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Prostate cancer is one of the most heritable cancers. Hundreds of germline polymorphisms have been linked to prostate cancer diagnosis and prognosis. Polygenic risk scores can predict genetic risk of a prostate cancer diagnosis. Although these scores inform the probability of developing a tumor, it remains unknown how germline risk influences the tumor molecular evolution. We cultivated a cohort of 1250 localized European-descent patients with germline and somatic DNA profiling. Men of European descent with higher genetic risk were diagnosed earlier and had less genomic instability and fewer driver genes mutated. Higher genetic risk was associated with better outcome. These data imply a polygenic “two-hit” model where germline risk reduces the number of somatic alterations required for tumorigenesis. These findings support further clinical studies of polygenic risk scores as inexpensive and minimally invasive adjuncts to standard risk stratification. Further studies are required to interrogate generalizability to more ancestrally and clinically diverse populations.

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However, the impact of this instability on the noncoding genome and its three-dimensional organization to aid progression is unclear. Using primary benign and tumor tissue, we find a high concordance in higher-order three-dimensional genome organization. This concordance argues for constraints to the topology of prostate tumor genomes. Nonetheless, we identified changes in focal chromatin interactions, typical of loops bridging noncoding cis-regulatory elements, and showed how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events resulted in opposing differential expression of genes found at antipodes of rearrangements. Collectively, these results argue that changes to focal chromatin interactions, as opposed to higher-order genome organization, allow for aberrant gene regulation and are repeatedly mediated by structural variants in primary prostate cancer. Significance: This work showcases how the noncoding genome can be hijacked by focal insults to its three-dimensional organization that contribute to prostate cancer oncogenesis.

Intratumoral heterogeneity can occur via phenotype transitions, often after chronic exposure to targeted anticancer agents. This process, termed lineage plasticity, is associated with acquired independence to an initial oncogenic driver, resulting in treatment failure. In non-small cell lung cancer (NSCLC) and prostate cancers, lineage plasticity manifests when the adenocarcinoma phenotype transforms into neuroendocrine (NE) disease. The exact molecular mechanisms involved in this NE transdifferentiation remain elusive. In small cell lung cancer (SCLC), plasticity from NE to nonNE phenotypes is driven by NOTCH signaling. Herein we review current understanding of NE lineage plasticity dynamics, exemplified by prostate cancer, NSCLC, and SCLC.

Inherited genetic variation profoundly influences cancer risk and outcome. While the impact of germline single nucleotide polymorphisms has been well-studied in several cancer types, the effects of germline structural variants (gSVs) on cancer biology and clinical outcomes is largely unknown. From our cohort of 300 men with localized, intermediate risk prostate cancer, we identified 6,003 gSVs present in at least 3% of patients; 48 were associated with recurrent somatic alterations or clinical outcome. Of these, approximately 50% were associated with expression of nearby genes or intersected with exons or regulatory regions. Using external cohorts, we validated three gSVs that were strongly associated with poor clinical outcomes, including an inversion at chr14q24.1 present in ~20% of patients. Notably, a strong synergistic effect on outcome was observed in patients with somatic TP53 alterations or high genomic instability, defining a new aggressive prostate cancer subtype with chr14INV as a novel, recurrent biomarker. Citation Format: Nicholas K. Wang, Alexandre Rouette, Kathleen E. Houlahan, Takafumi N. Yamaguchi, Julie Livingstone, Chol-Hee Jung, Peter Georgeson, Michael Fraser, Yu-Jia Shiah, Cindy Q. Yao, Vincent Huang, Natalie S. Fox, Natalie Kurganovs, Katayoon Kasaian, Veronica Y. Sabelnykova, Jay Jayalath, Kenneth Weke, Helen Zhu, Theodorus van der Kwast, Tony Papenfuss, Housheng H. He, Niall M. Corcoran, Robert G. Bristow, Alexandre R. Zlotta, Christopher Hovens, Paul C. Boutros. Germline structural variants shape prostate cancer clinical and molecular evolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4305.

Replicative immortality is a hallmark of cancer, and can be achieved through telomere lengthening and maintenance. Although the role of telomere length in cancer has been well studied, its association to genomic features is less well known. Here, we report the telomere lengths of 392 localized prostate cancer tumours and characterize their relationship to genomic, transcriptomic and proteomic features. Shorter tumour telomere lengths are associated with elevated genomic instability, including single-nucleotide variants, indels and structural variants. Genes involved in cell proliferation and signaling are correlated with tumour telomere length at all levels of the central dogma. Telomere length is also associated with multiple clinical features of a tumour. Longer telomere lengths in non-tumour samples are associated with a lower rate of biochemical relapse. In summary, we describe the multi-level integration of telomere length, genomics, transcriptomics and proteomics in localized prostate cancer., Despite the known role of telomere length in cancer, its association with genomic features remains unclear. Here, the authors integrate telomere length, genomics, transcriptomics and proteomics in localized prostate cancer and reveal links between telomere maintenance, disease drivers and clinical outcomes.

Oncogenesis is driven by germline, environmental and stochastic factors. It is unknown how these interact to produce the molecular phenotypes of tumors. We therefore quantified the influence of germline polymorphisms on the somatic epigenome of 589 localized prostate tumors. Predisposition risk loci influence a tumor’s epigenome, uncovering a mechanism for cancer susceptibility. We identified and validated 1,178 loci associated with altered methylation in tumoral but not nonmalignant tissue. These tumor methylation quantitative trait loci influence chromatin structure, as well as RNA and protein abundance. One prominent tumor methylation quantitative trait locus is associated with AKT1 expression and is predictive of relapse after definitive local therapy in both discovery and validation cohorts. These data reveal intricate crosstalk between the germ line and the epigenome of primary tumors, which may help identify germline biomarkers of aggressive disease to aid patient triage and optimize the use of more invasive or expensive diagnostic assays. Genetic variants in the germ line modulate DNA methylation in tumors and contribute to the aggressiveness of prostate cancer.

Background The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer. Methods Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines (DU145, PC3, LNCAP). Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia. Results Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells. Conclusion Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer.

Purpose: At diagnosis, colorectal cancer presents with synchronous peritoneal metastasis in up to 10% of patients. The peritoneum is poorly characterized with respect to its superspecialized microenvironment. Our aim was to describe the differences between peritoneal metastases and their matched primary tumors excised simultaneously at the time of surgery. Also, we tested the hypothesis of these differences being present in primary colorectal tumors and having prognostic capacity. Experimental Design: We report a comprehensive analysis of 30 samples from peritoneal metastasis with their matched colorectal cancer primaries obtained during cytoreductive surgery. We tested and validated the prognostic value of our findings in a pooled series of 660 colorectal cancer primary samples with overall survival (OS) information and 743 samples with disease-free survival (DFS) information from publicly available databases. Results: We identified 20 genes dysregulated in peritoneal metastasis that promote an early increasing role of “stemness” in conjunction with tumor-favorable inflammatory changes. When adjusted for age, gender, and stage, the 20-gene peritoneal signature proved to have prognostic value for both OS [adjusted HR for the high-risk group (vs. low-risk) 2.32 (95% confidence interval, CI, 1.69–3.19; P < 0.0001)] and for DFS [adjusted HR 2.08 (95% CI, 1.50–2.91; P < 0.0001)]. Conclusions: Our findings indicated that the activation of “stemness” pathways and adaptation to the peritoneal-specific environment are key to early stages of peritoneal carcinomatosis. The in silico analysis suggested that this 20-gene peritoneal signature may hold prognostic information with potential for development of new precision medicine strategies in this setting.

