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PDF file - 395KB, Figure S1. Pre-surgery plasma ddPCR analysis for PIK3CA mutations. Figure S2. Positive and negative controls for droplet thresholds and counts. Figure S3. Lower Limit of Detection for PIK3CA Exon 9. Supplementary Table S1: Primers used to amplify and sequence FFPE specimens. Supplementary Table S2: Primers and probes used for ddPCR on FFPE samples. Supplementary Table S3: Primers and probes for ddPCR of plasma DNA. Supplementary Table S4: Fractional abundance and 95% confidence intervals of positive ptDNA samples.

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection.Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR.Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery.Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

ObjectivesRadiation therapy (RT) is an integral part of treatment of head/neck cancer (HNC) but is associated with many toxicities. We sought to evaluate sociodemographic, pathologic, and clinical factors associated with emergency department (ED) visits, hospital admissions (HA), and RT breaks in HNC patients undergoing curative-intent RT.MethodsWe completed a Level 3 (Oxford criteria for evidence-based medicine) analysis of a cohort of HNC patients who underwent curative-intent RT at our institution from 2013 to 2017. We collected demographic characteristics and retrospectively assessed for heavy opioid use, ED visits or HA during RT as well as RT breaks. Treatment breaks were defined as total days to RT fractions ratio ≥1.6. Multivariable stepwise logistic regression analyses were done to determine the association of various sociodemographic, pathologic, and clinical characteristics with ED visits, HA and RT treatment breaks.ResultsThe cohort included 376 HNC patients (294 male, 82 female, median age 61). On multivariable analysis, significant factors associated with ED visits during RT were heavy opioid use and black race. Receipt of concomitant chemotherapy was the only factor associated with hospital admissions during RT. Advanced age, lower socioeconomic class, glandular site, and receipt of chemotherapy were all independently associated with RT breaks. Lower cancer stage and lack of substance abuse history were independently associated with lack of treatment breaks.ConclusionHNC patients with factors such as heavy opioid use, Black race, receipt of concomitant chemotherapy, and lower socioeconomic class may require closer monitoring during RT.

Background Coronavirus has serially overtaken our metropolitan hospitals. At peak, patients with acute respiratory distress syndrome may outnumber mechanical ventilators. In our Miami Hospital System, COVID‐19 cases have multiplied for 4 weeks and elective surgery has been suspended. Methods An Otolaryngologic Triage Committee was created to appropriately allocate resources to patients. Hospital ethicists provided support. Our tumor conference screened patients for nonsurgical options. Patients were tested twice for coronavirus before performing urgent contaminated operations. N95 masks and protective equipment were conserved when possible. Patients with low‐grade cancers were advised to delay surgery, and other difficult decisions were made. Results Hundreds of surgeries were canceled. Sixty‐five cases screened over 3 weeks are tabulated. Physicians and patients expressed discomfort regarding perceived deviations from standards, but risk of COVID‐19 exposure tempered these discussions. Conclusions We describe the use of actively managed surgical triage to fairly balance our patient's health with public health concerns.

Objective The objective of this study is to contrast the predictive ability of targeted muscle groups as radiographic proxies of sarcopenia on computerized tomography (CT) with body mass index (BMI) in head and neck cancer patients (HNCP) undergoing radiation at a safety net hospital, and to evaluate sarcopenia with survival, local progression, toxicities and treatment delays. Methods A retrospective review included 52 HNCP treated between 2017-2019. The posterior neck muscles (PN), sternocleidomastoids (SCM), and their summed volume (AM) were contoured at C3 on patients' pre-treatment CT scans, then normalized to obtain skeletal muscle index (MI) values. Pre-treatment BMI was also evaluated. Cutoffs for sarcopenia were determined by receiver operating characteristic curves. Overall survival and local recurrence-free survival were evaluated by Kaplan-Meier. Acute grade 3 or higher toxicities were evaluated by binomial logistic regression. Results Using all neck muscles (AM-MI) produced the best model for predicting outcomes, outperforming individual muscle groups and BMI. Local progression-free survival was worse in sarcopenic patients at 25.81 months versus 35.40 months (p=0.026). Acute grade 3 or higher toxicities were associated with sarcopenia (p=0.005). Conclusions In this small, retrospective single-institution experience at a safety net hospital, a single axial slice of the combined sternocleidomastoids and paravertebral muscles at C3 performed better than either muscle group alone or pre-treatment BMI at predicting oncologic outcomes.

