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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.

This file provides supplementary data on in vivo overall radiotoxicity (Table S1), in vitro radiosensitizing effect (Figure S1), histological images (Figures S2, S3, S5-S9) corresponding to the data presented in Figures 4 and 5 in the text, the changes in body weight in healthy and tumor-harboring mice (Figure S4), and additional tumor growth delay curves (Figure S10).

Supplementary Figure 1. (A) There is no effect on cell proliferation in RKO cells. (B) There is no effect on S-phase fraction in RKO and paired HCT116 cells. (C) 50-65% Clonogenic cell survival in RKO and paired HCT116 cells. (D) BER mRNA expression is unchanged in RKO cells. qRT-PCR analyses show no significant effects of prolonged hypoxia (72 h x 0.2% O2) on the mRNA levels of selected BER genes in RKO cells. Carbonic anhydrase IX (CAIX) is shown as a positive control for hypoxia.

There is a compelling need to develop anticancer therapies that target cancer cells and tissues. Arising from innovative femtomedicine studies, a new class of non–platinum-based halogenated molecules (called FMD molecules) that selectively kill cancer cells and protect normal cells in treatments of multiple cancers has been discovered. This article reports the first observation of the radiosensitizing effects of such compounds in combination with ionizing radiation for targeted radiotherapy of a variety of cancers. We present in vitro and in vivo studies focused on combination with radiotherapy of cervical, ovarian, head and neck, and lung cancers. Our results demonstrate that treatments of various cancer cells in vitro and in vivo mouse xenograft models with such compounds led to enhanced efficiencies in radiotherapy, while the compounds themselves induced no or little radiotoxicity toward normal cells or tissues. These compounds are therefore effective radiosensitizers that can be translated into clinical trials for targeted radiotherapy of multiple types of cancer. This study also shows the potential of femtomedicine to bring breakthroughs in understanding fundamental biologic processes and to accelerate the discovery of novel drugs for effective treatment or prevention of a variety of cancers. Mol Cancer Ther; 15(4); 640–50. ©2016 AACR.

Detected chromatin interactions in all 12 tumour and 5 benign samples.

Sequencing metrics as calculated by HiCUP for all Hi-C libraries generated in this study.

Supplementary Figure 10

Supplementary Figure 2 - PDF file 71K, Construction of biopsy TMA blocks. Four millimeter length of the needle biopsy core (previously delimited by pathologist) is cut out from the original formalin fixed paraffin embedded tissue block. A scalpel blade is used to cut the exceeding wax on the opposite side of the biopsy tissue (opposite side marked in green) in order to reach a final 4 mm x 2 mm x 2 mm checker. These checkers are flipped 90 degrees (long axis of the prostate needle biopsy turns vertical) and placed within a stainless steel mold as a TMA template. The TMA block is completed by re-embedding the checkers by pouring hot wax into the mold. Last step on the right side shows an Hematoxylin and Eosin (H-E)-stained section from a biopsy TMA block (low power)

RAD51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. Because imatinib (Gleevec) has been reported to reduce RAD51 protein levels, we tested the clonogenic survival for RT112, H1299, PANC1, and PC3 tumor cell lines of varying p53 status and normal GM05757 normal fibroblasts after exposure to single agent imatinib (0–20 μmol/L; 0–72 hours). We also combined imatinib with DNA damaging agents that are toxic to RAD51-deficient cells, including ionizing radiation, gemcitabine, and mitomycin C. We observed decreased nuclear expression and chromatin binding of RAD51 protein following imatinib treatment. Imatinib also resulted in decreased error-free HR as determined by a flow cytometry–based integrated direct repeat-green fusion protein reporter system; this correlated to reduced RAD51 expression. Clonogenic survival experiments revealed increased cell kill for imatinib-treated cells in combination with ionizing radiation, gemcitabine, and mitomycin C, due in part to mitotic catastrophe. In experiments using imatinib and gemcitabine, tumor cell lines were sensitized to a greater extent than normal fibroblasts. This preservation of the therapeutic ratio was confirmed in vivo using PC3 xenograft growth delay and intestinal crypt cell clonogenic assays. HR inhibition may be an additional mechanism of action for the chemosensitization and radiosensitization of solid tumors with imatinib with preservation of the therapeutic ratio. [Mol Cancer Ther 2009;8(1):203–13]

SV breakpoints detected by Hi-C in each tumour sample.

