Real-world outcomes for first line next-generation hormonal agents in metastatic prostate cancer: a systematic review.

Aim: This review aims to summarize published evidence on the real-world (RW) outcomes of abiraterone or enzalutamide in first-line metastatic castration-resistant prostate cancer. Materials & methods: Studies reporting on RW effectiveness, safety, economic and/or health-related quality of life outcomes were identified by systematic literature review (2011-2021, incl. Embase®, MEDLINE®) and presented in a qualitative synthesis. Risk of bias was assessed using ROBINS-I or the Molinier checklist. Results: 88 studies (n = 83,427 patients) were included. Median progression-free (40 studies) and overall survival (38 studies) ranged from 3.7 to 20.9 months and 9.8 to 45 months, respectively. Survival, safety and economic outcomes were similar across individual treatments, while limited health-related quality of life evidence suggested improvements with abiraterone. Risk of bias was moderate to high. Conclusion: RW outcomes in first-line metastatic castration-resistant prostate cancer remain poor despite treatment, highlighting an unmet need for new regimens. This review was supported by AstraZeneca and Merck Sharp & Dohme.

Impact of tumor heterogeneity and tissue sampling for genetic mutation testing: a systematic review and post hoc analysis.

OBJECTIVE: The objective of the study was to identify guidelines to assist systematic reviewers or clinical researchers in identifying sampling bias due to tumor heterogeneity (TH) in solid cancers assayed for somatic mutations. We also assessed current reporting standards to determine the impact of TH on sample bias. STUDY DESIGN AND SETTING: We conducted a systematic review searching 13 databases (to January 2019) to identify guidelines. A post hoc analysis was performed using 12 prostate tumor somatic mutation data sets from a previous systematic review to assess reporting on TH. RESULTS: Searches identified 2,085 records. No formal guidelines were identified. Forty publications contained incidental recommendations across five major themes: using multiple tumor samples (n = 29), sample purity thresholds (n = 14), using specific sequencing methods (n = 8), using liquid biopsies (n = 4), and microdissection (n = 4). In post hoc analyses, 50% (6 of 12) clearly reported pathology methods. Forty-two percent (5 of 12) did not report pathology results. Forty-two percent (5 of 12) confirmed the pathology of the sample by direct diagnosis rather than inference. Forty-two percent (5 of 12) used multiple samples per patient. Fifty-eight percent (7 of 12) reported on tumor purity (reported ranges 10% to 100%). CONCLUSIONS: As precision medicine progresses to the clinic, guidelines are required to help evidence-based decision makers understand how TH may impact sample bias. Authors need to clearly report pathology methods and results and tumor purity methods and results.

Effect of DNA damage response mutations on prostate cancer prognosis: a systematic review.

The prognosis of men with prostate cancer (PC) with mutations in DNA damage response (DDR) genes undergoing different treatments is unclear. This systematic review compared clinical outcomes in PC patients with DDR mutations (DDR+) versus no mutations (DDR-). 14 resources plus gray literature were searched for studies in PC and subgroups (castration-resistant PC, metastatic PC and metastatic castration-resistant PC) by DDR gene (ATM, ATR, BRCA1, BRCA2, CHEK2, FANCA, MLH1, MRE11A, NBN, PALB2, RAD51C) mutation status. From 11,648 records, 26 studies were included. For mCRPC, six studies reported comparative efficacy for key outcomes. Improvements in several clinical outcomes were observed for DDR+ (vs DDR-) after PARP inhibitor therapy or immunotherapy. DDR+ PC patients may have improved outcomes depending on the treatment they undergo.

A systematic review of the prevalence of DNA damage response gene mutations in prostate cancer.

