Genotype-Directed Synthetic Cytotoxicity of ATR Inhibition with Radiotherapy.

PURPOSE: The importance of the DNA damage response in mediating effects of radiotherapy (RT) has galvanized efforts to target this pathway with radiosensitizers. Yet early clinical trials of this approach have failed to yield a benefit in unselected populations. We hypothesized that ataxia-telangiectasia mutated (Atm)-null tumors would demonstrate genotype-specific synergy between RT and an inhibitor of the DNA damage response protein ataxia-telangiectasia and Rad3-related (ATR) kinase. EXPERIMENTAL DESIGN: We investigated the synergistic potential of the ATR inhibitor (ATRi) RP-3500 and RT in two Atm-null and isogenic murine models, both in vitro and in vivo. Staining of γ-H2AX foci, characterization of the immune response via flow cytometry, and tumor rechallenge experiments were performed to elucidate the mechanism of interaction. To examine genotype specificity, we tested the interaction of ATRi and RT in a Brca1-null model. Finally, patients with advanced cancer with ATM alterations were enrolled in a phase I/II clinical trial to validate preclinical findings. RESULTS: Synergy between RP-3500 and RT was confirmed in Atm-null lines in vitro, characterized by an accumulation of DNA double-strand breaks. In vivo, Atm-null tumor models had higher rates of durable control with RT and ATRi than controls. In contrast, there was no synergy in tumors lacking Brca1. Analysis of the immunologic response indicated that efficacy is largely mediated by cell-intrinsic mechanisms. Lastly, early results from our clinical trial showed complete responses in patients. CONCLUSIONS: Genotype-directed radiosensitization with ATRi and RT can unleash significant therapeutic benefit and could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments.

Large-scale copy number alterations are enriched for synthetic viability in BRCA1/BRCA2 tumors.

BACKGROUND: Pathogenic BRCA1 or BRCA2 germline mutations contribute to hereditary breast, ovarian, prostate, and pancreatic cancer. Paradoxically, bi-allelic inactivation of BRCA1 or BRCA2 (bBRCA1/2) is embryonically lethal and decreases cellular proliferation. The compensatory mechanisms that facilitate oncogenesis in bBRCA1/2 tumors remain unclear. METHODS: We identified recurrent genetic alterations enriched in human bBRCA1/2 tumors and experimentally validated if these improved proliferation in cellular models. We analyzed mutations and copy number alterations (CNAs) in bBRCA1/2 breast and ovarian cancer from the TCGA and ICGC. We used Fisher's exact test to identify CNAs enriched in bBRCA1/2 tumors compared to control tumors that lacked evidence of homologous recombination deficiency. Genes located in CNA regions enriched in bBRCA1/2 tumors were further screened by gene expression and their effects on proliferation in genome-wide CRISPR/Cas9 screens. A set of candidate genes was functionally validated with in vitro clonogenic survival and functional assays to validate their influence on proliferation in the setting of bBRCA1/2 mutations. RESULTS: We found that bBRCA1/2 tumors harbor recurrent large-scale genomic deletions significantly more frequently than histologically matched controls (n = 238 cytobands in breast and ovarian cancers). Within the deleted regions, we identified 277 BRCA1-related genes and 218 BRCA2-related genes that had reduced expression and increased proliferation in bBRCA1/2 but not in wild-type cells in genome-wide CRISPR screens. In vitro validation of 20 candidate genes with clonogenic proliferation assays validated 9 genes, including RIC8A and ATMIN (ATM-Interacting protein). We identified loss of RIC8A, which occurs frequently in both bBRCA1/2 tumors and is synthetically viable with loss of both BRCA1 and BRCA2. Furthermore, we found that metastatic homologous recombination deficient cancers acquire loss-of-function mutations in RIC8A. Lastly, we identified that RIC8A does not rescue homologous recombination deficiency but may influence mitosis in bBRCA1/2 tumors, potentially leading to increased micronuclei formation. CONCLUSIONS: This study provides a means to solve the tumor suppressor paradox by identifying synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers.

Increased Synthetic Cytotoxicity of Combinatorial Chemoradiation Therapy in Homologous Recombination Deficient Tumors.