The history of radiotherapy is intertwined with research on hypoxia. There is level 1a evidence that giving hypoxia-targeting treatments with radiotherapy improves locoregional control and survival without compromising late side-effects. Despite coming in and out of vogue over decades, there is now an established role for hypoxia in driving molecular alterations promoting tumour progression and metastases. While tumour genomic complexity and immune profiling offer promise, there is a stronger evidence base for personalising radiotherapy based on hypoxia status. Despite this, there is only one phase III trial targeting hypoxia modification with full transcriptomic data available. There are no biomarkers in routine use for patients undergoing radiotherapy to aid management decisions, and a roadmap is needed to ensure consistency and provide a benchmark for progression to application. Gene expression signatures address past limitations of hypoxia biomarkers and could progress biologically optimised radiotherapy. Here, we review recent developments in generating hypoxia gene expression signatures and highlight progress addressing the challenges that must be overcome to pave the way for their clinical application.

Functional impairment of the tumour suppressor PTEN is common in primary prostate cancer and has been linked to relapse post-radiotherapy (post-RT). Pre-clinical modelling supports elevated CXC chemokine signalling as a critical mediator of PTEN-depleted disease progression and therapeutic resistance. We assessed the correlation of PTEN deficiency with CXC chemokine signalling and its association with clinical outcomes. Gene expression analysis characterized a PTENLOW/CXCR1HIGH/CXCR2HIGH cluster of tumours that associates with earlier time to biochemical recurrence [hazard ratio (HR) 5.87 and 2.65, respectively] and development of systemic metastasis (HR 3.51). In vitro, CXCL signalling was further amplified following exposure of PTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2 signalling in PTEN-depleted cell-based models increased IR sensitivity. In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR to PTEN-deficient xenografts attenuated tumour growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL signalling and anti-apoptotic protein expression. Interventions targeting CXC chemokine signalling may provide an effective strategy to combine with RT in locally advanced prostate cancer patients with known presence of PTEN-deficient foci.

Purpose The National Comprehensive Cancer Network (NCCN) has recently endorsed the stratification of intermediate-risk prostate cancer (IR-PCa) into favorable and unfavorable subgroups and recommend the addition of androgen deprivation therapy (ADT) to radiation therapy (RT) for unfavorable IR-PCa. Recently, more accurate prognostication was demonstrated by integrating a 22-feature genomic classifier (GC) to the NCCN stratification system. Here, we test the utility of the GC to better identify patients with IR-PCa who are sufficiently treated by RT alone. Methods and Materials We identified a novel cohort comprising 121 patients with IR-PCa treated with dose-escalated image guided RT (78 Gy in 39 fractions) without ADT. GC scores were derived from tumors sampled in diagnostic biopsies. Multivariable analyses, including both NCCN subclassification and GC scores, were performed for biochemical failure (prostate-specific antigen nadir + 2 ng/mL) and metastasis occurrence. Results By NCCN subclassification, 33 (27.3%) and 87 (71.9%) of men were classified as having favorable and unfavorable IR-PCa, respectively (1 case unclassifiable). GC scores were high in 3 favorable IR-PCa and low in 60 unfavorable IR-PCa. Higher GC scores, but not NCCN risk subgroups, were associated with biochemical relapse (hazard ratio, 1.36; 95% confidence interval [CI], 1.09-1.71] per 10% increase; P = .007) and metastasis (hazard ratio, 2.05; 95% CI, 1.24-4.24; P = .004). GC predicted biochemical failure at 5 years (area under the curve, 0.78; 95% CI, 0.59-0.91), and the combinatorial NCCN + GC model significantly outperformed the NCCN alone model for predicting early-onset metastasis (area under the curve for 5-year metastasis of 0.89 vs 0.86 [GC alone] vs 0.54 [NCCN alone]). Conclusions We demonstrated the accuracy of the GC for predicting disease recurrence in IR-PCa treated with dose-escalated image guided RT alone. Our findings highlight the need to evaluate this GC in a prospective clinical trial investigating the role of ADT-RT in clinicogenomic-defined IR-PCa subgroups.

Functional impairment of the tumour-suppressorPTENis common in primary-prostate cancer and has been linked to relapse post-radiotherapy (RT). Pre-clinical modelling supports elevated CXC-chemokine signaling as a critical mediator ofPTEN-depleted disease progression and therapeutic resistance. We assessed the correlation ofPTEN-deficiency with CXC-chemokine signaling and its association with clinical outcomes. Gene expression analysis characterized aPTENLOW/CXCR1HIGH/CXCR2HIGHcluster of tumors that associates with earlier time-to-biochemical recurrence (HR 5.87 and HR 2.65 respectively) and development of systemic metastasis (HR 3.51).In vitro, CXCL-signaling was further amplified following exposure ofPTEN-deficient prostate cancer cell lines to ionizing radiation (IR). Inhibition of CXCR1/2-signaling inPTEN-depleted cell-based models increased IR-sensitivity.In vivo, administration of a CXCR1/2-targeted pepducin (x1/2pal-i3), or CXCR2-specific antagonist (AZD5069), in combination with IR toPTEN-deficient xenografts attenuated tumor growth and progression compared to control or IR alone. Post-mortem analysis confirmed that x1/2pal-i3 administration attenuated IR-induced CXCL-signaling and anti-apoptotic protein expression. Interventions targeting CXC-chemokine signaling may provide an effective strategy to combine with radiotherapy, in both locally-advanced and oligometastatic-prostate cancers, with known presence ofPTEN-deficient foci.

Background The development of noninvasive tests for the early detection of aggressive prostate tumors is a major unmet clinical need. miRNAs are promising noninvasive biomarkers: they play essential roles in tumorigenesis, are stable under diverse analytical conditions, and can be detected in body fluids. Methods We measured the longitudinal stability of 673 miRNAs by collecting serial urine samples from 10 patients with localized prostate cancer. We then measured temporally stable miRNAs in an independent training cohort (n = 99) and created a biomarker predictive of Gleason grade using machine-learning techniques. Finally, we validated this biomarker in an independent validation cohort (n = 40). Results We found that each individual has a specific urine miRNA fingerprint. These fingerprints are temporally stable and associated with specific biological functions. We identified seven miRNAs that were stable over time within individual patients and integrated them with machine-learning techniques to create a novel biomarker for prostate cancer that overcomes interindividual variability. Our urine biomarker robustly identified high-risk patients and achieved similar accuracy as tissue-based prognostic markers (area under the receiver operating characteristic = 0.72, 95% confidence interval = 0.69 to 0.76 in the training cohort, and area under the receiver operating characteristic curve = 0.74, 95% confidence interval = 0.55 to 0.92 in the validation cohort). Conclusions These data highlight the importance of quantifying intra- and intertumoral heterogeneity in biomarker development. This noninvasive biomarker may usefully supplement invasive or expensive radiologic- and tissue-based assays.

The Radiation Therapy Committee of SWOG periodically evaluates its strategic plan in an effort to maintain a current and relevant scientific focus, and to provide a standard platform for future development of protocol concepts. Participants in the 2017 Strategic Planning Workshop included leaders in cancer basic sciences, molecular theragnostics, pharmaceutical and technology industries, clinical trial design, oncology practice, and statistical analysis. The committee discussed high-priority research areas, such as optimization of combined modality therapy, radiation oncology–specific drug design, identification of molecular profiles predictive of radiation-induced local or distant tumor responses, and methods for normal tissue-specific mitigation of radiation toxicity. The following concepts emerged as dominant questions ready for national testing: (i) what is the role of radiotherapy in the treatment of oligometastatic, oligorecurrent, and oligoprogressive disease? (ii) How can combined modality therapy be used to enhance systemic and local response? (iii) Can we validate and optimize liquid biopsy and other biomarkers (such as novel imaging) to supplement current response criteria to guide therapy and clinical trial design endpoints? (iv) How can we overcome deficiencies of randomized survival endpoint trials in an era of increasing molecular stratification factors? And (v) how can we mitigate treatment-related side effects and maximize quality of life in cancer survivors? The committee concluded that many aspects of these questions are ready for clinical evaluation and example protocol concepts are provided that could improve rates of cancer cure and quality of survival. Clin Cancer Res; 24(15); 3500–9. ©2018 AACR.