Study outcomes of re-irradiation after salvage total laryngectomy (TL) for patients with recurrent laryngeal cancer. Determine overall survival (OS) and progression-free survival (PFS). Determine the incidence of severe complications associated with re-irradiation with a focus on carotid blowout (CB). Patients previously irradiated, with recurrent or second primary cancer of the larynx status-post salvage TL who received a second course of radiation from 2000 to 2017 were identified. Toxicities were measured using the CTCAE 4.0. Major toxicities were defined as grade 3+. Survival data and Kaplan-Meier curves were computed in SAS 9.4. Twenty-six patients were included in the analysis. Seventeen had progression of disease and 16 died during the follow-up period. Nine patients had no evidence of disease (NED) at last follow-up. One-, two-, and five-year OS were 65.8%, 44.2%, and 23.2%. One-, two-, and five-year PFS were 48.4%, 24.7%, and 18.5%. The most common severe toxicities were fibrosis (61.5%), dysphagia (53.8%), and wound healing complication (23.1%). Four patients (15.4%) suffered carotid blowouts. Two blowouts were preceded by manipulation of the aerodigestive tract. Patients undergoing re-irradiation for recurrent laryngeal cancer have a poor prognosis with high risk of progression or recurrence. Re-irradiation may offer survival benefit at the expense of significant toxicities. The incidence of severe toxicity is high enough to warrant pretreatment counseling. Carotid blowout was seen in 15% of patients unrelated to cancer progression. Caution should be exercised in re-irradiated patients before instrumentation of the aerodigestive tract.

Multiomic single nucleus RNA- and ATACseq profiling reveals regulators of glioma cell state diversity. The extensive intra- and intertumoral heterogeneity observed in glioma reflects the resistance to therapy and poor prognosis observed clinically. Single-cell sequencing studies have highlighted that glioma heterogeneity reflects the co-existence of cell subpopulations with distinct cell states. Prior studies have also shown that EGFR-amplifying extrachromosomal DNA (ecDNA) elements in IDH-wild-type gliomas can contribute to heterogeneity by driving oncogene amplification through long range chromatin contacts. However, single cell studies have largely focused on analyses of transcriptional profiles, and the epigenetic mechanisms underlying the contribution of ecDNA elements to tumor cell state diversity remain poorly understood. To further our understanding of the regulatory programs that contribute to transcriptional diversity and mediate the distribution of tumor cell states, we profiled primary-recurrent tumor pairs from 18 patient samples with multiomic single-nucleus RNA- and ATACseq, resulting in 86,135 cells identified with linked chromatin accessibility and gene expression profiles. Integrative clustering of the tumor cells identified tumor cell states ranging from a stem-like to differentiated- phenotype that were also associated with differences in chromatin accessibility and inferred transcription factor binding activity. Analyses of chromatin accessibility resulted in the identification of ecDNA, and integrative clustering of ecDNA+ cells highlighted distinct cell states with increased copy number burden, oncogene amplification, and differential chromatin accessibility. These results suggest that a better understanding of extrachromosomal contributions to tumor diversity would aid in development of more efficient therapies.