PURPOSE: Homologous recombination deficient (HRD) tumors are exquisitely sensitive to platinum-based chemotherapy and when combined with radiation therapy (RT), leads to improved overall survival in multiple cancer types. Whether a subset of tumors with distinct molecular characteristics demonstrate increased benefit from cisplatin and RT (c-RT) is unclear. We hypothesized that HRD tumors, whether associated with BRCA mutations or genomic scars of HRD, exhibit exquisite sensitivity to c-RT, and that HRD may be a significant driver of c-RT benefit. METHODS AND MATERIALS: Sensitivity to c-RT was examined using isogenic and sporadic breast cancer cell lines. HRD was assessed using 4 assays: RT-induced Rad51 foci, a DR-GFP reporter assay, a genomic scar score (large-scale state transitions [LST]), and clonogenic survival assays. Whole-genome sequencing of 4 breast tumors from a phase 2 clinical trial of neoadjuvant c-RT in triple-negative breast cancer was performed and HRD was defined using HRDetect. RESULTS: BRCA1/2 deficient cell lines displayed functional HRD based on the Rad51 functional assay, with c-RT to RT or cisplatin interaction ratios (IR) of 1.11 and 26.84 for the BRCA1 isogenic pair at 2 µM cisplatin and 6 Gy, respectively. The highest LST lines demonstrated HRD and synthetic cytotoxicity to c-RT with IR at 2 Gy and cisplatin 20 µM of 7.50, and the lowest LST line with IR of 0.65. Of 4 evaluable patients in the phase 2 trial, one achieved a pathologic complete response with corresponding HRD based on multiple genomic scar scores including HRDetect and LST scores, compared with patients without a pathologic complete response. CONCLUSIONS: HRD breast cancers, whether identified by BRCA1/2 mutation status, functional tests, or mutational signatures, appear to be significantly more sensitive to c-RT compared with isogenic controls or tumors without HRD mutational signatures. HRD tumors may be exquisitely sensitive to c-RT which warrants further clinical investigation to guide a precision oncology approach.

Most large structural variants in cancer genomes can be detected without long reads.

Short-read sequencing is the workhorse of cancer genomics yet is thought to miss many structural variants (SVs), particularly large chromosomal alterations. To characterize missing SVs in short-read whole genomes, we analyzed 'loose ends'-local violations of mass balance between adjacent DNA segments. In the landscape of loose ends across 1,330 high-purity cancer whole genomes, most large (>10-kb) clonal SVs were fully resolved by short reads in the 87% of the human genome where copy number could be reliably measured. Some loose ends represent neotelomeres, which we propose as a hallmark of the alternative lengthening of telomeres phenotype. These pan-cancer findings were confirmed by long-molecule profiles of 38 breast cancer and melanoma cases. Our results indicate that aberrant homologous recombination is unlikely to drive the majority of large cancer SVs. Furthermore, analysis of mass balance in short-read whole genome data provides a surprisingly complete picture of cancer chromosomal structure.

Long-molecule scars of backup DNA repair in BRCA1- and BRCA2-deficient cancers.

Homologous recombination (HR) deficiency is associated with DNA rearrangements and cytogenetic aberrations1. Paradoxically, the types of DNA rearrangements that are specifically associated with HR-deficient cancers only minimally affect chromosomal structure2. Here, to address this apparent contradiction, we combined genome-graph analysis of short-read whole-genome sequencing (WGS) profiles across thousands of tumours with deep linked-read WGS of 46 BRCA1- or BRCA2-mutant breast cancers. These data revealed a distinct class of HR-deficiency-enriched rearrangements called reciprocal pairs. Linked-read WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long-molecule data. Whereas one (cis) outcome corresponded to the copying and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10 kb) duplications at each junction. We propose an HR-independent replication-restart repair mechanism to explain the full spectrum of reciprocal pair outcomes. Linked-read WGS also identified single-strand annealing as a repair pathway that is specific to BRCA2 deficiency in human cancers. Integrating these features in a classifier improved discrimination between BRCA1- and BRCA2-deficient genomes. In conclusion, our data reveal classes of rearrangements that are specific to BRCA1 or BRCA2 deficiency as a source of cytogenetic aberrations in HR-deficient cells.

ATM Germline-Mutated Gastroesophageal Junction Adenocarcinomas: Clinical Descriptors, Molecular Characteristics, and Potential Therapeutic Implications.

