Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis.

Peritoneal carcinomatosis (PC) present a ubiquitous clinical conundrum in all intra-abdominal malignancies. Via functional and transcriptomic experiments of ascites-treated PC cells, we identify STAT3 as a key signaling pathway. Integrative analysis of publicly available databases and correlation with clinical cohorts (n = 7,359) reveal putative clinically significant activating ligands of STAT3 signaling. We further validate a 3-biomarker prognostic panel in ascites independent of clinical covariates in a prospective study (n = 149). Via single-cell sequencing experiments, we uncover that PAI-1, a key component of the prognostic biomarker panel, is largely secreted by fibroblasts and mesothelial cells. Molecular stratification of ascites using PAI-1 levels and STAT3 activation in ascites-treated cells highlight a therapeutic opportunity based on a phenomenon of paracrine addiction. These results are recapitulated in patient-derived ascites-dependent xenografts. Here, we demonstrate therapeutic proof of concept of direct ligand inhibition of a prognostic target within an enclosed biological space.

NEAR trial: A single-arm phase II trial of neoadjuvant apalutamide monotherapy and radical prostatectomy in intermediate- and high-risk prostate cancer.

OBJECTIVE: Treatment efficacy of androgen deprivation therapy with radical prostatectomy for intermediate- to high-risk prostate cancer is less well-studied. The NEAR trial is a single-arm, phase II investigation of neoadjuvant apalutamide monotherapy and radical prostatectomy (RP) in the treatment of D'Amico intermediate- and high-risk prostate cancer (NCT03124433). MATERIALS AND METHODS: Patients with histologically-proven, D'Amico intermediate- to high-risk prostate adenocarcinoma received apalutamide 240 mg once-daily for 12 weeks followed by RP + /-lymphadenectomy. Primary outcome was pathological complete response (pCR) rate. Secondary outcomes included rate of biochemical response (defined by PSA < 0.03 ng/mL at week 24 from starting apalutamide without subsequent PSA relapse), treatment-related adverse events, and RP complication rates. Correlative biomarker analyses were performed to examine for molecular predictors of treatment responses. RESULTS: From 2017 to 2019, 30 patients were recruited, of which 20 and 10 were high and intermediate risk, respectively; 25 completed treatment as per-protocol. We did not observe any pCR on trial; median reduction of cancer burden was 41.7% (IQR: 33.3%-60.0%). 18 out of 25 patients were classified as having a biochemical response (4 did not achieve PSA of <0.03 ng/mL at week 24 and 3 developed PSA relapse subsequently). Dry skin (N = 16; 53.3%), fatigue (N = 10; 33.3%) and skin rash (N = 9; 30.0%) were the most common adverse events, and there was no major peri-operative complication. We observed an association between tumours of low androgen receptor activity and PAM50 basal status with biochemical non-responders, albeit these molecular phenotypes were not associated with pathological response. CONCLUSIONS: A 12-week course of neoadjuvant apalutamide prior to RP did not meet the primary endpoint of pCR in this trial. Tumours with low androgen receptor activity or of the PAM50 basal subtype may have a reduced response to apalutamide.

An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours.

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

Molecular insights into paediatric breast fibroepithelial tumours.

AIMS: This study aims to examine the molecular genetics of paediatric breast fibroepithelial tumours through the targeted sequencing of 50 genes. METHODS AND RESULTS: Formalin-fixed paraffin-embedded tissues of fibroepithelial tumours diagnosed in a cohort of patients aged 18 years and below were subjected to next generation sequencing using the Haloplex Target Enrichment System. Twenty-five conventional and 17 juvenile fibroadenomas were studied, with MED12 mutations found in 53.8 and 35% of the tumours, respectively. There was also one benign fibroepithelial neoplasm with hybrid features of juvenile papillomatosis and infarcted benign phyllodes tumour-like areas. Most tumours did not have mutations in well-known cancer driver genes, none harboured TERT promoter mutations, while 25.6% (11 of 43) showed no mutations. Metachronous and synchronous tumours were found to have mutational heterogeneity with some containing mutations in MED12; other genes or no mutations were detected at all. Four of eight giant fibroadenomas (size 5 cm or larger) had no mutations detected, suggesting that there are other molecular mechanisms driving their growth. Tumours with MED12 mutations incidentally had a significantly higher stromal mitotic count compared with those without. CONCLUSION: While paediatric fibroepithelial lesions can have cellular stroma potentially raising concern for phyllodes tumour, their lack of TERT promoter and cancer driver mutations is reassuring. The absence of mutations in a significant proportion of tumours, especially the giant fibroadenomas, warrants investigation of pathogenetic mechanisms beyond those involving the 50 genes.

Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion.

AIMS: Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically. METHODS AND RESULTS: By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression. CONCLUSIONS: The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.

Aristolochic acids and their derivatives are widely implicated in liver cancers in Taiwan and throughout Asia.

Many traditional pharmacopeias include Aristolochia and related plants, which contain nephrotoxins and mutagens in the form of aristolochic acids and similar compounds (collectively, AA). AA is implicated in multiple cancer types, sometimes with very high mutational burdens, especially in upper tract urothelial cancers (UTUCs). AA-associated kidney failure and UTUCs are prevalent in Taiwan, but AA's role in hepatocellular carcinomas (HCCs) there remains unexplored. Therefore, we sequenced the whole exomes of 98 HCCs from two hospitals in Taiwan and found that 78% showed the distinctive mutational signature of AA exposure, accounting for most of the nonsilent mutations in known cancer driver genes. We then searched for the AA signature in 1400 HCCs from diverse geographic regions. Consistent with exposure through known herbal medicines, 47% of Chinese HCCs showed the signature, albeit with lower mutation loads than in Taiwan. In addition, 29% of HCCs from Southeast Asia showed the signature. The AA signature was also detected in 13 and 2.7% of HCCs from Korea and Japan as well as in 4.8 and 1.7% of HCCs from North America and Europe, respectively, excluding one U.S. hospital where 22% of 87 "Asian" HCCs had the signature. Thus, AA exposure is geographically widespread. Asia, especially Taiwan, appears to be much more extensively affected, which is consistent with other evidence of patterns of AA exposure. We propose that additional measures aimed at primary prevention through avoidance of AA exposure and investigation of possible approaches to secondary prevention are warranted.