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Patient demographic of low and high quartiles of HMOX1 expressing tumors from the METABRIC dataset

Purpose: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting immune checkpoint molecules. This preclinical study investigates heme oxygenase-1 (HO-1), an immunosuppressive enzyme that is expressed in a wide variety of cancers, as a potential immune checkpoint target in the context of a chemotherapy-elicited antitumor immune response. We evaluate repurposing tin mesoporphyrin (SnMP), which has demonstrated safety and efficacy targeting hepatic HO in the clinic for the treatment of hyperbilirubinemia, as an immune checkpoint blockade therapy for the treatment of cancer.Experimental Design: SnMP and genetic inactivation of myeloid HO-1 were evaluated alongside 5-fluorouracil in an aggressive spontaneous murine model of breast cancer (MMTV-PyMT). Single-cell RNA sequencing analysis, tumor microarray, and clinical survival data from breast cancer patients were used to support the clinical relevance of our observations.Results: We demonstrate that SnMP inhibits immune suppression of chemotherapy-elicited CD8+ T cells by targeting myeloid HO-1 activity in the tumor microenvironment. Microarray and survival data from breast cancer patients reveal that HO-1 is a poor prognostic factor in patients receiving chemotherapy. Single-cell RNA-sequencing analysis suggests that the myeloid lineage is a significant source of HO-1 expression, and is co-expressed with the immune checkpoints PD-L1/2 in human breast tumors. In vivo, we therapeutically compare the efficacy of targeting these two pathways alongside immune-stimulating chemotherapy, and demonstrate that the efficacy of SnMP compares favorably with PD-1 blockade in preclinical models.Conclusions: SnMP could represent a novel immune checkpoint therapy, which may improve the immunological response to chemotherapy. Clin Cancer Res; 24(7); 1617–28. ©2018 AACR.

Supplementary Figure S1â",HO-1 expression in MMTV-PyMT tumors; Supplementary Figure S2â",Effects of SnMP and chemotherapy combination therapy; Supplementary Figure S3â",HO-1+ TAMs in murine models of breast cancer; Supplementary Figure S4â",Genetic inactivation and therapeutic inhibition of myeloid HO-1 in the MMTV-PyMT model; Supplementary Figure S5â",HO-1 in human cancer; Supplementary Figure S6â",tSNE clustering identifies stromal and epithelial cell types from breast cancer patient single-cell RNA sequencing data; Supplementary Figure 7â", Single cell RNA-Seq gene expression for checkpoint molecules grouped by inferred cell type; Supplementary Figure S8â",HO-1 protein in myeloid cells in human cancer; Supplementary Figure S9â", Comparative efficacies of therapeutically targeting HO-1 and PD-1 alongside paclitaxel treatment in 4T1 tumors; Supplementary Figure S10â",Improving checkpoint blockade therapy using SnMP.

Metastatic and high-risk localized prostate cancer respond to hormone therapy but outcomes vary. Following a pre-specified statistical plan, we used Cox models adjusted for clinical variables to test associations with survival of multi-gene expression-based classifiers from 781 patients randomized to androgen deprivation with or without abiraterone in the STAMPEDE trial. Decipher score was strongly prognostic (p-5) and identified clinically-relevant differences in absolute benefit, especially for localized cancers. In metastatic disease, classifiers of proliferation, PTEN or TP53 loss and treatment-persistent cells were prognostic. In localized disease, androgen receptor activity was protective whilst interferon signaling (that strongly associated with tumor lymphocyte infiltration) was detrimental. Post-Operative Radiation-Therapy Outcomes Score was prognostic in localized but not metastatic disease (interaction p=0.0001) suggesting the impact of tumor biology on clinical outcome is context-dependent on metastatic state. Transcriptome-wide testing has clinical utility for advanced prostate cancer and identified worse outcomes for localized cancers with tumor-promoting inflammation.