Several ongoing international prostate cancer (PC) clinical trials are exploring therapies that target the DNA damage response (DDR) pathway. This systematic review summarizes the prevalence of DDR mutation carriers in the unselected (general) PC and familial PC populations. A total of 11 electronic databases, 10 conference proceedings, and grey literature sources were searched from their inception to December 2017. Studies reporting the prevalence of somatic and/or germline DDR mutations were summarized. Metastatic PC (mPC), castration­resistant PC (CRPC) and metastatic CRPC (mCRPC) subgroups were included. A total of 11,648 records were retrieved, and 80 studies (103 records) across all PC populations were included; 59 records were of unselected PC and 13 records of familial PC. Most data were available for DDR panels (n=12 studies), ataxia telangiectasia mutated (ATM; n=13), breast cancer susceptibility gene (BRCA)1 (n=14) and BRCA2 (n=20). ATM, BRCA2 and partner and localizer of BRCA2 (PALB2) had the highest mutation rates (≥4%). Median prevalence rates for DDR germline mutations were 18.6% in PC (range, 17.2­19%; three studies, n=1,712), 11.6% in mPC (range, 11.4­11.8%; two studies, n=1,261) and 8.3% in mCRPC (range, 7.5­9.1%; two studies, n=738). Median prevalence rates for DDR somatic mutations were 10.7% in PC (range, 4.9­22%; three studies, n=680), 13.2% in mPC (range, 10­16.4%; two studies, n=105) and not reported (NR) in mCRPC. The prevalence of DDR germline and/or somatic mutations was 27% in PC (one study, n=221), 22.67% in mCRPC (one study, n=150) and NR in mPC. In familial PC, median mutation prevalence was 12.1% (range, 7.3­16.9%) for germline DDR (two studies, n=315) and 3.7% (range, 1.3­7.9%) for BRCA2 (six studies, n=945). In total, 88% of studies were at a high risk of bias. The prevalence of DDR gene mutations in PC varied widely within somatic subgroups depending on study size, genetic screening techniques, DDR mutation definition and PC diagnosis; somatic and/or germline DDR mutation prevalence was in the range of 23­27% in PC. These findings support DDR mutation testing for all patients with PC (including those with mCRPC). With the advent of the latest clinical practice PC guidelines highlighting the importance of DDR mutation screening, and ongoing mCRPC clinical trials evaluating DDR mutation­targeted drugs, future larger epidemiological studies are warranted to further quantify the international burden of DDR mutations in PC.

A systematic review of the validity of patient derived xenograft (PDX) models: the implications for translational research and personalised medicine.

Patient-derived xenograft (PDX) models are increasingly being used in oncology drug development because they offer greater predictive value than traditional cell line models. Using novel tools to critique model validity and reliability we performed a systematic review to identify all original publications describing the derivation of PDX models of colon, prostate, breast and lung cancer. Validity was defined as the ability to recapitulate the disease of interest. The study protocol was registered with the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES). Searches were performed in Embase, MEDLINE and Pubmed up to July 2017. A narrative data synthesis was performed. We identified 105 studies of model validations; 29 for breast, 29 for colon, 25 for lung, 23 for prostate and 4 for multiple tissues. 133 studies were excluded because they did not perform any validation experiments despite deriving a PDX. Only one study reported following the ARRIVE guidelines; developed to improve the standard of reporting for animal experimentation. Remarkably, half of all breast (52%) and prostate (50%) studies were judged to have high concern, in contrast to 16% of colon and 28% of lung studies. The validation criteria that most commonly failed (evidence to the contrary) were: tissue of origin not proven and histology of the xenograft not comparable to the parental tumour. Overall, most studies were categorized as unclear because one or more validation conditions were not reported, or researchers failed to provide data for a proportion of their models. For example, failure to demonstrate tissue of origin, response to standard of care agents and to exclude development of lymphoma. Validation tools have the potential to improve reproducibility, reduce waste in research and increase the success of translational studies.

A systematic review of the asymmetric inheritance of cellular organelles in eukaryotes: A critique of basic science validity and imprecision.

We performed a systematic review to identify all original publications describing the asymmetric inheritance of cellular organelles in normal animal eukaryotic cells and to critique the validity and imprecision of the evidence. Searches were performed in Embase, MEDLINE and Pubmed up to November 2015. Screening of titles, abstracts and full papers was performed by two independent reviewers. Data extraction and validity were performed by one reviewer and checked by a second reviewer. Study quality was assessed using the SYRCLE risk of bias tool, for animal studies and by developing validity tools for the experimental model, organelle markers and imprecision. A narrative data synthesis was performed. We identified 31 studies (34 publications) of the asymmetric inheritance of organelles after mitotic or meiotic division. Studies for the asymmetric inheritance of centrosomes (n = 9); endosomes (n = 6), P granules (n = 4), the midbody (n = 3), mitochondria (n = 3), proteosomes (n = 2), spectrosomes (n = 2), cilia (n = 2) and endoplasmic reticulum (n = 2) were identified. Asymmetry was defined and quantified by variable methods. Assessment of the statistical reliability of the results indicated only two studies (7%) were judged to have low concern, the majority of studies (77%) were 'unclear' and five (16%) were judged to have 'high concerns'; the main reasons were low technical repeats (<10). Assessment of model validity indicated that the majority of studies (61%) were judged to be valid, ten studies (32%) were unclear and two studies (7%) were judged to have 'high concerns'; both described 'stem cells' without providing experimental evidence to confirm this (pluripotency and self-renewal). Assessment of marker validity indicated that no studies had low concern, most studies were unclear (96.5%), indicating there were insufficient details to judge if the markers were appropriate. One study had high concern for marker validity due to the contradictory results of two markers for the same organelle. For most studies the validity and imprecision of results could not be confirmed. In particular, data were limited due to a lack of reporting of interassay variability, sample size calculations, controls and functional validation of organelle markers. An evaluation of 16 systematic reviews containing cell assays found that only 50% reported adherence to PRISMA or ARRIVE reporting guidelines and 38% reported a formal risk of bias assessment. 44% of the reviews did not consider how relevant or valid the models were to the research question. 75% reviews did not consider how valid the markers were. 69% of reviews did not consider the impact of the statistical reliability of the results. Future systematic reviews in basic or preclinical research should ensure the rigorous reporting of the statistical reliability of the results in addition to the validity of the methods. Increased awareness of the importance of reporting guidelines and validation tools is needed for the scientific community.