BACKGROUND: Gastroesophageal junction (GEJ) adenocarcinoma is a rare cancer associated with poor prognosis. The genetic factors conferring predisposition to GEJ adenocarcinoma have yet to be identified. METHODS: We analyzed germline testing results from 23 381 cancer patients undergoing tumor-normal sequencing, of which 312 individuals had GEJ adenocarcinoma. Genomic profiles and clinico-pathologic features were analyzed for the GEJ adenocarcinomas. Silencing of ATM and ATR was performed using validated short-interfering RNA species in GEJ, esophageal, and gastric adenocarcinoma cell lines. All statistical tests were 2-sided. RESULTS: Pathogenic or likely pathogenic ATM variants were identified in 18 of 312 patients (5.8%), and bi-allelic inactivation of ATM through loss of heterozygosity of the wild-type allele was detected in all (16 of 16) samples with sufficient tumor content. Germline ATM-mutated GEJ adenocarcinomas largely lacked somatic mutations in TP53, were more likely to harbor MDM2 amplification, and harbored statistically significantly fewer somatic single nucleotide variants (2.0 mutations/Mb vs 7.9 mutations/Mb; P < .001). A statistically significantly higher proportion of germline ATM-mutated than ATM-wild-type GEJ adenocarcinoma patients underwent a curative resection (10 [100%] vs 92 [86.8%], P = .04; Fisher's exact test.), A synthetic lethal interaction between short-interfering RNA silencing of ATM and ATR was observed in the models analyzed. CONCLUSIONS: Our results indicate that germline pathogenic variants in ATM drive oncogenesis in GEJ adenocarcinoma and might result in a distinct clinical phenotype. Given the high prevalence of germline ATM-mutated GEJ adenocarcinomas, genetic testing for individuals with GEJ adenocarcinomas may be considered to better inform prognostication, treatment decisions, and future cancer risk.

Germline RAD51B variants confer susceptibility to breast and ovarian cancers deficient in homologous recombination.

Pathogenic germline mutations in the RAD51 paralog genes RAD51C and RAD51D, are known to confer susceptibility to ovarian and triple-negative breast cancer. Here, we investigated whether germline loss-of-function variants affecting another RAD51 paralog gene, RAD51B, are also associated with breast and ovarian cancer. Among 3422 consecutively accrued breast and ovarian cancer patients consented to tumor/germline sequencing, the observed carrier frequency of loss-of-function germline RAD51B variants was significantly higher than control cases from the gnomAD population database (0.26% vs 0.09%), with an odds ratio of 2.69 (95% CI: 1.4-5.3). Furthermore, we demonstrate that tumors harboring biallelic RAD51B alteration are deficient in homologous recombination DNA repair deficiency (HRD), as evidenced by analysis of sequencing data and in vitro functional assays. Our findings suggest that RAD51B should be considered as an addition to clinical germline testing panels for breast and ovarian cancer susceptibility.

Synthetic Lethality in Cancer Therapeutics: The Next Generation.

Synthetic lethality (SL) provides a conceptual framework for tackling targets that are not classically "druggable," including loss-of-function mutations in tumor suppressor genes required for carcinogenesis. Recent technological advances have led to an inflection point in our understanding of genetic interaction networks and ability to identify a wide array of novel SL drug targets. Here, we review concepts and lessons emerging from first-generation trials aimed at testing SL drugs, discuss how the nature of the targeted lesion can influence therapeutic outcomes, and highlight the need to develop clinical biomarkers distinct from those based on the paradigms developed to target activated oncogenes. SIGNIFICANCE: SL offers an approach for the targeting of loss of function of tumor suppressor and DNA repair genes, as well as of amplification and/or overexpression of genes that cannot be targeted directly. A next generation of tumor-specific alterations targetable through SL has emerged from high-throughput CRISPR technology, heralding not only new opportunities for drug development, but also important challenges in the development of optimal predictive biomarkers.

Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.

Immune checkpoint blockade (ICB) has improved outcomes for patients with advanced cancer, but the determinants of response remain poorly understood. Here we report differential effects of mutations in the homologous recombination genes BRCA1 and BRCA2 on response to ICB in mouse and human tumors, and further show that truncating mutations in BRCA2 are associated with superior response compared to those in BRCA1. Mutations in BRCA1 and BRCA2 result in distinct mutational landscapes and differentially modulate the tumor-immune microenvironment, with gene expression programs related to both adaptive and innate immunity enriched in BRCA2-deficient tumors. Single-cell RNA sequencing further revealed distinct T cell, natural killer, macrophage, and dendritic cell populations enriched in BRCA2-deficient tumors. Taken together, our findings reveal the divergent effects of BRCA1 and BRCA2-deficiency on ICB outcome, and have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy.

