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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)

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

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

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.

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

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 81K, Supplementary Legends

Background: Preclinical data suggest that TMPRSS2-ERG gene fusions, present in about 50% of prostate cancers, may be a surrogate for DNA repair status and therefore a biomarker for DNA-damaging agents. To test this hypothesis, we examined whether TMPRSS2-ERG status was associated with biochemical failure after clinical induction of DNA damage following image-guided radiotherapy (IGRT).Methods: Pretreatment biopsies from two cohorts of patients with intermediate-risk prostate cancer [T1/T2, Gleason score (GS) < 8, prostate-specific antigen (PSA) < 20 ng/mL; >7 years follow-up] were analyzed: (i) 126 patients [comparative genomic hybridization (CGH) cohort] with DNA samples assayed by array CGH (aCGH) for the TMPRSS2-ERG fusion; and (ii) 118 patients [immunohistochemical (IHC) cohort] whose biopsy samples were scored within a defined tissue microarray (TMA) immunostained for ERG overexpression (known surrogate for TMPRSS2-ERG fusion). Patients were treated with IGRT with a median dose of 76 Gy. The potential role of TMPRSS2-ERG status as a prognostic factor for biochemical relapse-free rate (bRFR; nadir + 2 ng/mL) was evaluated in the context of clinical prognostic factors in multivariate analyses using a Cox proportional hazards model.Results:TMPRSS2-ERG fusion by aCGH was identified in 27 (21%) of the cases in the CGH cohort, and ERG overexpression was found in 59 (50%) patients in the IHC cohort. In both cohorts, TMPRSS2-ERG status was not associated with bRFR on univariate or multivariate analysis.Conclusions: In two similarly treated IGRT cohorts, TMPRSS2-ERG status was not prognostic for bRFR, in disagreement with the hypothesis that these prostate cancers have DNA repair defects that render them clinically more radiosensitive. TMPRSS2-ERG is therefore unlikely to be a predictive factor for IGRT response. Clin Cancer Res; 19(18); 5202–9. ©2013 AACR.

Supplementary Material - PDF file 121K, Supplementary figue legends and supplementary tables

Supplementary Figure 4 - PDF file 122K, Univariate Kaplan-Meier plots of biochemical relapse-free survival in a subgroup of patients treated with androgen deprivation therapy (ADT) in the (A) aCGH- and (B) IHC-cohorts

Current risk-stratification systems for prostate cancer (PCa) do not sufficiently reflect the disease heterogeneity. Genomic classifiers (GC) enable improved risk stratification after surgery, but less data exist for patients treated with definitive radiation therapy (RT) or RT in oligo-/metastatic disease stages. To guide future perspectives of GCs for RT, we conducted (1) a systematic review on the evidence of GCs for patients treated with RT and (2) a survey of experts using the Delphi method, addressing the role of GCs in personalized treatments to identify relevant fields of future clinical and translational research. We performed a systematic review and screened ongoing clinical trials on ClinicalTrials.gov. Based on these results, a multidisciplinary international team of experts received an adapted Delphi method survey. Thirty-one and 30 experts answered round 1 and round 2, respectively. Questions with ≥75% agreement were considered relevant and included in the qualitative synthesis. Evidence for GCs as predictive biomarkers is mainly available to the postoperative RT setting. Validation of GCs as prognostic markers in the definitive RT setting is emerging. Experts used GCs in patients with PCa with extensive metastases (30%), in postoperative settings (27%), and in newly diagnosed PCa (23%). Forty-seven percent of experts do not currently use GCs in clinical practice. Expert consensus demonstrates that GCs are promising tools to improve risk-stratification in primary and oligo-/metastatic patients in addition to existing classifications. Experts were convinced that GCs might guide treatment decisions in terms of RT-field definition and intensification/deintensification in various disease stages. This work confirms the value of GCs and the promising evidence of GC utility in the setting of RT. Additional studies of GCs as prognostic biomarkers are anticipated and form the basis for future studies addressing predictive capabilities of GCs to optimize RT and systemic therapy. The expert consensus points out future directions for GC research in the management of PCa. ispartof: Int J Radiat Oncol Biol Phys vol:116 issue:3 pages:503-520 ispartof: location:United States status: published