PurposeTo assess the performance of a proton-specific knowledge-based planning (KBP) model in the creation of robustly optimized intensity-modulated proton therapy (IMPT) plans for treatment of advanced head and neck (HN) cancer patients.MethodsSeventy-three patients diagnosed with advanced HN cancer previously treated with volumetric modulated arc therapy (VMAT) were selected and replanned with robustly optimized IMPT. A proton-specific KBP model, RapidPlanPT (RPP), was generated using 53 patients (20 unilateral cases and 33 bilateral cases). The remaining 20 patients (10 unilateral and 10 bilateral cases) were used for model validation. The model was validated by comparing the target coverage and organ at risk (OAR) sparing in the RPP-generated IMPT plans with those in the expert plans. To account for the robustness of the plan, all uncertainty scenarios were included in the analysis.ResultsAll the RPP plans generated were clinically acceptable. For unilateral cases, RPP plans had higher CTV_primary V100 (1.59% ± 1.24%) but higher homogeneity index (HI) (0.7 ± 0.73) than had the expert plans. In addition, the RPP plans had better ipsilateral cochlea Dmean (−5.76 ± 6.11 Gy), with marginal to no significant difference between RPP plans and expert plans for all other OAR dosimetric indices. For the bilateral cases, the V100 for all clinical target volumes (CTVs) was higher for the RPP plans than for the expert plans, especially the CTV_primary V100 (5.08% ± 3.02%), with no significant difference in the HI. With respect to OAR sparing, RPP plans had a lower spinal cord Dmax (−5.74 ± 5.72 Gy), lower cochlea Dmean (left, −6.05 ± 4.33 Gy; right, −4.84 ± 4.66 Gy), lower left and right parotid V20Gy (left, −6.45% ± 5.32%; right, −6.92% ± 3.45%), and a lower integral dose (−0.19 ± 0.19 Gy). However, RPP plans increased the Dmax in the body outside of CTV (body-CTV) (1.2 ± 1.43 Gy), indicating a slightly higher hotspot produced by the RPP plans.ConclusionIMPT plans generated by a broad-scope RPP model have a quality that is, at minimum, comparable with, and at times superior to, that of the expert plans. The RPP plans demonstrated a greater robustness for CTV coverage and better sparing for several OARs.

Human papilloma virus (HPV) infection is a key risk factor and etiology for oropharyngeal squamous cell carcinoma (OPSCC). HPV-induced OPSCC is rapidly increasing in incidence, with men experiencing increased mortality. When identified at an early stage, HPV-induced OPSCC can be successfully treated. Diagnosis of HPV-related OPSCC relies on an expert physical examination and invasive biopsy. Since saliva bathes the oropharyngeal mucosa and can be collected noninvasively, saliva obtained via salivary risings is an attractive body fluid for early detection of HPV-induced OPSCC. A plethora of DNA, RNA, and protein salivary biomarkers have been explored. This review discusses these markers and their robustness for detecting oncogenic HPV in OPSCC saliva samples. Methods detecting HPV DNA were more reliable than those detecting RNA, albeit both require time-consuming analyses. Salivary HPV proteomics are a new, promising focus of HPV detection research, and while more practical, lag behind nucleic acid detection methods in their development.

BACKGROUND We had previously analyzed the variables that determine the rates of opioid use at 1-year postradiotherapy in patients with head and neck cancer. Here we analyze the variables associated with opioid abstinence during and in the 12 months after radiotherapy at our institution. METHODS We identified a cohort of patients with head and neck cancer who received radiotherapy as part of curative treatment at our institution. Logistic regression analyses were performed to determine socioeconomic and clinical factors associated with opioid abstinence. RESULTS The cohort included 376 patients. On multivariable analysis, patients from an upper-income class (p = 0.004), black race (p = 0.004), older (p = 0.008), with dependent children (p

