - 287 +/- 52 N m(-2), PC-3 - 1401 +/- 162 N m(-2) and BPH - 2797 +/- 491 N m(-2)) which were determined using AFM and the Hertz model.

Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells.

The fundamental changes which predispose for renal cell carcinoma (RCC) are poorly characterized. It is hypothesized that "cancer stem cells" may be influential in carcinogenesis, and the epithelial side population (SP) is enriched for stemlike cells in other epithelial cancers. In this study, we have isolated and characterized the SP and non-SP (NSP) populations from normal (NK) and malignant (RCC) human kidney tissue. NK specimens were taken from patients undergoing non-renal cancer surgery and paired malignant and macroscopically normal tissue samples were taken from patients undergoing surgery for RCC. The Hoechst 33342 dye efflux technique was used to isolate epithelial SP and NSP from normal and malignant human renal tissue. Cellular subpopulations were phenotyped for lineage, cell cycle, and putative stem cell markers, and functionally characterized using in vitro colony-forming and proliferation assays. The SP constituted 3.8 +/- 0.4 and 5.9 +/- 0.9% of epithelial cells in NK and RCC, respectively, of which 14.1 +/- 3.5 and 13.2 +/- 3.6% were shown to be in G(0). SP cells demonstrated greater proliferative potential in colony-forming efficiency, long-term culture, and spheroids assays and were shown to be maintained upon tissue culture passage. We have shown that the renal SP is enriched for quiescent cells, with a high proliferative capacity and stemlike properties. The population is, however, heterogeneous, confirming that the terms "SP cell" and "stem cell" cannot be used interchangeably.

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.

Discrimination of prostate cancer cells by reflection mode FTIR photoacoustic spectroscopy.

In this communication reflection mode Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) is used to obtain IR spectra of four prostate and prostate cancer cell line types (CaP) allowing their differentiation by principal components analysis.

The combined application of FTIR microspectroscopy and ToF-SIMS imaging in the study of prostate cancer.

At present. a prognosis for prostate cancer (CaP) is determined by its accurate assessment of disease grade and stage. Histopathological typing using the Gleason grading system is the most universally accepted approach for grading CaP and provides an indication as to the aggressiveness of the tumour at the time of presentation. However, this system is based upon a visual criterion of pattern recognition that is operator dependent and subject to intra- and inter-observer variability, which can result in inappropriate patient management. Thus, there is a need for a molecular based diagnostic technique to grade tissue samples in a reliable and reproducible manner. In this paper we report a prototype diagnostic classifier for Gleason graded CaP tissue, based upon the integration of FTIR microspectroscopy with linear discriminant analysis (LDA). Blind testing of this model demonstrates 80% agreement of FTIR-LDA grade to histology, for the specimens analysed. We also study the effects of connective tissue absorption upon the area ratio of peaks at A1030 cm(-1)/A1080(cm(-1) which we use as a criterion to biospectroscopically map and distinguish areas of benign from malignant tissue. In addition, imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been applied to study freeze-dried, freeze-fractured prostate cancer cells in vitro. Preliminary results demonstrate localisation of various species including K, Ca and Mg within the cytoplasm that are present at millimolar concentrations and vital to cell physiology. The soft ionisation technique employed also permits for molecular information to be obtained and this has been used to evaluate chemically, different fracture planes within the analysis area.