We herein present a new concept to produce dual-color photoconvertible probes based on a mechanism called Directed Photooxidation Induced Conversion (DPIC). As a support of this mechanism, styryl-coumarins (SCs) bearing Aromatic Singlet Oxygen Reactive Moieties (ASORM) like furan and pyrrole have been synthesized. SCs are bright fluorophores, which undergo a hypsochromic conversion upon visible light irradiation due to directed photooxidation of the ASORM that leads to the disruption of conjugation. SC-P, a yellow emitting probe bearing a pyrrole moiety, converts to a stable blue emitting coumarin with a 68 nm shift allowing the photoconversion and tracking of lipid droplet in live cells. This new approach might pave the way to a new generation of photoconvertible dyes for advanced bioimaging applications.

Purpose The safety and effectiveness of moderately hypofractionated post-operative radiation therapy for breast cancer were demonstrated by several trials. This study aimed to evaluate the current patterns of practice and to assess possible aspects that affect the decision-making process regarding the use of fractionation in breast cancer patients in Latin America and the Caribbean (LAC). Methods Radiation oncologists from LAC were invited to contribute to this this study. A 38-question survey was used to evaluate their opinions. Results A total of 173 radiation oncologists from 13 countries answered the questionnaire. The majority of respondents (84.9%) preferred moderately hypofractionated post-operative radiation therapy as their first choice in cases of whole breast irradiation. For whole breast plus regional nodal irradiation, post-mastectomy (chest wall and regional nodal irradiation) without reconstruction, and post-mastectomy (chest wall and regional node irradiation) with reconstruction hypofractionated post-operative radiation therapy was preferred by 72.2% 71.1%, and 53.7% of respondents, respectively. Breast cancer stage, and flap-based breast reconstruction were the factors associated with absolute contraindications for the use of hypofractionated schedules. Conclusion Even though moderately hypofractionated post-operative radiation therapy for breast cancer is considered a new standard to the vast majority of the patients, its unrestricted application in clinical practice across LAC still faces reluctance.

Mastitis is a most common disease of dairy cows and causes tremendous economic loss to the dairy industry worldwide. Somatic cell counts (SCC) reflect the inflammatory response to infections and is a metric used as key indicator in mastitis screening programs, typically within the framework of national milk recording schemes. Besides the determination of total SCC, the differentiation of cell types has been described to be beneficial for a more definite description of the actual udder health status of dairy cows. Differential somatic cell count (DSCC) represents the combined proportion of polymorphonuclear leukocytes (PMN) and lymphocytes expressed as a percentage of the total. The aim of this study was to study the relationship between SCC and differential somatic cell count (DSCC) in individual quarter milk samples collected at different time points: at dry-off, after calving and at the lactation peak. We used individual quarter data from farms representing the specialized production system of Parmigiano Reggiano cheese in Northern Italy. Average DSCC values ranged between 44.9% and 56.3%, with higher values (60.4%-72.1%) in milk samples with ≥ 1 million SCC/ml (where the proportion of samples with DSCC > 70% can be as high as 0.73). Moderate overall correlations between DSCC and log(SCC) were estimated (Pearson=0.42, Spearman=0.38), with a clear increasing trend with parity and around the lactation peak (e.g. Pearson=0.59 at 60 DIM in parity 4).Taking SCC values as indicators of subclinical mastitis, DSCC would diagnose mastitis with 0.75 accuracy. Data editing criteria do have an impact on results, with stricter filtering leading to lower correlations between log(SCC) and DSCC. In conclusion DSCC and SCC provide different descriptions of the udder health status of dairy cows which, at least to some extent, are independent. DSCC alone doesn’t provide more accurate information than SCC atquarter level but, used in combination with SCC, can be of potential interest within the framework of milk recording programs, especially in the context of selective dry-cow therapy (SDCT). However, this needs further investigation and updated threshold values need to be selected and validated.