Homologous recombination deficiency: how genomic signatures are generated.

Cancer genomes harbor mutational and structural rearrangements that are jointly shaped by DNA damage and repair mechanisms. Accumulating evidence suggests that genetic alterations in DNA repair-defective tumors reflect the scars caused by the use of backup DNA repair mechanisms needed to maintain cellular viability. Detailed analysis of the patterns of mutations and structural rearrangements present in BRCA1/2-deficient tumors has allowed for the delineation of genomic signatures that reflect alternative repair with inactive homologous recombination (HR). Here we aim to summarize recent advances in the analysis of genomic signatures associated with HR-deficiency and examine recent studies that have shed light on the backup repair mechanisms responsible for genomic scarring in HR-deficient tumors.

Pathogenic ATM Mutations in Cancer and a Genetic Basis for Radiotherapeutic Efficacy.

BACKGROUND: Radiation therapy is one of the most commonly used cancer therapeutics but genetic determinants of clinical benefit are poorly characterized. Pathogenic germline variants in ATM are known to cause ataxia-telangiectasia, a rare hereditary syndrome notable for marked radiosensitivity. In contrast, somatic inactivation of ATM is a common event in a wide variety of cancers, but its clinical actionability remains obscure. METHODS: We analyzed 20 107 consecutively treated advanced cancer patients who underwent targeted genomic sequencing as part of an institutional genomic profiling initiative and identified 1085 harboring a somatic or germline ATM mutation, including 357 who received radiotherapy (RT). Outcomes of irradiated tumors harboring ATM loss-of-function (LoF) mutations were compared with those harboring variants of unknown significance. All statistical tests were 2-sided. RESULTS: Among 357 pan-cancer patients who received 727 courses of RT, genetic inactivation of ATM was associated with improved radiotherapeutic efficacy. The 2-year cumulative incidence of irradiated tumor progression was 13.2% vs 27.5% for tumors harboring an ATM LoF vs variant of unknown significance allele, respectively (hazard ratio [HR] = 0.51, 95% confidence interval [CI] = 0.34 to 0.77, P = .001). The greatest clinical benefit was seen in tumors harboring biallelic ATM inactivation (HR = 0.19, 95% CI = 0.06 to 0.60, P = .005), with statistically significant benefit also observed in tumors with monoallelic ATM inactivation (HR = 0.57, 95% CI = 0.35 to 0.92, P = .02). Notably, ATM LoF was highly predictive of outcome in TP53 wild-type tumors but not among TP53-mutant tumors. CONCLUSIONS: We demonstrate that somatic ATM inactivation is associated with markedly improved tumor control following RT. The identification of a radio-sensitive tumor phenotype across multiple cancer types offers potential clinical opportunities for genomically guided RT.

Distinct Classes of Complex Structural Variation Uncovered across Thousands of Cancer Genome Graphs.

Cancer genomes often harbor hundreds of somatic DNA rearrangement junctions, many of which cannot be easily classified into simple (e.g., deletion) or complex (e.g., chromothripsis) structural variant classes. Applying a novel genome graph computational paradigm to analyze the topology of junction copy number (JCN) across 2,778 tumor whole-genome sequences, we uncovered three novel complex rearrangement phenomena: pyrgo, rigma, and tyfonas. Pyrgo are "towers" of low-JCN duplications associated with early-replicating regions, superenhancers, and breast or ovarian cancers. Rigma comprise "chasms" of low-JCN deletions enriched in late-replicating fragile sites and gastrointestinal carcinomas. Tyfonas are "typhoons" of high-JCN junctions and fold-back inversions associated with expressed protein-coding fusions, breakend hypermutation, and acral, but not cutaneous, melanomas. Clustering of tumors according to genome graph-derived features identified subgroups associated with DNA repair defects and poor prognosis.

Moving beyond PARP Inhibition in ATM-Deficient Prostate Cancer.

DNA repair defects are found in primary and metastatic prostate cancer. Alterations in the ATM gene are the second most common defect after BRCA2, but their sensitivity to PARP inhibitors has been questioned by recent clinical literature. The work by Rafiei and colleagues in this issue of Cancer Research now supports this observation with genetically engineered cells and quantitative responses. ATR inhibitors have not yet found a clear role in the clinic, but the new work suggests that ATM-deficient cancers may be more vulnerable to ATR inhibition rather than PARP inhibitors, which is a testable hypothesis for clinical trials.See related article by Rafiei et al., p. 2094.