5076 Background: Treatment intensification with docetaxel or abiraterone improved survival for advanced prostate cancer starting androgen deprivation therapy (ADT) in the Systemic Therapy in Advancing or Metastatic Prostate Cancer: Evaluation of Drug Efficacy (STAMPEDE, NCT00268476) trial. However, survival and time-to-progression is highly variable on ADT, introducing the risk of unnecessary toxicity from additional treatments for some patients. Here we test the prognostic association of proliferation index using Ki67 scores in the control arm of the STAMPEDE population of high-risk localised (M0) and metastatic (M1) prostate cancer. Methods: Pre-ADT diagnostic needle biopsies were obtained from 517 men randomized in STAMPEDE arm A between 2006 and 2015. These were assessed for proliferation using an analytically optimised Ki67 immunohistochemistry assay. Ki67 was tested for associations with baseline clinico-pathological variables (Grade group, pre-ADT serum PSA and imaging metastatic burden) in univariable linear-regression models, and for associations with survival outcomes in multivariable Cox-regression models adjusted for these and additional confounding variables. Primary outcome measure was overall survival, secondary outcomes were prostate cancer-specific, failure-free, progression-free and metastatic progression-free survival. Results: Ki67 was available for 475 patients who received ADT only for at least 2 years ± radiotherapy. Of 202 M0, 74 were node positive. Of 273 M1, 116, 127 and 30 were respectively low, high and unknown radiological M1 volume. Ki67 score associated with higher Gleason (p=7.15x10-11) and presence of extra-pelvic metastases (p=1.41x10-8). Increasing Ki67 scores showed a strong linear association with poorer overall survival, with an estimated 2% increase in the hazard of death per percentage increase in the score (adjusted HR=1.02, 95% CI 1.01-1.02; p=1.04x10-5). There was also strong evidence that Ki67 associated positively with all secondary outcomes, including prostate cancer-specific survival (adjusted p=5.50x10-6) and metastatic progression-free survival (adjusted p=3.50x10-9). Conclusions: Ki67 immuno-score is strongly prognostic in clinically advanced prostate cancer independent of Gleason score and the other clinicopathological variables tested in this study. Ki67 is a clinically scalable assay that could improve selection for treatment intensification and provide a tool for screening patients most likely to benefit from further molecular investigation.

5021 Background: Men with advanced hormone-sensitive prostate cancer (HSPC) starting long term androgen deprivation therapy (ADT) follow a highly variable clinical course. Treatment intensification with docetaxel or AR targeted therapies improves outcomes but there is a risk of overtreatment, especially in non-metastatic (M0) or metastatic (M1) low volume disease. We established a framework for biomarker evaluation in the STAMPEDE trial. We aimed to evaluate the feasibility and clinical utility of assessing the burden of copy number (CN) aberrations in newly diagnosed advanced HSPC. We hypothesised that increased percentage genome altered (PGA) would associate with higher disease burden and worse prognosis. Methods: We implemented a scalable strategy using low coverage whole genome sequencing (lpWGS) of formalin fixed paraffin embedded (FFPE) diagnostic core biopsies from STAMPEDE participants randomised to the standard of care ADT arm, between 2005 and 2016. Tissue was retrieved from 136 trial sites. 315 cases were randomly selected, aiming for a biomarker population of 300, anticipating an assay failure rate ̃5%. We defined 40% as the minimum histopathologically determined tumor cellularity (TC) for inclusion. We performed a survival analysis investigating PGA at diagnosis as a continuous measure with fractional polynomial specification in Cox models adjusting for disease burden, Gleason grade, pre-ADT PSA (log-transformed), age at randomisation and TC. We pre-specified that all hypothesis tests required evidence at the 5% significance level to consider rejecting the null hypothesis. Results: We successfully CN profiled 300/315 cases. There were no significantly different baseline clinico-pathological features between the full trial comparison n = 3106 and final biomarker population n = 300, 290/300 cases were de novo presentations. PGA in the core with highest Gleason grade and TC was median 18% (range 0%-75%; n = 300). PGA was significantly higher in M1 (n = 169) compared to M0 (n = 131) cases (median: 21% vs 14%; p = 0.00006). 284/300 were subclassified by disease burden into M0 node negative and node positive, and M1 low and high volume. PGA was significantly associated with increased disease burden (p = 0.00002). Increased PGA was significantly and non-linearly associated with an increased hazard of failure-free survival (p = 0.004), progression-free survival (p = 0.002), metastatic progression-free survival (p = 0.003), overall survival (p = 0.045) and prostate cancer-specific survival (p = 0.011). Conclusions: Evaluation of the burden of CN aberrations in archival, poor quality FFPE diagnostic tissue from men randomised in the STAMPEDE trial is feasible using lpWGS and has potential clinical utility to identify better prognosis advanced HSPC patients, who may not require treatment intensification.