Purpose To describe and compare outcomes in men with initially presumed indolent prostate cancer receiving definitive radiation therapy after active surveillance (AS) versus those in a risk-matched cohort undergoing up-front radiation therapy. Methods and Materials Men prospectively enrolled in an AS program between 1992 and 2014 and subsequently undergoing curative radiation therapy (ie, image guided radiation therapy [IGRT] or low-dose-rate brachytherapy [LDR-BT]) were identified. Biochemical relapse–free rate (bRFR), metastasis-free rate (mFR), and overall survival (OS) were compared against a cohort of men treated up front, matched by age, clinical prognostic indices (risk group, prostate-specific antigen, cT category, Gleason score, percentage of involved biopsy cores), and radiation therapy modality. Results Of 1070 patients in the AS registry, 200 underwent definitive radiation therapy (143 IGRT and 57 LDR-BT) after a median of 32.9 (interquartile range [IQR] 20.6-59.8) months on surveillance. Main reasons for treatment were grade and volume upgrading (57.5% and 26%, respectively). Median follow-up after radiation therapy was 4.9 (IQR 3.1-7.5) years. At 5 years the bRFR, mFR, and OS were, respectively, 97%, 99%, and 98.5%. No patient died of prostate cancer. Adequate risk-matching was confirmed in an independent cohort comprising 359 patients receiving up-front IGRT (71%) or LDR-BT (29%) and followed for a median of 9 (IQR 3.1-7.5) years. There was no difference in the disease-specific outcomes (bRFR, mFR) between the 2 cohorts (Gray's P value of .257 and .934, respectively). In multivariate analyses, timing of radical radiation therapy (deferred vs up-front) was not correlated to biochemical relapse or metastases occurrence. Conclusions Curative-intent radiation therapy (ie, dose-escalated IGRT or LDR-BT) after a period of AS renders excellent oncologic outcomes at 5 years. Deferring radical therapy after a period of AS does not seem to result in inferior oncologic outcomes compared with patients with similar risk characteristics undergoing up-front treatment.

Cell-free circulating tumor DNA (ctDNA) or circulating tumor cell (CTC) assays are potentially powerful in the treatment of metastatic castration-resistant prostate cancer (mCRPC). A new study suggests that mutation of TP53 supersedes AR in predicting mCRPC survival. A role for TP53 mutation as a driver for mCRPC remains unexplored. See related article by De Laere et al., p. 1766

433 Background: Prior randomized controlled trials (RCT) comparing bladder preservation to radical cystectomy (RC) for muscle invasive bladder cancer (MIBC) closed early due to lack of accrual. Given that no future RCTs are foreseen, and in the absence of level 1 data, we aimed to provide the best evidence possible on outcomes of matched cohorts comparing trimodality therapy (TMT, maximal transurethral resection of bladder tumor followed by concurrent chemoradiation) to RC in order to guide management. Methods: This retrospective analysis included 703 patients with MIBC clinical stage T2-T3/4aN0M0 MIBC urothelial carcinoma of the bladder, 421 RC and 282 TMT who would have been eligible for both TMT or RC, treated at the Massachusetts General Hospital, Boston; Princess Margaret Cancer Centre, Toronto; and University of Southern California, Los Angeles between 2005-2017. To compare homogeneous cohorts, all patients included in this analysis had solitary tumors < 7 cm, no or unilateral hydronephrosis, and no multifocal carcinoma in situ. Treatment propensity scores were estimated using logistic regression, and patients were matched 3:1 with replacement. Covariates included age, sex, clinical T stage, hydronephrosis, (neo)adjuvant chemotherapy, body mass index, smoking history, and ECOG status. Overall survival (OS) was estimated with adjusted Cox models; cancer-specific survival (CSS), distant failure-free survival, pelvic nodal failure-free survival and metastasis-free survival (combined distant and pelvic nodal failure) were estimated with adjusted competing risk models. Our primary endpoint of interest was metastasis-free survival. The analysis was performed as intent-to-treat. Results: The 3:1 matched cohort comprised of 1,116 patients (834 RC vs 282 TMT). After matching, age (71.3 vs 71.6), cT2 clinical stage (88 vs 90%), presence of hydronephrosis (12 vs 10%), and use of (neo)adjuvant chemotherapy (60 vs 65%) were similar between RC and TMT cohorts. Salvage cystectomy was performed in 38 patients (13%) treated by TMT. At 5 years, metastasis-free (73 vs 78%, p = 0.07), distant failure-free (78 vs 82%, p = 0.14), and pelvic nodal failure-free (96 vs 94%, p = 0.33) survival were not statistically different between RC and TMT, whereas CSS and OS favored TMT (78 vs 85%, p = 0.02; 70 vs 78%, p < 0.001). Outcomes for RC and TMT were not different among centers. Final pT stage in the RC patients was: pT0 14%, pT1 7%, pT2 29%, pT3/4 42% and N+ 24%. Peri RC mortality was 2.1% and median number of nodes removed was 40. NMIBC recurrence occurred in 57/278 (20.5%) TMT patients. Conclusions: This large multi-institutional contemporary study provides the best evidence to date, in the absence of randomized trials, supporting TMT for select patients with MIBC. Oncologic outcomes seem to be equivalent between TMT and RC, affirming the position that TMT should be offered as an effective alternative.

Genomic instability is underpinned by defects in the DNA damage response and DNA repair pathways. Subsequent clonal selection and adaption can lead to a mutator phenotype and tumor aggression. Importantly, tumor cell sensitivity to chemotherapy and radiotherapy can depend highly on the cellular capacity to repair DNA damage within and between tumor types. Annotation of functional defects in DNA damage response and DNA repair function may allow for the development of novel prognostic biomarkers. This information could also be used to predict therapeutic response, including predicting responses following inhibition of DNA repair. Herein, we highlight the increasing potential for annotating and targeting DNA repair defects in patients undergoing precision radiotherapy.

Autophagy is a survival mechanism that allows recycling of cellular breakdown products, particularly in stressed cells. Here we evaluate the hypotheses that up-regulation of autophagy is a common mechanism of resistance to chemotherapy, and that drug resistance can be reversed by inhibiting autophagy with a proton pump inhibitor. We exposed human PC3, LNCaP and MCF7 cells to seven clinically-used chemotherapy drugs ± pantoprazole, examined the up-regulation of autophagy and the effect on cellular proliferation by Western Blots, MTS assay and colony-forming assay. The distribution of drug effects and of autophagy was quantified in LNCaP tumor sections in relation to blood vessels and hypoxia by immunohistochemistry using γH2AX, cleaved caspase-3 and p62. All anticancer drugs led to up-regulation of autophagy in cultured tumor cells. Pantoprazole inhibited the induction of autophagy in a time- and dose-dependent manner, and sensitized cancer cells to the seven anti-cancer drugs. Treatment of LNCaP xenografts with paclitaxel induced both DNA damage and autophagy; autophagy was inhibited and markers of toxicity were increased by pantoprazole. Induction of autophagy is a general mechanism associated with resistance to anticancer drugs and that its inhibition is a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes.