Supplementary Figure 5 - PDF file 61K, Univariate Kaplan-Meier plots of biochemical relapse-free survival in a subgroup of patients treated without androgen deprivation therapy (ADT) in the (A) aCGH- and (B) IHC-cohorts

Supplementary Video

Purpose: Proteomic profiling of patients undergoing intensity-modulated radiotherapy (IMRT) for prostate cancer can identify unique biomarkers that reflect acute toxicity in normal tissues. Our objectives were to measure inflammatory cytokine proteins during IMRT and assess the variability of individual proteomic signatures.Experimental Design: Forty-two patients with intermediate-risk prostate cancer were recruited as follows: group 1, definitive IMRT (78 Gy in 39 fractions, n = 22), and group 2, IMRT postprostatectomy (66 Gy in 33 fractions, n = 20). Blood/urine samples were collected at baseline and weekly during IMRT. Acute toxicity was graded weekly during radiotherapy using CTC-AE v3.0 criteria. Multiplexed immunoassays were used to quantify cytokines including granulocyte macrophage colony-stimulating factor, IFN-γ, tumor necrosis factor-α, interleukin (IL)-1α, IL-2, IL6, IL-8, IL-10, and IL-12p70.Results: We observed positive correlations between cytokine expression between serum and plasma, but not between serum/plasma and urine. The Mann-Whitney test showed a significant increase in IFN-γ and IL-6 during IMRT (P = 0.0077, 0.0035). Increasing IL-2 and IL-1 expression were associated with increased probability of acute gastrointestinal and genitourinary toxicity, respectively.Conclusions: Determination of radiation-response signatures is feasible using multiplexed immunoassays and is a promising predictive early biomarker of toxicity outcomes. (Clin Cancer Res 2009;15(17):5576–83)

PDF file - 41K, Percentage genome alteration (PGA) levels are displayed for patient biopsies categorized as per their NKX3.1/T-category status (a), NKX3.1/PSA status (b), NKX3.1/Gleason-score status (c), NKX3.1/c-MYC/T-category status (d) and NKX3.1/c-MYC/PSA status (e). The median PGA for each group is represented by the black line. (GS=Gleason-score, *=significantly different). Patients with NKX3.1 allelic loss have a significant increase in PGA as compared to patients with a normal status of NKX3.1 in T-category groups pT1=0.00045 and pT210=0.0012 (b). Patients with NKX3.1 allelic loss have a significant increase in PGA as compared to patients with a normal status of NKX3.1 in Gleason-scores pGS6=0.0015 and pGS710=0.001 (e).

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Simple and complex SVs reconstructed from SV breakpoints.

Purpose:PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported.Experimental Design: Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN+/+, DU145-PTEN+/−, PC3-PTEN−/−) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression.Results:PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H2O2, and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel.Conclusions: These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials. Clin Cancer Res; 18(4); 1015–27. ©2011 AACR.

Supplementary Figure Legends 1-2, Tables 1-6 from NKX3.1 Haploinsufficiency Is Prognostic for Prostate Cancer Relapse following Surgery or Image-Guided Radiotherapy

PDF file 97K, Table S1 shows the clinical demographics of the patient cohort treated with radiotherapy for prostate cancer

Supplementary Figure 3 - PDF file 164K, A) Univariate Kaplan-Meier plots of biochemical relapse-free rates (bRFR) according to PSA levels in the aCGH-cohort. B) Univariate Kaplan-Meier plots of biochemical relapse-free rates (bRFR) according to T-category in the aCGH-cohort. C) Univariate Kaplan-Meier plots of biochemical relapse-free rates (bRFR) according to Gleason score in the aCGH-cohort. D) Univariate Kaplan-Meier plots of biochemical relapse-free rates (bRFR) according PSA levels in the IHC-cohort. E) Univariate Kaplan-Meier plots of biochemical relapse-free rates (bRFR) according to T category in the IHC-cohort. F) Univariate Kaplan-Meier plots of biochemical relapse-free rates (bRFR) according to Gleason score in the IHC-cohort

Supplementary Figure 7 - PDF file 106K, Univariate Kaplan-Meier plots of bRFR versus time to recurrence for the Memorial Sloan Kettering Prostate Cancer cohort of patients who underwent surgery with differential ERG status based on mRNA expression

Supplementary Figure 1 - PDF file 49K, Schematic representation of TMPRSS2-ERG fusion allele in the chromosome 21q22.2/21q22.3. Gene positions are based on NCBI36/hg18 human genome reference

Compartmentalization changes in tumours is not associated with widespread differential gene expression.