The Landscape of Somatic Genetic Alterations in Breast Cancers from CHEK2 Germline Mutation Carriers.

Pathogenic germline variants in checkpoint kinase 2 (CHEK2), which plays pivotal roles in DNA damage response and cell cycle regulation, confer an increased breast cancer (BC) risk. Here, we investigated the phenotypic and genomic characteristics of 33 BCs from CHEK2 germline mutation carriers (16 high-risk variants and 17 low-risk p.Ile157Thr variants). CHEK2-associated BCs from patients with high-risk germline variants were largely hormone receptor-positive (87%, 13/15), and 81% (13/16) exhibited loss of heterozygosity (LOH) of the CHEK2 wild-type allele. Conversely, CHEK2-associated BCs from patients with the low-risk p.Ile157Thr variant displayed less-frequent loss of heterozygosity (5/17, 29%) and higher levels of CHEK2 protein expression than those with high-risk germline variants. CHEK2-associated BCs lacked a dominant mutational signature 3, a genomics feature of homologous recombination DNA repair deficiency (HRD). Our findings indicate that CHEK2-associated BCs are generally hormone receptor-positive and lack HRD-related mutational signatures, recapitulating the features of ATM-associated BCs. Specific CHEK2 germline variants may have a distinct impact on tumor biology.

The therapeutic significance of mutational signatures from DNA repair deficiency in cancer.

Cancer is fundamentally a disease of the genome and inherited deficiencies in DNA repair pathways are well established to increase lifetime cancer risk. Computational analysis of pan-cancer data has identified signatures of mutational processes thought to be responsible for the pattern of mutations in any given cancer. These analyses identified altered DNA repair pathways in a much broader spectrum of cancers than previously appreciated with significant therapeutic implications. The development of DNA repair deficiency biomarkers is critical to the implementation of therapeutic targeting of repair-deficient tumors, using either DNA damaging agents or immunotherapy for the personalization of cancer therapy.

A new role for a tumor-suppressing protein.

In addition to its role in preventing tumors, the protein p53 appears to participate in a DNA repair process known as the replication-stress response.

Predictive modeling of outcomes following definitive chemoradiotherapy for oropharyngeal cancer based on FDG-PET image characteristics.

In this study, we investigate the use of imaging feature-based outcomes research ('radiomics') combined with machine learning techniques to develop robust predictive models for the risk of all-cause mortality (ACM), local failure (LF), and distant metastasis (DM) following definitive chemoradiation therapy (CRT). One hundred seventy four patients with stage III-IV oropharyngeal cancer (OC) treated at our institution with CRT with retrievable pre- and post-treatment 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) scans were identified. From pre-treatment PET scans, 24 representative imaging features of FDG-avid disease regions were extracted. Using machine learning-based feature selection methods, multiparameter logistic regression models were built incorporating clinical factors and imaging features. All model building methods were tested by cross validation to avoid overfitting, and final outcome models were validated on an independent dataset from a collaborating institution. Multiparameter models were statistically significant on 5 fold cross validation with the area under the receiver operating characteristic curve (AUC) = 0.65 (p = 0.004), 0.73 (p = 0.026), and 0.66 (p = 0.015) for ACM, LF, and DM, respectively. The model for LF retained significance on the independent validation cohort with AUC = 0.68 (p = 0.029) whereas the models for ACM and DM did not reach statistical significance, but resulted in comparable predictive power to the 5 fold cross validation with AUC = 0.60 (p = 0.092) and 0.65 (p = 0.062), respectively. In the largest study of its kind to date, predictive features including increasing metabolic tumor volume, increasing image heterogeneity, and increasing tumor surface irregularity significantly correlated to mortality, LF, and DM on 5 fold cross validation in a relatively uniform single-institution cohort. The LF model also retained significance in an independent population.

Genomic analysis of exceptional responders to radiotherapy reveals somatic mutations in ATM.