Radiotherapy has been optimized over the last decades not only through technological advances, but also through the translation of biological knowledge into clinical treatment schedules. Optimization of fractionation schedules and/or the introduction of simultaneous combined systemic treatment have significantly improved tumour cure rates in several cancer types. With modern techniques, we are currently able to measure factors of radiation resistance or radiation sensitivity in patient tumours; the definition of new biomarkers is expected to further enable personalized treatments. In this Review article, we overview important translation paths and summarize the quality requirements for preclinical and translational studies that will help to avoid bias in trial results., Translation of preclinical radiobiological research into clinical radiotherapy has largely improved treatment outcomes. Indicatively, based on the results of current biological research, fractionation schedules have been optimized, and combined treatments have been developed. In this Review, we discuss translation paths that have substantially improved radiotherapy and summarize what quality requirements within preclinical and translational studies will ensure the development of unbiased clinical trials.

Background: New technologies and techniques in radiation oncology and imaging offer opportunities to enhance the benefit of loco-regional treatments, expand treatment to new patient populations such as those with oligometastatic disease and decrease normal tissue toxicity. Furthermore, novel agents have become available which may be combined with radiation therapy, and identification of radiation-related biomarkers can be studied to refine treatment prescriptions. Finally, the use of artificial intelligence (AI) capabilities may also improve treatment quality assurance or the ease with which radiation dosing is prescribed. All of these potential advances present both opportunities and challenges for academic clinical researchers. Methods: Recently, the European Organisation for Research and Treatment of Cancer addressed these topics in a meeting of multiple stakeholders from Europe and North America. The following five themes radiobiology-based biomarkers, new technologies - particularly proton beam therapy, combination systemic and radiation therapy, management of oligometastatic disease and AI opportunities in radiation oncology were discussed in a State of Science format to define key controversies, unanswered questions and propose clinical trial priorities for development. Conclusions: Priorities for clinical trials implementing new science and technologies have been defined. Solutions to integrate the multidimensional complexity of data have been explored. New types of platforms and partnerships can support innovative approaches for clinical research in radiation oncology. (C) 2020 The Authors. Published by Elsevier Ltd.

Prostate cancer is the second most commonly diagnosed malignancy among men worldwide. Recurrently mutated in primary and metastatic prostate tumors, FOXA1 encodes a pioneer transcription factor involved in disease onset and progression through both androgen receptor-dependent and androgen receptor-independent mechanisms. Despite its oncogenic properties however, the regulation of FOXA1 expression remains unknown. Here, we identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic single-nucleotide variants in primary prostate tumors. We find that deletion and repression of these cis-regulatory elements significantly decreases FOXA1 expression and prostate cancer cell growth. Six of the ten single-nucleotide variants mapping to FOXA1 regulatory plexus significantly alter the transactivation potential of cis-regulatory elements by modulating the binding of transcription factors. Collectively, our results identify cis-regulatory elements within the FOXA1 plexus mutated in primary prostate tumors as potential targets for therapeutic intervention., FOXA1 pioneer transcription factor is recurrently mutated in primary and metastatic prostate tumors. Here, authors identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic SNVs in primary prostate tumors and characterize their role in regulating FOXA1 expression and prostate cancer cell growth.

DNA sequencing has identified recurrent mutations that drive the aggressiveness of prostate cancers. Surprisingly, the influence of genomic, epigenomic, and transcriptomic dysregulation on the tumor proteome remains poorly understood. We profiled the genomes, epigenomes, transcriptomes, and proteomes of 76 localized, intermediate-risk prostate cancers. We discovered that the genomic subtypes of prostate cancer converge on five proteomic subtypes, with distinct clinical trajectories. ETS fusions, the most common alteration in prostate tumors, affect different genes and pathways in the proteome and transcriptome. Globally, mRNA abundance changes explain only ∼10% of protein abundance variability. As a result, prognostic biomarkers combining genomic or epigenomic features with proteomic ones significantly outperform biomarkers comprised of a single data type.

Understanding the mechanistic basis for transition to late stage aggressive disease is vital for both assigning patient risk status in the localised setting and also identifying novel treatment strategies to prevent progression. Sub-regions of intratumoral hypoxia are found in all solid tumours and are associated with many biologic drivers of tumour progression. Crucially, more recent findings show the co-presence of hypoxia and genomic instability can confer a uniquely adverse prognosis in prostate cancer. In-depth informatic and functional studies suggests a role for hypoxia in co-operating with oncogenic drivers (e.g. loss of PTEN) and suppressing DNA repair capacity. To delineate a direct relationship between hypoxia and PTEN loss, we interrogated the impact of chronic hypoxia (1% O2, 0.2% O2; 72 h) on PTEN oncogenic signalling and chromosome instability. Quantitative immunofluorescence of centrosome aberrations, using antibodies to phospho-Histone H3 and Centrin-1, show significant increases in centriole numbers (p < 0.05) in mitotic cells under chronic hypoxic conditions and an increase in the incidence of micronuclei (p < 0.005) specifically in chronic hypoxic cells with PTEN loss. Hypoxic suppression of homologous recombination represents a "contextual lethal" vulnerability in hypoxic prostate tumours which could extend the application of existing DNA repair targeting agents such as poly-ADP ribose polymerase inhibitors. Additional mechanistic studies using hTERT-immortalized, primary prostate epithelial cells isogenic for c-MYC, BRCA2, or TP53/RB (SV40-transduced) were studied as to the relationship between hypoxia, inherent versus acquired genetic instability and DNA repair capacity in-vitro and in-situ. Chronic hypoxia (20 generation doublings at 0.2% O2) was sufficient to drive increased copy number alterations (CNAs) in selected clones by up to 30% in TP53/RB null cells. This finding is now being quantitated with a clonal evolution model and genotype-phenotype associations are being validated using functional transformation and invasion assays. Further investigation is now required to assess this relationship on the background of existing genomic alterations relevant to prostate and other cancers and also characterise the role of hypoxia in driving early metastatic spread. Supported by core funding from Cancer Research UK Manchester Institute and grants within the Cancer Research UK Manchester RadNet Unit, NIHR Manchester Biomedical Research Centre and a Prostate Cancer UK-Movember Centre of Excellence award. Citation Format: Robert G. Bristow. Genomic instability and hypoxia as co-drivers of prostate cancer aggression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY37-01.

Prostate cancer is the most commonly diagnosed cancer in the UK. Increased levels of prostate hypoxia have been shown to drive tumor aggression as an adverse prognostic factor following surgery or radiotherapy, especially when combined with tumor genetic instability. Our recent collaborative bioinformatic studies (Bhandari et al, Nat. Genetics 2019; Nat. Communications 2020) have shown various copy number changes to be enriched in hypoxic tumors, with mono- or bi-allelic loss of the PTEN being the most significant. To delineate a direct relationship between hypoxia and PTEN loss, we interrogated the impact of chronic hypoxia (1% O2, 0.2% O2; 72 h) on PTEN oncogenic signaling and asked whether this translates to improved fitness and survival in non-transformed and transformed PTEN isogenic cell lines. Despite increased oncogenic signaling (increased phospho-AKT) of PTEN-null 22Rv1 cells and hTERT-immortalized prostate epithelial cells (GHM) under chronic hypoxic gassing conditions, this did not lead to increased adaption and tumor clone survival. Differential survival for the PTEN isogenic cell lines following prostate cancer therapies (i.e. IR, PARPi, cDDP, taxols) under oxia and hypoxia are underway to further this work and determine a potential translational importance to clinical treatment. Independent evidence also points to hypoxia/PTEN loss in deregulating centrosome biology as a potential mechanism for genetic instability under hypoxia. Hence, we also hypothesized that the relationship between PTEN and hypoxia might be unearthed if aberrant unstable mitoses following deregulation of spindle poles under hypoxia selectively adapt and have a survival advantage. Quantitative immunofluorescence of centrosome aberrations, using antibodies to phospho-Histone H3 and Centrin-1, show significant increases in centriole numbers (p < 0.05) in mitotic cells under chronic hypoxic conditions and an increase in the incidence of micronuclei (p < 0.005), specifically in chronic hypoxic cells with PTEN loss, supporting our hypothesis. Current studies are investigating in situ staining of centrosomes as a readout of chromosomal instability (CIN) in primary prostate cancer tissues with variable PTEN status as a new biomarker for aggressiveness to link hypoxia-associated genetic instability with novel clinical trial development. (Funded by Cancer Research UK as a core grant to RGB). Citation Format: Alexandru Suvac, Richard Rebello, Stephen Lyons, Robert G. Bristow. Understanding the role of hypoxia and PTEN loss in driving prostate cancer progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-035.