Supplementary Fig. S1 from Targeting homologous recombination using imatinib results in enhanced tumor cell chemosensitivity and radiosensitivity

Supplementary Figures and descriptions for Supplementary Tables

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Supplementary Material

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Purpose: Tumor hypoxia is a negative prognostic factor in multiple cancers, due in part to its role in causing resistance to radiotherapy. Hypoxia arises in tumor regions distal to blood vessels as oxygen is consumed by more proximal tumor cells. Reducing the rate of oxygen consumption is therefore a potential strategy to reduce tumor hypoxia. We hypothesized that the anti-diabetic drug metformin, which reduces oxygen consumption through inhibition of mitochondrial complex I, would improve radiation response by increasing tumor oxygenation.Experimental Design: Tumor hypoxia was measured in xenografts before and after metformin treatment using 2-nitroimidazole hypoxia markers quantified by immunohistochemistry (IHC), flow cytometry, and positron emission tomography (PET) imaging. Radiation response was determined by tumor growth delay and clonogenic survival in xenografts with and without administration of metformin. The impact of metformin use on outcome was assessed in 504 patients with localized prostate cancer treated with curative-intent, image-guided radiotherapy (IGRT) from 1996 to 2012. Three-year biochemical relapse-free rates were assessed using the Kaplan–Meier method.Results: Metformin treatment significantly improved tumor oxygenation in two xenograft models as measured by IHC, flow cytometry, and PET imaging. Metformin also led to improved radiotherapy responses when mice were administered metformin immediately before irradiation. Clinically, metformin use was associated with an independent and significant decrease in early biochemical relapse rates (P = 0.0106).Conclusion: Our data demonstrate that metformin can improve tumor oxygenation and response to radiotherapy. Our study suggests that metformin may represent an effective and inexpensive means to improve radiotherapy outcome with an optimal therapeutic ratio. Clin Cancer Res; 19(24); 6741–50. ©2013 AACR.

Supplementary Figure 5

PDF file 209K, Figure S1 illustrates the method for IHC analysis. It also shows a reduction in hypoxic fraction after metformin exposure in POP-92S and LNCaP xenografts

PDF file 80K, Figure S2 shows data from in vitro and ex vivo clonogenic assays of metformin treatment in combination with radiation

Supplementary Figure 6

Structural variant detection from Hi-C data.

Supplementary Figure 8

Assembly of structural variants involving enhancer-hijacking events.

Supplementary Figure 6 - PDF file 113K, Univariate Kaplan-Meier plots of bRFR versus time to recurrence for the Memorial Sloan Kettering Prostate Cancer cohort of patients who underwent surgery with differential TMPRSS2-ERG fusion status based on array Comparative Genome Hybridization (aCGH)

PDF file 54K, Figure S5 shows blood glucose levels in mice after metformin treatment

Supplementary Figure 2

Supplementary Data from Longitudinal Cytokine Expression during IMRT for Prostate Cancer and Acute Treatment Toxicity

PDF file 92K, Figure S4 shows the effect of metformin on bRFR in the matched cohort of 172 prostate cancer patients

Background: Despite the use of prostate specific antigen (PSA), Gleason-score, and T-category as prognostic factors, up to 40% of patients with intermediate-risk prostate cancer will fail radical prostatectomy or precision image-guided radiotherapy (IGRT). Additional genetic prognosticators are needed to triage these patients toward intensified combination therapy with novel targeted therapeutics. We tested the role of the NKX3.1 gene as a determinant of treatment outcome given its reported roles in tumor initiating cell (TIC) renewal, the DNA damage response, and cooperation with c-MYC during prostate cancer progression.Methods: Using high-resolution array comparative genomic hybridization (aCGH), we profiled the copy number alterations in TIC genes using tumor DNA from frozen needle biopsies derived from 126 intermediate-risk patients who underwent IGRT. These data were correlated to biochemical relapse-free rate (bRFR) by the Kaplan–Meier method and Cox proportional hazards models.Results: A screen of the aCGH-IGRT data for TIC genes showed frequent copy number alterations for NKX3.1, PSCA, and c-MYC. NKX3.1 haploinsufficiency was associated with increased genomic instability independent of PSA, T-category, and Gleason-score. After adjusting for clinical factors in a multivariate model, NKX3.1 haploinsufficiency was associated with bRFR when tested alone (HR = 3.05, 95% CI: 1.46–6.39, P = 0.0030) or when combined with c-MYC gain (HR = 3.88, 95% CI: 1.78–8.49, P = 0.00067). A similar association was observed for patients following radical prostatectomy with a public aCGH database. NKX3.1 status was associated with positive biopsies post-IGRT and increased clonogen radioresistance in vitro.Conclusions: Our results support the use of genomic predictors, such as NKX3.1 status, in needle biopsies for personalized approaches to prostate cancer management. Clin Cancer Res; 18(1); 308–16. ©2011 AACR.

Individual TAD calls in all 12 tumour and 5 benign samples.

PDF file 81K, Supplementary Legends

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Sample processing and TAD similarity between samples.

CCR Translation for This Article from PTEN Deletion in Prostate Cancer Cells Does Not Associate with Loss of RAD51 Function: Implications for Radiotherapy and Chemotherapy

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Summary statistics for TAD counts in all 12 tumour and 5 benign samples, across multiple window sizes.