PURPOSE/OBJECTIVE(S) Females with HPV-negative head and neck squamous cell carcinoma (HNSCC) have worse outcomes compared to men in The Cancer Genome Atlas (TCGA). Our primary aim was to determine if females have an increased frequency of destructive TP53 mutations and divergent mRNA gene expression of immune markers. Our secondary aim was to determine if females have poor progression-free survival (PFS) and overall survival (OS) after controlling for TP53 mutations, immune markers, and clinicopathologic factors. MATERIALS/METHODS We identified 461 patients in TCGA with non-metastatic HNSCC (stages I-IVB) treated with radiotherapy (RT) +/- chemotherapy (CT) (N = 51) or surgery +/- adjuvant RT and CT (N = 410). We performed descriptive statistics on sex, TP53 mutations, and immune markers. We used Mann-Whitney U and Fisher's exact tests to evaluate differences in continuous log2 transformed mRNA counts for immune markers and the presence of destructive TP53 mutations, respectively. We performed Cox regression on OS and PFS according to sex, presence of destructive TP53 mutations, and a gene signature of immune markers (as a weighted sum of genes). We controlled for demographic and clinicopathologic factors. All tests were two-tailed. Final model was derived from primary covariates and confounders with P-value thresholds of < 0.05 and < 0.10, respectively. Data were extracted using open-source software packages and analyzed using statistical software. RESULTS In females, we observed increased mRNA gene expression of PD1, PDL1, IDO1, CXCL11, TIGIT, and TIM3 (all P-values < 0.05). We did not observe differences in TP53 mutations or L1CAM, SAA1, CTLA4, and LAG3 mRNA gene expression. On adjusted Cox regression for OS, female sex (HR: 1.7, CI: 1.12-1.74, P = 0.01), destructive TP53 mutations (HR: 1.63, CI: 1.03-2.60, P = 0.03), ENE (HR: 2.22, CI: 1.40-3.50, P < 0.001), ≥ four lymph nodes (HR: 1.71, CI: 1.05-2.78, P = 0.03), positive margins (HR: 2.63, CI: 1.51-4.58, P < 0.001), and a signature of immune markers (HR: 2.72, CI: 1.44-5.13, P = 0.002) were all associated with decreased OS. Adjuvant RT (HR: 0.60, CI: 0.36-0.99, P = 0.048) and CT (HR: 0.59, CI: 0.37-0.93, P = 0.02) were associated with increased OS. On adjusted Cox regression for PFS, female sex (HR: 1.36, CI: 1.00-1.87, P = 0.048), ENE (HR: 2.44, CI: 1.78-3.45, P < 0.001), ≥ four lymph nodes (HR: 1.46, CI: 1.04-2.05, P = 0.03), and positive margins (HR: 1.57, CI: 1.07-2.32, P = 0.02) were all associated with decreased PFS. We did not observe covariate interactions in either model. CONCLUSION Females with HPV-negative HNSCC had increased mRNA gene expression of specific immune markers and worse outcomes, even after controlling for relevant factors. Integration of TP53 mutations and immune marker expression may help improve health disparities research in HNSCC. Validation of our findings is ongoing using institutional data.

