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Clinical prognostic groupings for localised prostate cancers are imprecise, with 30–50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1–9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65–0·76]) and radical prostatectomy (4·0 [1·6–9·7]; p=0·0024; AUC 0·57 [0·52–0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2–12]; p=0·019; AUC 0·67 [0·61–0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0–19]; p=0·0015; AUC 0·74 [95% CI 0·65–0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4–6·0]; p=0·0039; AUC 0·68 [95% CI 0·63–0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.

BackgroundThe combined use of radiation therapy and chemotherapy is commonly being used in cancer treatment. The side effects of the treatment can be further minimized through targeted delivery of anticancer drugs and local enhancement of the radiation dose. Gold nanoparticles (GNPs) can play a significant role in this regard since GNPs can be used as radiation dose enhancers and anticancer drug carriers. Anticancer drug, bleomycin, was chosen as the model drug, since it could be easily conjugated onto GNPs through the gold–thiol bond.MethodsGold nanoparticles of size 10 nm were synthesized using the citrate reduction method. The surface of The GNPs was modified with a peptide sequence (CKKKKKKGGRGDMFG) containing the RGD domain and anticancer drug, bleomycin. Human breast cancer cells (MDA-MB-231) were incubated with 0.3 nM concentration of GNP–drug complex for 16 h prior to irradiation with a 2 Gy single fraction of 6 MV X-rays. After the treatment, cells were trypsinized and seeded in 60 mm dishes for clonogenic assay. Damage to DNA was probed using immunofluorescence assay.ResultsCancer cells internalized with the GNP–drug complex had a 32 ± 9% decrease in cell survival and statistically significant enhancement in DNA (deoxyribonucleic acid) damage as compared to control cells (irradiated with no GNPs) after receiving a radiation dose of 2 Gy with 6 MV photons.ConclusionsThe experimental results demonstrate that GNP-mediated chemoradiation has the potential to improve cancer care in the near future through enhancement of the local radiation dose and controlled delivery of anticancer drugs.

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Identifying changes in DNA copy number can pinpoint genes that may be involved in tumor development. Here we have defined the smallest overlapping regions of imbalance (SORI) in testicular germ cell tumors other than the 12p region, which has been previously investigated. Definition of the regions was achieved through comparative genomic hybridization (CGH) analysis of a 4559 cDNA clone microarray. A total of 14 SORI were identified, which involved at least five of the 11 samples analysed. Many of these refined regions were previously reported using chromosomal or allelic imbalance studies. The SORI included gain of material from the regions 4q12, 17q21.3, 22q11.23 and Xq22, and loss from 5q33, 11q12.1, 16q22.3 and 22q11. Comparison with parallel chromosomal CGH data supported involvement of most regions. The various SORI span between one and 20 genes and highlight potential oncogenes/tumor suppressor genes to be investigated further (Supplementary material is available at http://www.crukdmf.icr.ac.uk/array/array.html ).

Paul Boutros, Robert Bristow and colleagues report a molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer. They find that multifocal tumors are highly heterogeneous, and they identify a novel recurrent amplification of MYCL1 . Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL , which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Hypoxia exists in all solid tumors and leads to clinical radioresistance and adverse prognosis. We hypothesized that hypoxia and cellular localization of gold nanoparticles (AuNPs) could be modifiers of AuNP-mediated radiosensitization. The possible mechanistic effect of AuNPs on cell cycle distribution and DNA double-strand break (DSB) repair postirradiation were also studied. Clonogenic survival data revealed that internalized and extracellular AuNPs at 0.5 mg/mL resulted in dose enhancement factors of 1.39 ± 0.07 and 1.09 ± 0.01, respectively. Radiosensitization by AuNPs was greatest in cells under oxia, followed by chronic and then acute hypoxia. The presence of AuNPs inhibited postirradiation DNA DSB repair, but did not lead to cell cycle synchronization. The relative radiosensitivity of chronic hypoxic cells is attributed to defective DSB repair (homologous recombination) due to decreased (RAD51)-associated protein expression. Our results support the need for further study of AuNPs for clinical development in cancer therapy since their efficacy is not limited in chronic hypoxic cells.

All invasive testicular germ cell tumors of adolescents and adults (TGCTs), that is, seminomas and nonseminomas, show gain of 12p sequences, mostly as isochromosomes. Although several candidate genes have been suggested, the relevant gene(s) have not been identified yet. About 10% of testicular seminomas, however, show a more restricted amplification of the 12p11.2–p12.1 region, in which the various amplicons show an apparent overlap, allowing for the shortest region of amplification overlap approach, aiming at the identification of pathogenetically relevant sequences residing in this region. Here we report on a high-resolution 12p-amplicon architecture analysis using microarray-based comparative genomic hybridization, the results of which were subsequently confirmed by fluorescent in situ hybridization studies. The 12p-specific microarray contained 63 positionally selected BAC clones, which are more or less evenly distributed over the short arm of chromosome 12 (average spacing: less than 500 Kb), including 20 clones within the region of amplification. Out of a series of 17 seminomas, seven seminomas showed amplification of the whole amplicon region, of which three showed a dip in T / R value in the center of the amplified area. A more complex amplification pattern was found in the other 10 seminomas: three showed predominant amplification at the centromeric border; one mainly at the telomeric border; six showed a balanced amplification of both the centromeric and telomeric regions. The only nonseminoma investigated showed a structure in which the centromeric border was only amplified. These data support a mechanistic model in which at least two 12p genes, situated at the border regions of the amplicon, are positional candidates capable of actively supporting tumor progression in TGCTs.

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Further-more, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is ∼8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/ RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.

The CCAAT boxes of the β‐like globin genes interact with three proteins: NF‐Y, GATA‐1 and NFE‐6. We demonstrate that NFE‐6 contains C/EBPγ, and address its role in globin gene regulation by erythroid overexpression of C/EBPγ, and a dominant‐negative form C/EBPγΔB, in mice. Elevated levels of C/EBPγ, but not C/EBPγΔB, increase expression of the (fetal) γ‐globin relative to the (adult) β‐globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPγ and C/EBPγΔB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant‐negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.