Systematic review and mixed treatment comparison of intravitreal aflibercept with other therapies for diabetic macular edema (DME).

BACKGROUND: This was an indirect comparison of the effectiveness of intravitreal aflibercept (IVT-AFL) 2 mg every 8 weeks after 5 initial monthly doses (or if different periods, after an initial monthly dosing period) (2q8) and other diabetic macular edema (DME) therapies at doses licensed outside the USA. METHODS: A comprehensive search was undertaken to source relevant studies. Feasibility networks were prepared to identify viable comparisons of 12-month outcomes between IVT-AFL 2q8 and therapies licensed outside the USA, which were assessed for clinical and statistical homogeneity. Pooled effect sizes (mean difference [MD] and relative risk/risk ratio [RR]) were calculated using fixed- and random-effects models. Indirect comparisons were performed using Bucher analysis. If at least one 'head-to-head' study was found then a mixed treatment comparison (MTC) was performed using Bayesian methods. Two 12-month comparisons could be undertaken based on indirect analyses: IVT-AFL 2q8 versus intravitreal ranibizumab (IVR) 0.5 mg as needed (PRN) (10 studies) and IVT-AFL 2q8 versus dexamethasone 0.7 mg implants (three studies). RESULTS: There was an increase in mean best-corrected visual acuity (BCVA) with IVT-AFL 2q8 over IVR 0.5 mg PRN by 4.67 letters [95% credible interval (CrI): 2.45-6.87] in the fixed-effect MTC model (10 studies) and by 4.82 letters [95% confidence interval (CI): 2.52-7.11] in the Bucher indirect analysis (four studies). IVT-AFL 2q8 doubled the proportion of patients gaining ≥ 10 Early Treatment Diabetic Retinopathy Study letters at 12 months compared with dexamethasone 0.7 mg implants (RR = 2.10 [95% CI: 1.29-3.40]) in the fixed-effect model. There were no significant differences in safety outcomes between IVT-AFL 2q8 and IVR 0.5 mg PRN or dexamethasone 0.7 mg implants. CONCLUSIONS: Studies of IVT-AFL 2q8 showed improved 12-month visual acuity measures compared with studies of IVR 0.5 mg PRN and dexamethasone 0.7 mg implants based on indirect comparisons. These analyses are subject to a number of limitations which are inherent in indirect data comparisons.

Stromal upregulation of lateral epithelial adhesions: gene expression analysis of signalling pathways in prostate epithelium.

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture: a role for actin/cadherin dynamics.

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFß2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFß2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFß2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Modeling the prostate stem cell niche: an evaluation of stem cell survival and expansion in vitro.

The goal of this work was to engineer a clinically relevant in vitro model of human prostate stem cells (PSCs) that could be used to interrogate the mechanisms of stem cell control. We, therefore, compared the growth potential of stem cells in 3D culture (where the conditions would favor a quiescent state) with monolayer culture that has previously been demonstrated to induce PSC division. We found a fundamental difference between cultures of primary, adult PSCs grown as monolayers compared to those grown as spheres. The first supported the expansion and maintenance of PSCs from single cells while the latter did not. In an attempt to determine the mechanisms governing stem cell control, several known stem cell activators (including IFNalpha, FGF2, anti-TGFbeta, and dihydrotestosterone) were studied. However, cell division was not observed. CD133+ cells derived from a prostate cell line did not grow as spheres from single cells but did grow from aggregates. We conclude that PSCs can be expanded and maintained in monolayer culture from single cells, but that PSCs are growth quiescent when grown as spheres. It is likely that the physical arrangement of cells in monolayer provides an injury-type response, which can activate stem cells into cycle.

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions.