Prostate cancer (PCa) is the 2nd most prevalent cancer in men and has poor patient outcome upon transition from localized disease to the castration-resistant and metastatic state. Numerous studies have indicated intratumoral hypoxia as a key driver in disease progression across multiple cancers and in prostate cancer. Hypoxia is associated with local recurrence, metastatic capacity, androgen independence and neuroendocrine differentiation (Ashton and Bristow, Brit.J.Rad, 2020). Crucially, among localized PCa patients, hypoxia and genomic instability have an independent and additive effect on biochemical relapse and bioinformatic studies suggest that hypoxia co-segregates mutation signatures representing defects in homologous recombination (HR) and mismatch repair (MMR) DNA repair pathways (Bhandari et al., Nat Comms, 2020). In order to investigate whether hypoxia directly drives genome instability in prostate cancer, mechanistic studies using hTERT-immortalized, primary prostate epithelial cells isogenic for c-MYC, BRCA2, or TP53/RB (SV40-transduced as PNT2 cells) were studied as to the relationship between hypoxia, inherent versus acquired genetic instability, and DNA repair capacity in-vitro and in-situ. Chronic hypoxia (20 generation doublings at 0.2% O2) was sufficient to drive increased copy number alterations (CNAs; based on low-pass whole genome sequencing - WGS) in selected clones by up to 30% in TP53/RB null cells, but not hTERT immortalized models. This finding is now being quantitated within a clonal evolution model and genotype-phenotype associations are being validated using functional transformation and invasion assays. Associated mechanistic studies have revealed that the hypoxic suppression of homologous recombination (HR) factor transcript and protein abundance (e.g up to ~60% reduction of RAD51/BRCA2 in PC3, DU145 and PNT2 cells) and associated with reduced RAD51 nuclear foci induction or defective DR-GFP reconstitution, is highly-dependent on cell density. This suggests that reduced HR and MMR repair capacity may exist in situ within hypoxic tumor oxygen gradients with close cell-cell interactions; in situ studies of RAD51 and other DNA repair proteins across hypoxic gradients in primary human tumors will be presented. These translational studies will accompany the HYPROGEN trial in Manchester in which patients who present with treatment-naïve M1 bone metastatic disease are given oral pimonidazole prior to prostate and bone metastasis biopsies to directly study genomic instability (using WGS) and hypoxia (pimonidazole staining and RNA hypoxic signatures) to understand these relationships in paired primary-metastatic samples. This data will provide a mechanistic basis for the adverse prognosis seen in patients whose tumors have both hypoxia and genome instability, as well as fresh evidence for stratification and treatment intensification of localized PCa patients with hypoxic tumors. (Supported by a core grant from Cancer Research UK to RGB) Citation Format: Jack Ashton, Richard Rebello, Stephen Lyons, Robert G. Bristow. Mechanistic studies of hypoxia as a driver of genomic instability in prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-032.

Many primary tumor sub-regions have low levels of molecular oxygen, termed hypoxia. Hypoxic tumors are at elevated risk for local failure and distant metastasis, but the molecular hallmarks of tumor hypoxia remain poorly defined. To fill this gap, we quantified hypoxia in 8,006 tumors across 19 tumor types. In ten tumor types, hypoxia was associated with elevated genomic instability. In all 19 tumor types, hypoxic tumors exhibited characteristic driver mutation signatures. We observed widespread hypoxia-associated dysregulation of miRNAs across cancers and functionally validated miR-133a-3p as a hypoxia-modulated miRNA. In localized prostate cancer, hypoxia was associated with elevated rates of chromothripsis, allelic loss of PTEN and shorter telomeres. These associations are particularly enriched in polyclonal tumors, representing a constellation of features resembling tumor nimbosus – an aggressive cellular phenotype. Overall, this work establishes that tumor hypoxia may drive aggressive molecular features across cancers and shape the clinical trajectory of individual tumors. Citation Format: Vinayak Bhandari, Christianne Hoey, Lydia Liu, Emilie Lalonde, Jessica Ray, Julie Livingstone, Robert Lesurf, Yu-Jia Shiah, Tina Vujcic, Xiaoyong Huang, Shadrielle M.G. Espiritu, Lawrence E. Heisler, Fouad Yousif, Vincent Huang, Takafumi N. Yamaguchi, Cindy Q. Yao, Veronica Y. Sabelnykova, Michael Fraser, Melvin L.K. Chua, Theodorus van der Kwast, Stanley K. Liu, Paul C. Boutros, Robert G. Bristow. Molecular landmarks of tumor hypoxia across cancer types [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-018.

Understanding the mechanistic basis for transition to late stage aggressive disease is vital for both assigning patient risk status in the localized setting and also identifying novel treatment strategies to prevent progression. Subregions of intratumoral hypoxia are found in all solid tumors and are associated with many biologic drivers of tumor progression. Crucially, more recent findings show the co-presence of hypoxia and genomic instability can confer a uniquely adverse prognosis. In-depth informatic and functional studies suggests a role for hypoxia in co-operating with oncogenic drivers (e.g. loss of PTEN) and suppressing DNA repair capacity to alter clonal adaption and evolution and driving an aggressive mutator phenotype. More specifically, hypoxic suppression of homologous recombination represents a "contextual lethal" vulnerability in hypoxic prostate tumors which could extend the application of existing DNA repair targeting agents such as poly-ADP ribose polymerase inhibitors. Further investigation is now required to assess this relationship on the background of existing genomic alterations relevant to prostate and other cancers, and also characterize the role of hypoxia in driving early metastatic spread. Supported by core funding from Cancer Research UK Manchester Institute and grants within the Cancer Research UK Manchester RadNet Unit, NIHR Manchester Biomedical Research Centre and a Prostate Cancer UK-Movember Centre of Excellence award. Citation Format: Robert G. Bristow. Genomic architecture and targets in hypoxic tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-001.

Gold nanoparticles (AuNPs) and cisplatin have been explored in concomitant chemoradiotherapy, wherein they elicit their effects by distinct and overlapping mechanisms. Cisplatin is one of the most frequently utilized radiosensitizers in the clinical setting; however, the therapeutic window of cisplatin-aided chemoradiotherapy is limited by its toxicity. The goal of this study was to determine whether AuNPs contribute to improving the treatment response when combined with fractionated cisplatin-based chemoradiation in both in vitro and in vivo models of triple-negative breast cancer (MDA-MB-231Luc+). Cellular-targeting AuNPs with receptor-mediated endocytosis (AuNP-RME) in vitro at a noncytotoxic concentration (0.5 mg/ml) or cisplatin at IC25 (12 μM) demonstrated dose enhancement factors (DEFs) of 1.25 and 1.14, respectively; the combination of AuNP-RME and cisplatin resulted in a significant DEF of 1.39 in vitro. Transmission electron microscopy (TEM) images showed effective cellular uptake of AuNPs at tu...

