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In recent years, there has been growing evidence for the involvement of stem cells in cancer initiation. As a result of their long life span, stem cells may have an increased propensity to accumulate genetic damage relative to differentiated cells. Therefore, stem cells of normal tissues may be important targets for radiation-induced carcinogenesis.Knowledge of the effects of ionizing radiation (IR) on normal stem cells and on the processes involved in carcinogenesis is very limited. The influence of high doses of IR (>5 Gy) on proliferation, cell cycle and induction of senescence has been demonstrated in stem cells. There have been limited studies of the effects of moderate (0.5–5 Gy) and low doses (This review will provide an overview of the current knowledge of radiation-induced effects on normal stem cells, with particular focus on low and moderate doses of IR.

Purpose The biological optimization of proton therapy can be achieved only through a detailed evaluation of relative biological effectiveness (RBE) variations along the full range of the Bragg curve. The clinically used RBE value of 1.1 represents a broad average, which disregards the steep rise of linear energy transfer (LET) at the distal end of the spread-out Bragg peak (SOBP). With particular attention to the key endpoint of cell survival, our work presents a comparative investigation of cell killing RBE variations along monoenergetic (pristine) and modulated (SOBP) beams using human normal and radioresistant cells with the aim to investigate the RBE dependence on LET and intrinsic radiosensitvity. Methods and Materials Human fibroblasts (AG01522) and glioma (U87) cells were irradiated at 6 depth positions along pristine and modulated 62-MeV proton beams at the INFN-LNS (Catania, Italy). Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and the local effect model (LEM). Results We observed significant cell killing RBE variations along the proton beam path, particularly in the distal region showing strong dose dependence. Experimental RBE values were in excellent agreement with the LEM predicted values, indicating dose-averaged LET as a suitable predictor of proton biological effectiveness. Data were also used to validate a parameterized RBE model. Conclusions The predicted biological dose delivered to a tumor region, based on the variable RBE inferred from the data, varies significantly with respect to the clinically used constant RBE of 1.1. The significant RBE increase at the distal end suggests also a potential to enhance optimization of treatment modalities such as LET painting of hypoxic tumors. The study highlights the limitation of adoption of a constant RBE for proton therapy and suggests approaches for fast implementation of RBE models in treatment planning.

R-Spondin (RSPO) proteins bind to LGR receptors and potentiate Wnt/β-catenin signaling. We have identified a therapeutic anti-RSPO3 antibody targeting the RSPO-LGR pathway. In preclinical studies, RSPO3 gene expression has shown correlation with anti-RSPO3 antibody efficacy in multiple solid tumor types. A qPCR-based RSPO3 assay has been developed as a predictive biomarker for response to the anti-RSPO3 antibody. In addition, RSPO gene fusions may play a role in the activation of Wnt signaling. A gene fusion detection workflow consisting of a RSPO3 CLIA assay, a RSPO3 RUO assay and next generation sequencing (NGS) has also been developed. We designed 6 qPCR-based assays for the RSPO3 CLIA assay development and 2 assays for the RUO assay. These assays were designed to span exon-exon junctions or target microarray probe set sequences. Amplification sensitivity and specificity were assessed for assay selection. The analytic performance of the candidate RSPO3 CLIA assay and quality control measures were established in a validation study. The validation study included: 1) performance specifications of the RSPO3 assay including analytical sensitivity, linearity, and precision, 2) determination of a reportable range, 3) establishment of a cut-off for the RSPO3 CLIA assay for patient selection, and 4) establishment of quality control procedures. 104 human cancer tissues and 24 independent patient-derived tumor xenografts (PDX) were used in these studies. To evaluate the fusion detection workflow, the RUO assay was performed on samples that tested above the CLIA assay cut-off. The delta Ct difference between the CLIA and RUO assays was calculated to identify potential fusions. The limit of quantification was established for the RSPO3 CLIA assay. The 95% reference interval was estimated to be (-2.44, 16.02) with 90% confidence interval for the lower bound (-3.45, -2.12) and upper bound (15.26, 16.57). The delta Ct cut-off for the RSPO3 CLIA assay was set based on sensitivity, specificity and prevalence. No statistically significant difference in the total variance across the tested samples was observed. A549 and OV56 were identified to be cell line controls with established acceptable delta Ct limits. Using NGS, RSPO3 fusions were identified in 6 PDX tumors with delta Ct RUO - delta Ct CLIA>7, including a novel fusion. This cut-off was further refined with NGS of 9 clinical samples. Prevalence of the RSPO3 expression and fusions will be presented. A qPCR based RSPO3 assay was developed and CLIA-validated for use as a potential predictive biomarker for response to anti-RSPO3 therapy. This RSPO3 CLIA assay, together with the fusion detection workflow, will be evaluated in a Phase 1a/b dose escalation study of anti-RSPO3 (OMP-131R10) in advanced solid tumors and in combination with FOLFIRI in metastatic colorectal cancer (NCT02482441). Citation Format: Chun Zhang, Yuwang Liu, Min Wang, Gilbert OYoung, Joy Kavanagh, Cheryl McFarlane, Fiore Cattaruzza, Pete Yeung, Jennifer Cain, Wan-Ching Yen, Marcus Fischer, Belinda Cancilla, Edwina Dobbin, Michelle McCarthy, Austin Gurney, Leonardo Faoro, John Lewicki, Tim Hoey, Ann M. Kapoun. Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 404.

The human breast is a radiosensitive tissue and radiation exposure is proposed to contribute to mammary gland carcinogenesis through changes to stem and progenitor cells that result in aberrant proliferation, differentiation and genome instability. However, little is known about the low dose radiation response (LD IR) under conditions of relevance to environmental and occupational and medical imaging exposures. This study utilises 2-D and 3-D breast models derived from two normal breast cell lines (MCF10A and HME1) and primary human mammary epithelial cells from reduction mammoplasty surgery, to investigate the mechanisms underpinning sensitivity of normal breast to LD IR and elucidate its role in breast cancer initiation and progression. We have shown that DNA damage can be detected in both cell lines after doses Using antibodies to cell surface markers suggested to be putative breast stem cell markers, we have performed Fluoresence Activated Cell Sorting (FACS) to isolate subpopulations from the MCF10A cell line. CD49fhi, EpCAM-, MUC1- cells thought to be progenitor like cells were found to be more resistant to LD IR than the EpCAM+, MUC1+ population. As the pathways that govern survival/proliferation of stem and progenitor cells versus differentiation are interconnected, we have investigated if radiation resistance is also correlated with changes to breast cell differentiation. It was observed that LD IR modulated breast cell lineage markers in 2D cultures of both cell lines. We subsequently found that radiation induces a decrease in MUC1 mRNA expression levels which coincides with increased Lef1 expression. Lef1 has been shown to promote Mre11 expression thereby conferring elevated DNA repair and radiation resistance in tumour cells. Based on our findings, we hypothesize that progenitor cell populations within normal breast cell lines may be more efficient at detecting DNA DSBs resulting from LD IR leading to a pro-survival response. We are currently investigating these effects in primary mammary epithelial cell samples, using FACS to isolate progenitor subpopulations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-14.