Radiation therapy is a mainstay of cancer treatment, yet the molecular determinants of clinical response are poorly understood. We identified exceptional responders to radiotherapy based on clinical response, and investigated the associated tumor sequencing data in order to identify additional patients with similar mutations. Among head and neck squamous cell cancer patients receiving palliative radiotherapy at our institution, we identified one patient with documented complete metabolic response. Targeted sequencing analysis of the tumor identified a somatic frame-shift mutation in ATM, a gene known to be associated with radio-sensitivity in the germline. To validate the association of somatic ATM mutation with radiotherapy response, we identified eight patients with ATM truncating mutations who received radiotherapy, all of whom demonstrated excellent responses with a median local control period of 4.62 years. Analysis of 22 DNA repair genes in The Cancer Genome Atlas (TCGA) data revealed mutations in 15.9% of 9064 tumors across 24 cancer types, with ATM mutations being the most prevalent. This is the first study to suggest that exceptional responses to radiotherapy may be determined by mutations in DNA repair genes. Sequencing of DNA repair genes merits attention in larger cohorts and may have significant implications for the personalization of radiotherapy.

Patterns of nodal failure after intensity modulated radiotherapy for nasopharyngeal carcinoma.

OBJECTIVES: To evaluate the sites of nodal failure (NF) of nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT). STUDY DESIGN: Retrospective chart review. METHODS: We reviewed the records of 165 patients with nonmetastatic NPC treated with IMRT between July 1998 and April 2011 at our institution. Recurrent nodes were delineated on imaging and coregistered with the original treatment planning computed tomography. Failures were assessed as in-field, out-field, or marginal based on the relative volumes of the recurrent nodes covered by the original dose distribution. RESULTS: Ten patients had NF at a median follow-up of 70.4 months for surviving patients. The 3- and 5-year overall survival and NF rates were 88.7%, 76.0% and 5.8%, 7.7%, respectively. Six of the nodal failures were in-field, of which five occurred in level II; whereas four had out-field failures, all of which were in the protected parotid gland area. There were no recurrences in level 1b despite this region being protected. The cumulative 3- and 5-year failure rates in the parotid gland area were 2.2% and 3.1%, respectively. Three patients with parotid failure initially had subcentimeter, nonspecific nodules in the same locations of the parotid gland as the recurrent nodes. CONCLUSION: Nodal failure is uncommon after IMRT in NPC. Recurrence in the parotid gland region accounts for all of the out-field failures and 40% of NF in our study. Comprehensive assessment of nodules in or around the parotid gland is therefore a key aspect of treatment planning and follow-up. LEVEL OF EVIDENCE: 4. Laryngoscope, 2016 127:377-382, 2017.

Modeling Dose Response for Late Dysphagia in Patients With Head and Neck Cancer in the Modern Era of Definitive Chemoradiation.

PURPOSE: To develop personalized multivariate dose-response models for late dysphagia in patients with head and neck cancer treated in the modern era of combined chemotherapy with intensity-modulated radiation therapy. PATIENTS AND METHODS: The analysis included 424 patients (oropharyngeal cancer [n = 295] and nasopharyngeal, hypopharyngeal, or laryngeal cancer [n = 129]) who received definitive chemoradiation between January 2004 and April 2009. The superior, middle, and inferior pharyngeal constrictor muscles were contoured. We calculated generalized equivalent uniform dose (gEUD) for each and the total constrictor muscle volume, with the volume effect parameter a varying from log10 a = -1 to +1 in steps of 0.1. We used the National Cancer Institute Common Toxicity Criteria for Adverse Events (version 3.0) to grade late dysphagia and logistic regression to evaluate the correlation of gEUD( a) with grade 2 or higher (≥ G2) and grade 3 or higher (≥ G3) late dysphagia at each value of a. RESULTS: Median follow-up was 33.3 months (range, 6 to 69 months). There were 41 cases (10%) of ≥ G2 dysphagia and 22 cases (5%) of ≥ G3 dysphagia. Mean doses to the total constrictor ranged from 30.1 to 85.7 Gy (median, 61.2 Gy). The predicted rate of ≥ G2 dysphagia increased by approximately 3.4% per Gy at the mean dose, for which the probability of ≥ G2 dysphagia is 50%. The threshold mean total constrictor doses that limited rates of ≥ G2 and ≥ G3 dysphagia to < 5% were < 58 Gy and < 61 Gy, respectively. Other significant factors in the multivariate predictive model included disease site, mean dose to total constrictor muscle, and patient age. CONCLUSION: Incidences of both ≥ G2 and ≥ G3 dysphagia were dependent on the mean radiation dose to the total constrictor muscle volume, disease site, and patient age. Limiting the total volume of constrictor muscle to < 58 Gy could keep the predicted rate of ≥ G2 dysphagia to < 5%.