Purpose: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting immune checkpoint molecules. This preclinical study investigates heme oxygenase-1 (HO-1), an immunosuppressive enzyme that is expressed in a wide variety of cancers, as a potential immune checkpoint target in the context of a chemotherapy-elicited antitumor immune response. We evaluate repurposing tin mesoporphyrin (SnMP), which has demonstrated safety and efficacy targeting hepatic HO in the clinic for the treatment of hyperbilirubinemia, as an immune checkpoint blockade therapy for the treatment of cancer. Experimental Design: SnMP and genetic inactivation of myeloid HO-1 were evaluated alongside 5-fluorouracil in an aggressive spontaneous murine model of breast cancer (MMTV-PyMT). Single-cell RNA sequencing analysis, tumor microarray, and clinical survival data from breast cancer patients were used to support the clinical relevance of our observations. Results: We demonstrate that SnMP inhibits immune suppression of chemotherapy-elicited CD8+ T cells by targeting myeloid HO-1 activity in the tumor microenvironment. Microarray and survival data from breast cancer patients reveal that HO-1 is a poor prognostic factor in patients receiving chemotherapy. Single-cell RNA-sequencing analysis suggests that the myeloid lineage is a significant source of HO-1 expression, and is co-expressed with the immune checkpoints PD-L1/2 in human breast tumors. In vivo, we therapeutically compare the efficacy of targeting these two pathways alongside immune-stimulating chemotherapy, and demonstrate that the efficacy of SnMP compares favorably with PD-1 blockade in preclinical models. Conclusions: SnMP could represent a novel immune checkpoint therapy, which may improve the immunological response to chemotherapy. Clin Cancer Res; 24(7); 1617–28. ©2018 AACR.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with > 10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance. We thank Aida Murra, Naheed Shaikh, Justine Korteweg, Jeremy Tai, Eleanor Carlyle, Leonora Conneely, KimEdmonds, Karla Lingard, Karen O'Meara, Helen Breeze, Sarah Sarker, Lesley Cooper, Linda Shephard, Susie Slater, and Catherine Rogers for study support. We thank the patients and their families. S.T. and H.X. are funded by Cancer Research UK (CRUK) (C50947/A18176). S.T., T.C., J.L., and M.G. are funded by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) at the Royal Marsden Hospital and Institute of Cancer Research (A109). J.I.L. is funded by the Ministerio de Economia y Competitividad (MINECO, SAF2016-79847-R). M.S., A.S., J. Lynch, R.F., L.A., and L.S. are funded by the Royal Marsden Cancer Charity. K.L. is funded by UK Medical Research Council (MR/P014712/1). T.B.K.W. is funded by the European Union Seventh Framework Programme (FP7-People-2013-ITN). M.J.-H. is funded by the NIHR. N.M.L. is a Winton Group Leader in recognition of the Winton Charitable Foundation's support towards the establishment of The Francis Crick Institute. N.M.L. is additionally funded by a Wellcome Trust Joint Investigator Award (103760/Z/14/Z) and the MRC eMedLab Medical Bioinformatics Infrastructure Award (MR/L016311/1). M.K. is funded by the Danish Cancer Society grant (R90-A6213). P.C. is funded by the Wellcome Trust (WT088340MA). N.M. receives funding from CRUK, Rosetrees, and the NIHR BRC at University College London Hospitals. C.S. is a Royal Society Napier Research Professor. C.S. is funded by Cancer Research UK (TRACERx and CRUK Cancer Immunotherapy Catalyst Network), the CRUK Lung Cancer Centre of Excellence, Stand Up 2 Cancer (SU2C), the Rosetrees and Stoneygate Trusts, NovoNordisk Foundation (THESEUS), Marie Curie Network PloidyNet, the NIHR BRC at University College London Hospitals, and the CRUK University College London Experimental Cancer Medicine Centre. C.S., O.P., D.R., I.C., and Z.S. are funded by NovoNordisk Foundation (16584). O.P., D.R., and I.C. are funded by the National Research, Development and Innovation Office of Hungary (NVKP_16-1-20160004). This work was supported by CRUK (Clinical Scientist Fellowship to S.T., C50947/A18176), The Francis Crick Institute, which receives its core funding from Cancer Reseach UK (FC010110), the UK Medical Research Council (FC010110), the Wellcome Trust (FC010110), and NIHR BRC at the Royal Marsden Hospital and Institute of Cancer Research (A109). High-performance computing was supported by eMedLab. The results published here are in whole or part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.