Background/aim Carbonic anhydrase IX (CA9) catalyses the interconversion of carbon dioxide to carbonic acid and bicarbonate and is considered a putative biomarker of tumour hypoxia. We set out to evaluate the prognostic significance of CA9 in prostate cancer. Patients and methods Plasma samples were assessed from 68 men with high-risk localised prostate cancer treated with radical prostatectomy (RP) or radiotherapy (RT), and 20 men with castration-resistant prostate cancer (CRPC) treated with docetaxel chemotherapy between 2010 and 2012 at the Princess Margaret Cancer Centre, Canada. Results Of the 68 patients with high-risk localised prostate cancer, 57 underwent RP and 11 underwent RT. Baseline CA9 was not associated with recurrence or prostate-specific antigen in either group (p=0.98 and 0.20, respectively). CA9 levels before chemotherapy correlated with overall survival (r=-0.37; two-sided p=0.11). Conclusion Baseline CA9 in men with CRPC may portend a more aggressive prostate cancer phenotype with poorer survival.

BACKGROUND The role of hormone therapy (HT) with dose-escalated external-beam radiotherapy (DE-EBRT) in the treatment of intermediate-risk prostate cancer (IRPC) remains controversial. The authors report the long-term outcome of a phase 3 study of DE-EBRT with or without HT for patients with localized prostate cancer (LPC). METHODS From 1999 to 2006, 252 of an intended 338 patients with LPC were randomized to receive DE-EBRT with or without 5 months of neoadjuvant and concurrent bicalutamide 150 mg once daily. The study was closed early because of contemporary concerns surrounding bicalutamide. The primary outcome was biochemical failure (BF) incidence, and the secondary endpoints were overall survival (OS), local control (LC), and quality of life. The BF and OS rates were estimated using the cumulative incidence function and Kaplan-Meier methods and were compared using the Gray test and the log-rank test. RESULTS Eleven patients were excluded the from analysis. Characteristics were well balanced in each treatment arm. Ninety-five percent of patients had IRPC. The prescribed dose increased from 75.6 grays (Gy) in 42 fractions to 78 Gy in 39 fractions over the period. At a median follow-up of 9.1 years, 98 BFs occurred, with no significant effect of HT versus no HT on the BF rate (40% vs 47%; P = .32), the OS rate (82% vs 86%; P = .37), the LC rate (52% vs 48 %; P = .32) or quality of life, in the patients who completed the questionnaires. Dose escalation to 75.6 Gy versus >75.6 Gy reduced the BF rate by 26% (P = .004). CONCLUSIONS For patients who predominantly have IRPC, the addition of HT to DE-EBRT did not significantly affect BF, OS, or LC. Bicalutamide appeared to be well tolerated. The conclusions from the study are limited by incomplete recruitment. Cancer 2016. © 2016 American Cancer Society.

Prostate cancer (PCa) is a clinically heterogeneous disease and has poor patient outcome when tumours progress to castration-resistant and metastatic states. Understanding the mechanistic basis for transition to late stage aggressive disease is vital for both assigning patient risk status in the localised setting and also identifying novel treatment strategies to prevent progression. Subregions of intratumoral hypoxia are found in all solid tumours and are associated with many biologic drivers of tumour progression. Crucially, more recent findings show the co-presence of hypoxia and genomic instability can confer a uniquely adverse prognosis in localised PCa patients. In-depth informatic and functional studies suggests a role for hypoxia in co-operating with oncogenic drivers (e.g. loss of PTEN) and suppressing DNA repair capacity to alter clonal evolution due to an aggressive mutator phenotype. More specifically, hypoxic suppression of homologous recombination represents a “contextual lethal“ vulnerability in hypoxic prostate tumours which could extend the application of existing DNA repair targeting agents such as poly-ADP ribose polymerase inhibitors. Further investigation is now required to assess this relationship on the background of existing genomic alterations relevant to PCa, and also characterise the role of hypoxia in driving early metastatic spread. On this basis, PCa patients with hypoxic tumours can be better stratified into risk categories and treated with appropriate therapies to prevent progression.

Introduction Lung cancer is the most common cause of cancer-related death. Carcinogenic and endogenous processes driving somatic mutation acquisition in cancer can be extracted and defined as mutational signatures using whole genome sequencing (WGS). Tobacco smoke is the main aetiological cause of lung cancer, with mutational signature 4 representing the characteristic C>A transversions produced by smoking. Whilst smoking cessation has been shown to reduce lung cancer risk in epidemiological studies, there has been little exploration into the persistence of smoking 4 in NSCLC genomes after a patient has quit smoking. We investigated the extent and persistence of signature 4 in NSCLC genomes of current, ex- and never-smokers, correlating in particular with clinical history of smoking cessation. Methods 132 NSCLC samples were resected from 131 patients in Greater Manchester. These samples were submitted to the 100,000 Genomes Project (Genomics England). WGS was performed on tumour specimens and matched blood samples. Data generated was processed by a standard pipeline devised by Genomics England. Tumour mutational burden (TMB), mutational signatures and copy number variation (CNV) were obtained. Clinical data collected included: smoking status, date of diagnosis, TNM stage, date of relapse and date of death (where relevant). Fisher's exact tests and Kruskal-Wallis tests were used for statistical comparisons, with Kaplan-Meier plots for survival. Results Signature 4 was associated with a smoking history in 102/119 (85.7%) NSCLCs with a detailed smoking history available. In 17/119 (14.3%) patients with a smoking history but no signature 4 NSCLC, 15/17 (88.2%) patients quit smoking a median of 22 years ago (range 0.006 - 45 years). 6/7 (85.7%) never-smoker NSCLCs were non-signature 4 NSCLCs. 60/75 (80%) ex-smokers had sufficient smoking data to assess signature 4 persistence. Signature 4 endured in the lung tissue prior to tumour diagnosis for a median of 180 months (15 years) (range 1 - 600 months). There was no association between the time of smoking cessation and the time to NSCLC diagnosis (R2=0.0009, p=0.82). Non-signature 4 NSCLCs had a more diverse signature profile (signature 4: mean 4.36, 95% CI 4.13-4.58; non-signature 4: mean 5.52, 95% CI 4.95-6.09; p= Conclusion The genomic alterations introduced by smoking persist for many years after smoking cessation. NSCLCs arising from smoking carry a distinctive identity compared to those from never-smokers, with higher TMBs driven primarily by signature 4. Whilst survival analysis is limited in this cohort, the pervasive contributions from smoking suggest that lung cancer screening programmes should include all patients with a smoking history. Citation Format: Pantelis A. Nicola, Shereen Rafee, George Burghel, Andrew Wallace, Helene Schlecht, Eleanor Baker, Katie Baker, Lynsey Priest, Mathew Carter, Sharzad Moghadam, Jane Rogan, Robert G. Bristow, William Newman, Fiona H. Blackhall, Colin Lindsay. Persistence of smoking signature 4 in the non-small cell lung cancer genome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3810.