BACKGROUND: The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133+/alpha2beta1hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133-/alpha2beta1low) counterparts, resulting in an informative cancer stem cell gene-expression signature. RESULTS: Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor kappaB (NF-kappaB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-kappaB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches. CONCLUSION: We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.

Differentiation of prostate epithelial cell cultures by matrigel/ stromal cell glandular reconstruction.

Three-dimensional epithelial culture models are widely used to emulate a more physiologically relevant microenvironment for the study of genes and signaling pathways. Prostate epithelial cells can grow into solid cell masses or acinus-like spheroids in Matrigel. To test if the ability to form acinus-like spheroids in Matrigel is dependent on how undifferentiated a cell is or whether it is tumor or nontumor, we established six novel epithelial cell lines. Primary prostate epithelial cells were immortalized using HPV16 E6 gene transduction and were named Shmac 2, 3, and 6 (nontumor); Shmac 4, Shmac 5, and P4E6 (tumor). All cell lines were phenotyped in monolayer culture, and their ability to form acinus-like spheroids in Matrigel investigated. The cell lines exhibited a wide range of population doubling times and all showed an intermediate phenotype in monolayer culture ((luminal)CK(+)/(basal)CK(+)/CD44(+)/PSA(+)/AR(-)). Only Shmac 5 cells formed acinus-like spheroids when cultured in Matrigel. Co-culture of the spheroids with fibroblasts advanced differentiation by inducing androgen receptor expression and epithelial polarization. Our findings indicate that tumor cells can form acinus-like spheroids in Matrigel.

CD133, a novel marker for human prostatic epithelial stem cells.

Stem cells are clonogenic cells with self-renewal and differentiation properties, which may represent a major target for genetic damage leading to prostate cancer and benign prostatic hyperplasia. Stem cells remain poorly characterised because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying cells, which have a more restricted proliferative potential. Human CD133 antigen, also known as AC133, was recently identified as a haematopoietic stem cell marker. Here we show that a small population (approximately 1%) of human prostate basal cells express the cell surface marker CD133 and are restricted to the alpha(2)beta(1)(hi) population, previously shown to be a marker of stem cells in prostate epithelia. alpha(2)beta(1)(hi)/CD133(+) cells exhibit two important attributes of epithelial stem cells: they possess a high in vitro proliferative potential and can reconstitute prostatic-like acini in immunocompromised male nude mice.

Enhanced expression of vimentin in motile prostate cell lines and in poorly differentiated and metastatic prostate carcinoma.

BACKGROUND: The metastatic potential of a series of prostate cell lines was analysed by measuring motility and invasiveness, and further correlated to the expression of epithelial differentiation markers. METHODS: Invasion and motility were measured using in vitro assays. Immunohistochemistry of cell lines and tissues was used to identify expression of cytokeratins 8 and 1, 5, 10, 14, vimentin, prostate specific antigen, prostate specific membrane antigen, androgen receptor, desmoglein, E-cadherin, beta1 integrin, CD44, hmet, vinculin and actin. RESULTS: Expression of vimentin was the only marker to correlate with motility, no markers correlated to invasion. Lower vimentin expression was observed in cells with low motility (PNT2-C2) and high expression in cells with high motility (P4E6, PNT1a, PC-3). Vimentin expression was not detected in well differentiated tumours, moderately differentiated tumours contained vimentin positive cells (1/9 bone scan negative, 2/5 bone scan positive), but the majority of poorly differentiated cancers (4/11 bone scan negative, 9/14 bone scan positive) and bone metastases (7/8) had high vimentin expression in tumour cells. CONCLUSIONS: Motile prostate cancer cell lines express vimentin. In tissue sections, the presence of vimentin positive tumour cells correlated positively to poorly differentiated cancers and the presence of bone metastases.

Primary prostate stromal cells modulate the morphology and migration of primary prostate epithelial cells in type 1 collagen gels.

The effects of human primary prostatic stromal cells on the migration and morphogenesis of human prostatic epithelial cells, derived from tumor or benign prostatic hyperplasia tissue, were studied using a three-dimensional coculture system. Epithelial cells from tumor or benign tissue migrated efficiently into collagen gels populated with stromal cells from benign tissue. Only epithelial cells from benign prostate formed acinus-like structures that exhibited differentiated prostatic function and strong expression of membrane-associated E-cadherin. In gels populated by stromal cells from tumor tissue, migration of primary prostatic epithelial cells did not occur. In the absence of stromal cells, primary epithelial cells were unable to proliferate. This three-dimensional culture system allows closely controlled manipulation and analysis in vitro of interactions between prostatic epithelial and stromal cells.