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Key Points More than half of all patients with cancer receive radiation therapy. Normal tissue tolerance for radiation limits the dose of radiation that can safely be delivered, which can limit the probability of curing a tumour. As our knowledge of the mechanisms and signalling pathways that determine the response of tumour tissues and normal tissues to radiation increases, targeted drugs can be developed that selectively sensitize tumours or protect normal tissues. Promising approaches to selectively enhance tumour radiosensitivity include triggering synthetic lethality, inhibiting multiple targets to simultaneously block more than one signalling pathway and targeting the tumour microenvironment. More than half of all patients with cancer receive radiation therapy, but normal tissue tolerance to radiation often limits the ability to cure tumours with radiation therapy. Here, Moding, Kastan and Kirsch discuss current approaches and possible future directions for combining radiation therapy with targeted therapies to enhance the probability of tumour cure. Approximately 50% of all patients with cancer receive radiation therapy at some point during the course of their treatment, and the majority of these patients are treated with curative intent. Despite recent advances in the planning of radiation treatment and the delivery of image-guided radiation therapy, acute toxicity and potential long-term side effects often limit the ability to deliver a sufficient dose of radiation to control tumours locally. In the past two decades, a better understanding of the hallmarks of cancer and the discovery of specific signalling pathways by which cells respond to radiation have provided new opportunities to design molecularly targeted therapies to increase the therapeutic window of radiation therapy. Here, we review efforts to develop approaches that could improve outcomes with radiation therapy by increasing the probability of tumour cure or by decreasing normal tissue toxicity.

The tissue-specific incidence of cancers and their genetic basis are poorly understood. Although prior studies have shown global correlation across tissues for cancer risk single-nucleotide polymorphisms (SNPs) identified through genome-wide association studies (GWAS), any shared functional regulation of gene expression on a per SNP basis has not been well characterized. We set to quantify cis-mediated gene regulation and tissue sharing for SNPs associated with eight common cancers. We identify significant tissue sharing for individual SNPs and global enrichment for breast, colorectal, and Hodgkin lymphoma cancer risk SNPs in multiple tissues. In addition, we observe increasing tissue sharing for cancer risk SNPs overlapping with super-enhancers for breast cancer and Hodgkin lymphoma providing further evidence of tissue specificity. Finally, for genes under cis-regulation by breast cancer SNPs, we identify a phenotype characterized by low expression of tumor suppressors and negative regulators of the WNT pathway associated with worse freedom from progression and overall survival in patients who eventually develop breast cancer. Our results introduce a paradigm for functionally annotating individual cancer risk SNPs and will inform the design of future translational studies aimed to personalize assessment of inherited cancer risk across tissues.

To provide additional functional information for tumor characterization, we investigated the use of dual-energy computed tomography for imaging murine lung tumors. Tumor blood volume and vascular permeability were quantified using gold and iodine nanoparticles. This approach was compared with a single contrast agent/single-energy CT method. Ex vivo validation studies were performed to demonstrate the accuracy of in vivo contrast agent quantification by CT. Primary lung tumors were generated in LSL-Kras(G12D); p53(FL/FL) mice. Gold nanoparticles were injected, followed by iodine nanoparticles two days later. The gold accumulated in tumors, while the iodine provided intravascular contrast. Three dual-energy CT scans were performed-two for the single contrast agent method and one for the dual contrast agent method. Gold and iodine concentrations in each scan were calculated using a dual-energy decomposition. For each method, the tumor fractional blood volume was calculated based on iodine concentration, and tumor vascular permeability was estimated based on accumulated gold concentration. For validation, the CT-derived measurements were compared with histology and inductively-coupled plasma optical emission spectroscopy measurements of gold concentrations in tissues. Dual-energy CT enabled in vivo separation of gold and iodine contrast agents and showed uptake of gold nanoparticles in the spleen, liver, and tumors. The tumor fractional blood volume measurements determined from the two imaging methods were in agreement, and a high correlation (R(2) = 0.81) was found between measured fractional blood volume and histology-derived microvascular density. Vascular permeability measurements obtained from the two imaging methods agreed well with ex vivo measurements. Dual-energy CT using two types of nanoparticles is equivalent to the single nanoparticle method, but allows for measurement of fractional blood volume and permeability with a single scan. As confirmed by ex vivo methods, CT-derived nanoparticle concentrations are accurate. This method could play an important role in lung tumor characterization by CT.

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53 , loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3 + / + mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3 + /− and Phlda3 −/− littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Background The FDA approval of T cell receptor-engineered T cells (TCR-T) for synovial sarcoma demonstrates the potential for adoptive T cell therapies (ACTs) in solid tumors. However, the paucity of tumor-associated targets without expression in normal tissues remains a major bottleneck, especially in rare cancer subtypes. Methods We developed a comprehensive computational pipeline called SCAN-ACT that leverages single-cell RNA sequencing and multi-omics data from tumor and normal tissues to nominate and prioritize putative targets for both chimeric antigen receptor (CAR)- and TCR-T cells. For surface membrane targets, SCAN-ACT proposes monospecific targets and potential target pairs for bispecific Boolean logic-gated CAR T cells. For peptide-MHC targets, SCAN-ACT proposes intracellular peptides bound to a diverse set of human leukocyte antigens. Selected targets were validated experimentally by protein expression and for peptide-MHC binding. Results We applied the SCAN-ACT pipeline to soft tissue sarcoma (STS), analyzing 986,749 single cells to identify and prioritize 395 monospecific CAR-T targets, 14,192 bispecific CAR-T targets, and 5020 peptide-MHC targets for TCR-T cells. Proposed targets and target pairs reflected the mesenchymal, neuronal, and hematopoietic ontogeny of STS. We further validated SCAN-ACT in glioblastoma revealing its versatility. Conclusions This work provides a robust data repository along with a web-based and user-friendly set of analysis tools to accelerate ACT development for solid tumors ( https://scanact.stanford.edu/ ).

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors. The sensitivity of circulating tumor DNA detection is improved by identifying sequences with two or more mutations.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality 1 , 2 ; however, a small minority of eligible individuals undergo such screening in the United States 3 , 4 . The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq) 5 , a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed ‘lung cancer likelihood in plasma’ (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies. Circulating tumour DNA in blood is analysed to identify genomic features that distinguish early-stage lung cancer patients from risk-matched controls, and these are integrated into a machine-learning method for blood-based lung cancer screening.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed ‘epigenetic expression inference from cell-free DNA-sequencing’ (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential. EPIC-seq predicts expression of individual genes from cell-free DNA.

The site-specific recombinases Cre and Flp can mutate genes in a spatially and temporally restricted manner in mice. Conditional recombination of the tumor suppressor gene p53 using the Cre-loxP system has led to the development of multiple genetically engineered mouse models of human cancer. However, the use of Cre recombinase to initiate tumors in mouse models limits the utilization of Cre to genetically modify other genes in tumor stromal cells in these models. To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2-6 of the endogenous p53 gene in mice to generate a p53(FRT) allele that can be deleted by Flp recombinase. We show that FlpO-mediated deletion of p53 in mouse embryonic fibroblasts impairs the p53-dependent response to genotoxic stress in vitro. In addition, using FSF-Kras(G12D/+); p53(FRT/FRT) mice, we demonstrate that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. p53(FRT) mice will enable dual recombinase technology to study cancer biology because Cre is available to modify genes specifically in stromal cells to investigate their role in tumor development, progression and response to therapy.

Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.