Introduction: Kirsten rat sarcoma viral oncogene (KRAS) is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), occurring in approximately 30% of cases. Recent work suggests KRAS -mutant (KRASm) NSCLC is a microcosm for diverse immune checkpoint inhibitor responses, partly dictated by its co-mutation with TP53 (“KP” group=~1/3 cases, “immune hot”), STK11 (“KL” group=~1/3 cases, “immune cold”) or CDKN2A (“KC” group=~1/3 cases). Here we identify and describe genomic and clinical associations of a further subset of KRASm NSCLC defined by “triple WT” status for common KRASm co-mutations. Method: Sequencing results from 364 early-stage NSCLC cases from the Cancer Research UK TRACERx program (whole-exome sequencing) and the Genomics England 100,000 genome program (whole-genome sequencing) were analyzed for KRASm, associated co-mutations in TP53, STK11 and CDKN2A, copy number changes in the RAS-RAF-MEK-ERK axis, tumor mutational burden (TMB) and mutational signatures. Clinical demographics and factors including tumor size, nodal status and stage were ascertained and assessed for statistical associations with sequencing data using the Mann-Whitney and Fisher’s exact tests. Results: Overall, 143/364 lung cancer cases (39%) were KRASm with 65/143 (45%) tumors identified as “KP,” 26/143 (18%) as “KL,” and 3/143 (2%) as “KC.” There were 49/143 KRASm cases (34%) with no co-mutation in TP53, STK11 or CDKN2A, defined as “triple wild-type” (triple WT). Relative to the KP, KL and KC genotypes, there was a positive association of the KRAS G12D mutation with triple WT status: 10/49 cases, 20%; KP/KL/KC: 6/94 cases, 6%; p=0.022). In the 100,000-genome cohort, TMB was similar for the triple WT and KL groups but significantly lower than that observed for KP (KP: median 10.55 mut/Mb, 95% CI 8.8-15.33; triple WT: median 6.96, 95% CI 4.87-12.53; p=0.036). Despite this lower median TMB, smoking-associated mutational signature 4 was common in triple WT tumors (triple WT: 13/14 pts, 93%; overall 76/114 pts, 67%). In the triple WT group overall, there was a reduction of >50% in cancers with copy number changes of NF1 and NRAS. There was an inverse association between pathologic stage III tumors and KRASm triple WT genotype (p=0.0014). Conclusion: There are a significant proportion of KRASm NSCLC patients whose tumors are triple WT for TP53, STK11 and CDKN2A. Despite their high frequency of smoking-associated mutational signatures, these tumors are characterized by low TMB and are more common in early-stage disease. They also associate more commonly with KRAS G12D. Our observations suggest a discrete KRASm subset that may have implications for stratification in trials of immune checkpoint inhibitors and/or targeted therapeutics of KRASm tumors. Citation Format: Colin R. Lindsay, Pantelis Nicola, Mariam Jamal-Hanjani, Andrew Wallace, Gareth Wilson, George Burghel, Helene Schlecht, Katie Baker, Eleanour Baker, Lynsey Priest, Jane Rogan, Sharzad Moghadam, Mathew Carter, Caroline Dive, Robert G. Bristow, Charles Swanton, William Newman, Fiona Blackhall. “Triple wild-type” co-mutational profile in early-stage KRAS-mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B49.

Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients., Neuroendocrine prostate cancer (NEPC) is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, resulting in resistance to AR-targeted therapy. Here they report ONECUT2 to drive NEPC tumorigenesis via regulation of hypoxia signaling and tumor hypoxia, and find hypoxia directed therapy to be effective in NEPC.

Purpose: Hypoxia is associated with poor prognosis and is predictive of poor response to cancer treatments, including radiotherapy. Developing noninvasive biomarkers that both detect hypoxia prior to treatment and track change in tumor hypoxia following treatment is required urgently. Experimental Design: We evaluated the ability of oxygen-enhanced MRI (OE-MRI) to map and quantify therapy-induced changes in tumor hypoxia by measuring oxygen-refractory signals in perfused tissue (perfused Oxy-R). Clinical first-in-human study in patients with non–small cell lung cancer (NSCLC) was performed alongside preclinical experiments in two xenograft tumors (Calu6 NSCLC model and U87 glioma model). Results: MRI perfused Oxy-R tumor fraction measurement of hypoxia was validated with ex vivo tissue pathology in both xenograft models. Calu6 and U87 experiments showed that MRI perfused Oxy-R tumor volume was reduced relative to control following single fraction 10-Gy radiation and fractionated chemoradiotherapy (P < 0.001) due to both improved perfusion and reduced oxygen consumption rate. Next, evaluation of 23 patients with NSCLC showed that OE-MRI was clinically feasible and that tumor perfused Oxy-R volume is repeatable [interclass correlation coefficient: 0.961 (95% CI, 0.858–0.990); coefficient of variation: 25.880%]. Group-wise perfused Oxy-R volume was reduced at 14 days following start of radiotherapy (P = 0.015). OE-MRI detected between-subject variation in hypoxia modification in both xenograft and patient tumors. Conclusions: These findings support applying OE-MRI biomarkers to monitor hypoxia modification, to stratify patients in clinical trials of hypoxia-modifying therapies, to identify patients with hypoxic tumors that may fail treatment with immunotherapy, and to guide adaptive radiotherapy by mapping regional hypoxia.

Purpose Neoadjuvant radiation therapy (RT) improves disease control in various cancers and has become an established oncologic treatment strategy. During 2001 to 2004, we conducted a phase 1 pilot study assessing the role of short-course preoperative RT (PreORT) for men with unfavorable intermediate- and high-risk localized prostate cancer. Herein, we present long-term follow-up toxicity and oncologic outcomes. Methods and Materials Eligible patients had histologically proven prostate cancer, cT1-T2N0M0 disease, prostate-specific antigen >15 to 35 ng/mL regardless of Gleason score, or prostate-specific antigen 10 to 15 ng/mL with Gleason score ≥7. Patients received 25 Gy in 5 consecutive daily fractions (5 Gy per fraction) to the prostate only, followed by radical prostatectomy within 14 days after RT completion. Primary outcomes were intraoperative morbidity and late genitourinary (GU) and gastrointestinal toxicities. Results In total, 15 patients were enrolled; 14 patients completed PreORT followed by radical prostatectomy, which also included bilateral lymph node dissections in 13 cases. Median follow-up was 12.2 years (range, 6.7-16.3). Late GU toxicity was common, with 2 patients (13.3%) experiencing G2 toxicity and 6 patients (40%) G3 toxicity. There were no patients with G4 to G5 late GU toxicity. Late gastrointestinal toxicity was infrequent, with only 1 patient (6.7%) experiencing transient G2 proctitis. At last follow-up, 8 (53.3%) and 6 (40%) patients experienced biochemical and metastatic disease recurrence, respectively. Conclusions The use of PreORT in men with high-risk prostate cancer is associated with unexpected high rates of late GU toxicity. Future studies examining the role of RT preradical prostatectomy must cautiously select RT technique and dose schedule. Importantly, long-term follow-up data are essential to fully determine the therapeutic index of PreORT in the management of localized disease.

Hypoxia is a generic micro-environmental factor in most solid tumours. While most published literature focused on in vitro or single tumour type investigations, we carried out the first multi-omics pan cancer analysis of hypoxia with the aim of gaining a comprehensive understanding of its implication in tumour biology. A core set of 52 mRNAs were curated based on experimentally validated hypoxia gene sets from multiple cancer types. The 52 mRNAs collectively stratified high- and low-hypoxia tumours from The Cancer Genome Atlas (TCGA) database (9698 primary tumours) in each of the 32 cancer types available. High- hypoxia tumours had high expression of not only mRNA but also protein and microRNA markers of hypoxia. In a pan cancer transcriptomic analysis, ≥70% of the known cancer hallmark pathways were enriched in high-hypoxia tumours, most notably epithelial mesenchymal transition potential, proliferation (G2M checkpoint, E2F targets, MYC targets) and immunology response. In a multi-omics analysis, gene expression-determined high- hypoxia tumours had a higher non-silent mutation rate, DNA damage repair deficiency and leukocyte infiltration. The associations largely remained significant after correcting for confounding factors, showing a profound impact of hypoxia in tumour evolution across cancer types. High-hypoxia tumours determined using the core gene set had a poor prognosis in 16/32 cancer types, with statistical significances remaining in five after adjusting for tumour stage and omics biomarkers. In summary, this first comprehensive in vivo map of hypoxia in cancers highlights the importance of this micro-environmental factor in driving tumour progression.