153 Background: The PAM50 gene expression classifier stratifies localized prostate cancer into luminal A/B and basal subtypes: luminal A have superior prognosis compared to luminal B and basal. Drug response scores built using the NCI-60 cell lines predict luminal subtypes are more taxane sensitive compared to basal; luminal B is hypothesized to be the most proliferative and hormone responsive compared to the basal subtype. The STAMPEDE trial provides a framework to validate prognostic and predictive associations of the PAM50 test in tumors from men randomized to androgen deprivation therapy (ADT) alone or with docetaxel or abiraterone. We here present multi-region whole-transcriptome expression array data at completion of Stage I designed to confirm the feasibility of molecular subtyping STAMPEDE tumor blocks. Methods: In collaboration with Decipher Biosciences we generated clinical-grade whole-transcriptome expression array data (Human Exon 1.0 ST GeneChip) performed to CLIA standards on mRNA (from three 10 micron slides) from cancer-enriched areas and applied the PAM50 classifier. Results: As of January 2019, we retrieved blocks from 2012 men from an ITT population of 3879. For Stage I feasibility assessment, we sectioned diagnostic trans-rectal biopsies of the prostate from 50 randomly selected men treated with ADT. We extracted mRNA from 109 cores; > 92% cores (101) from > 94% patients (47/50) passed quality control. The prevalence of PAM50 subtypes was: 31 basal cases (62%), 18 luminal B (36%) and 1 luminal A (2%). 26 cases (52%) were identified as ETS-related gene (ERG) positive. More than one core was interrogated from 35/50 cases (range: 1-6 cores). ERG positive cores were homogeneous across cores from the same patient but PAM50 subtyping identified intra-patient variability across cores from the same case. Conclusions: We confirm the feasibility of expression profiling STAMPEDE tumor blocks and identify a low prevalence of luminal A subtype. Whereas ERG status is consistent across multiple cores, basal and luminal subtypes co-exist in the same prostate. Multi-region analysis will enable further refinement for individual patient classification.

e15129Background: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting the immune checkpoint molecules PD-1 and CTLA-4. However, as a significant number of patien...

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases. S.T. and H.X. are funded by Cancer Research UK (CRUK) (C50947/A18176). S.T., T.C., J.L., and M.G. are funded by the NIH Research (NIHR) Biomedical Research Centre (BRC) at the Royal Marsden Hospital and Institute of Cancer Research (A109). J.I.L. is funded by the Ministerio de Economia y Competitividad (MINECO, SAF2016-79847-R). M.S., A.S., J.L., R.F., L.A., and L.S. are funded by the Royal Marsden Cancer Charity. K.L. is funded by UK Medical Research Council (MR/P014712/1). N.M. receives funding from CRUK, Rosetrees, and the NIHR BRC at University College London Hospitals. C.S is Royal Society Napier Research Professor. C.S. is funded by Cancer Research UK (TRACERx and CRUK Cancer Immunotherapy Catalyst Network), the CRUK Lung Cancer Centre of Excellence, Stand Up 2 Cancer (SU2C), the Rosetrees and Stoneygate Trusts, NovoNordisk Foundation (ID 16584), the Breast Cancer Research Foundation (BCRF), the European Research Council (THESEUS), Marie Curie Network PloidyNet, the NIHR BRC at University College London Hospitals, and the CRUK University College London Experimental Cancer Medicine Centre. The work presented in this manuscript was funded by Cancer Research UK (grant reference number C50947/A18176), Ventana Medical Systems (grant reference numbers 10467 and 10530), the Kidney Cancer Fund of The Royal Marsden Cancer Charity, NIHR BRC at the Royal Marsden Hospital and Institute of Cancer Research (grant reference number A109), and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001202), the UK Medical Research Council (FC001202), and the Wellcome Trust (FC001202). In particular, we acknowledge the support of the Advanced Sequencing Facility and the High-Performance Computing at the Francis Crick Institute. This project was enabled through access to the MRC eMedLab Medical Bioinformatics infrastructure, supported by the Medical Research Council (grant number MR/L016311/1).