BACKGROUND: The Hedgehog (Hh) signaling pathway is active in esophageal adenocarcinoma (EAC). We used a patient-derived murine xenograft (PDX) model of EAC to evaluate tumour response to conventional treatment with radiation/chemoradiation with or without Hh inhibition. Our goal was to determine the potential radioresistance effects of Hh signaling and radiosensitization by Hh inhibitors.METHODS: PDX models were treated with radiation, chemotherapy or combined chemoradiation. Tumour response was measured by growth delay. Hh transcript levels (qRT-PCR) were compared among frozen tumours from treated and control mice. 5E1, a monoclonal SHH antibody, or LDE225, a clinical SMO inhibitor (Novartis®) inhibited Hh signaling.RESULTS: Precision irradiation significantly delayed xenograft tumour growth in all 7 PDX models. Combined chemoradiation further delayed growth relative to either modality alone in three of six PDX models. Following irradiation, two of three PDX models demonstrated sustained up-regulation of Hh transcripts. Combined LDE225 and radiation, and 5E1 alone delayed growth relative to either treatment alone in a Hh-responsive PDX model, but not in a non-responsive model.CONCLUSION: Hh signaling mediates the radiation response in some EAC PDX models, and inhibition of this pathway may augment the efficacy of radiation in tumours that are Hh dependent.

Many primary-tumor subregions have low levels of molecular oxygen, termed hypoxia. Hypoxic tumors are at elevated risk for local failure and distant metastasis, but the molecular hallmarks of tumor hypoxia remain poorly defined. To fill this gap, we quantified hypoxia in 8,006 tumors across 19 tumor types. In ten tumor types, hypoxia was associated with elevated genomic instability. In all 19 tumor types, hypoxic tumors exhibited characteristic driver-mutation signatures. We observed widespread hypoxia-associated dysregulation of microRNAs (miRNAs) across cancers and functionally validated miR-133a-3p as a hypoxia-modulated miRNA. In localized prostate cancer, hypoxia was associated with elevated rates of chromothripsis, allelic loss of PTEN and shorter telomeres. These associations are particularly enriched in polyclonal tumors, representing a constellation of features resembling tumor nimbosus, an aggressive cellular phenotype. Overall, this work establishes that tumor hypoxia may drive aggressive molecular features across cancers and shape the clinical trajectory of individual tumors.

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials. Postprint

Solid tumors contain regions of chronic and cyclic hypoxia. Chronic hypoxia can downregulate RAD51 and sensitize cells to PARP inhibition. Herein, we show that RAD51 expression, cell survival and toxicity to PARP inhibition is not affected under cyclic hypoxic conditions. This suggests that PARP inhibition may be selectively toxic in tumor sub-regions associated with chronic hypoxia.

Androgen suppression mediates transcriptional downregulation of DNA repair genes. Stimulation with supraphysiologic levels of dihydrotestosterone induces formation of lethal DNA breaks through recruitment of topoisomerase II enzymes to fragile DNA sites. Bipolar castration and stimulation that contributes to increasing DNA damage represents a novel strategy of sensitizing prostate cancer to cytotoxic therapies, including radiotherapy. Clin Cancer Res; 22(13); 3124–6. ©2016 AACR. See related article by Hedayati et al., p. 3310

Background Invasive cribriform and intraductal carcinoma (CR/IDC) is associated with adverse outcome of prostate cancer patients. The aim of this study was to determine the molecular aberrations associated with CR/IDC in primary prostate cancer, focusing on genomic instability and somatic copy number alterations (CNA). Methods Whole-slide images of The Cancer Genome Atlas Project (TCGA, N = 260) and the Canadian Prostate Cancer Genome Network (CPC-GENE, N = 199) radical prostatectomy datasets were reviewed for Gleason score (GS) and presence of CR/IDC. Genomic instability was assessed by calculating the percentage of genome altered (PGA). Somatic copy number alterations (CNA) were determined using Fisher-Boschloo tests and logistic regression. Primary analysis were performed on TCGA (N = 260) as discovery and CPC-GENE (N = 199) as validation set. Results CR/IDC growth was present in 80/260 (31%) TCGA and 76/199 (38%) CPC-GENE cases. Patients with CR/IDC and ≥ GS 7 had significantly higher PGA than men without this pattern in both TCGA (2.2 fold; p = 0.0003) and CPC-GENE (1.7 fold; p = 0.004) cohorts. CR/IDC growth was associated with deletions of 8p, 16q, 10q23, 13q22, 17p13, 21q22, and amplification of 8q24. CNAs comprised a total of 1299 gene deletions and 369 amplifications in the TCGA dataset, of which 474 and 328 events were independently validated, respectively. Several of the affected genes were known to be associated with aggressive prostate cancer such as loss of PTEN, CDH1, BCAR1 and gain of MYC. Point mutations in TP53, SPOP and FOXA1were also associated with CR/IDC, but occurred less frequently than CNAs. Conclusions CR/IDC growth is associated with increased genomic instability clustering to genetic regions involved in aggressive prostate cancer. Therefore, CR/IDC is a pathologic substrate for progressive molecular tumour derangement. Electronic supplementary material The online version of this article (10.1186/s12885-017-3976-z) contains supplementary material, which is available to authorized users.

BACKGROUND: Clinical grading systems using clinical features alongside nomograms lack precision in guiding treatment decisions in prostate cancer (PCa). There is a critical need for identification of biomarkers that can more accurately stratify patients with primary PCa. OBJECTIVE: To identify a robust prognostic signature to better distinguish indolent from aggressive prostate cancer (PCa). DESIGN, SETTING, AND PARTICIPANTS: To develop the signature, whole-genome and whole- transcriptome sequencing was conducted on five PCa patient-derived xenograft (PDX) models collected from independent foci of a single primary tumor and exhibiting variable metastatic phenotypes. Multiple independent clinical cohorts including an intermediate-risk cohort were used to validate the biomarkers. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The outcome measurement defining aggressive PCa was metastasis following radical prostatectomy. A generalized linear model with lasso regularization was used to build a 93-gene stroma-derived metastasis signature (SDMS). The SDMS association with metastasis was assessed using a Wilcoxon rank-sum test. Performance was evaluated using the area under the curve (AUC) for the receiver operating characteristic, and Kaplan-Meier curves. Univariable and multivariable regression models were used to compare the SDMS alongside clinicopathological variables and reported signatures. AUC was assessed to determine if SDMS is additive or synergistic to previously reported signatures. RESULTS AND LIMITATIONS: A close association between stromal gene expression and metastatic phenotype was observed. Accordingly, the SDMS was modeled and validated in multiple independent clinical cohorts. Patients with higher SDMS scores were found to have worse prognosis. Furthermore, SDMS was an independent prognostic factor, can stratify risk in intermediate-risk PCa, and can improve the performance of other previously reported signatures. CONCLUSIONS: Profiling of stromal gene expression led to development of an SDMS that was validated as independently prognostic for the metastatic potential of prostate tumors. PATIENT SUMMARY: Our stroma-derived metastasis signature can predict the metastatic potential of early stage disease and will strengthen decisions regarding selection of active surveillance versus surgery and/or radiation therapy for prostate cancer patients. Further-more, profiling of stroma cells should be more consistent than profiling of diverse cellular populations of heterogeneous tumors.