PURPOSE/OBJECTIVE(S) Improved stratification is needed in HPV-negative HNSCC patients with intermediate and select high-risk disease. Significant molecular and immune response features have not been well-integrated with clinicopathologic factors to predict outcomes in this population. We sought to develop and validate an integrated molecular and clinicopathologic machine learning-based predictor for OS and PFS in this population to inform a future trial design. We hypothesize that our predictor will stratify patients better than a predictor derived from standard techniques. MATERIALS/METHODS We included 253 patients from TCGA with pathologic stage III-IVB (excluded T4b; included N3a) HPV-negative HNSCC with microscopic ENE and intermediate-risk disease (close margins/LVSI/PNI), treated with surgery and RT +/- chemotherapy. We split the data into training (70%) and testing (30%) sets. We identified 29 relevant molecular features (genomic: mutation vs. no mutation, transcriptomic: ≥ vs. < upper third quartile of log2 transformed mRNA expression) associated with significant HNSCC pathways, including cellular proliferation, cellular differentiation, cell cycle control, adhesion and invasion, anti-tumor immune response, and apoptosis. We also identified 19 demographic, behavioral, and clinicopathologic factors. We performed random survival forest modeling on OS and PFS and validated the machine learning-based predictor on the testing set, using ROC/AUC. Variables of importance were identified using the "Janitza" method. We also built a predictor using standard "best" Cox proportional hazards modeling and compared AUC values for OS and PFS versus our machine learning-based values. RESULTS The median OS and PFS times were 4.4 years and 3 years, respectively. After accounting for pairwise correlations, we kept 38 variables for model building. Using the training set, significant variables of most importance for OS included age, female sex, PNI, LVSI, ≥ four pathologically involved lymph nodes, microscopic ENE, TP53 missense and nonsense mutations, and expression of PD1, LAG3, TIM3, L1CAM, CASP8, PIK3CA, E2F2, E2F4, FAT1, and NOTCH1 (all P-values < .05). Significant variables of importance for PFS included age, female sex, ≥ four pathologically involved lymph nodes, microscopic ENE, anatomic subsite, TP53 nonsense mutations, and expression of PD1, L1CAM, PIK3CA, and FAT1 (all P-values < .05). Using the testing set, we validated the 38-variable predictor on OS and PFS with AUC values of 0.82 and 0.75, respectively. In contrast, the "best" Cox models resulted in low AUC values for OS and PFS (0.65 and 0.66, respectively). CONCLUSION We developed and validated a machine learning-based predictor for OS and PFS that outperforms standard Cox predictors in HPV-negative HNSCC. This study provides a rationale to use the predictor as a stratifier in a prospective clinical trial of surgery, radiotherapy, and PD1 blockade in patients with microscopic ENE and intermediate-risk disease.

Purpose/Objective(s) The tumor suppressor gene TP53 is inactivated by mutation in a large fraction of cancers. In head and neck squamous cell carcinoma (HNSCC) patients, a subset of TP53 mutations are nonsense mutations that lead to premature termination of non-functional p53 translation. These mutations have shown worse overall survival in HNSCC yet we have not been able to use this information to optimize treatment. We hypothesized that destructive-type TP53 mutations are associated with worse overall survival (OS), even after controlling for important clinical characteristics. In addition, we sought to identify clinical factors associated with destructive-type TP53 mutations. Materials/Methods We abstracted 461 patients from The Cancer Genome Atlas (TCGA) with stages I-IVB HPV-negative HNSCC, treated with surgery +/- adjuvant therapy or with radiotherapy +/- chemotherapy. We identified TP53 mutations and categorized them as “destructive-type” versus all other mutations (e.g., missense, silent, splice site). We collected patient, tumor, and treatment information. We performed univariable (not shown) and multivariable logistic regressions (LR) to determine which factors are associated with destructive-type TP53 mutations. Then, we performed univariable (not shown) and multivariable Cox proportional hazards (CPH) regressions on OS as a function of TP53 mutations and clinical factors. Backward elimination was used for multivariable feature selection, and variables were kept in the final models if they met the P-value threshold of less than 0.05. Results 51 patients had destructive-type TP53 nonsense mutations and 211 had non-destructive type mutations. On multivariable LR, the presence of destructive-type TP53 mutations was associated with advanced age (OR:2.33, 95% CI:1.09-4.73, P = 0.022), ENE (OR:2.17, 95% CI:1.12-4.13, P = 0.019), and close (OR:2.72, 95% CI:1.15-6.00, P = 0.017) or positive margins (OR:2.36, 95% CI:1.02-5.13, P = 0.036). On multivariable CPH regression, destructive-type TP53 mutations were independently associated with worse OS (HR:1.56, 95% CI:1.02-2.37, P = 0.039), while controlling for significant clinical factors, including PNI (HR:1.43, 95% CI:1.03-1.98, P = 0.032), ≥4 lymph nodes (HR:1.62, 95% CI:1.13-2.34, P = 0.010), microscopic ENE (HR:2.10, 95% CI:1.41-3.14, P = 0.0003), gross ENE (HR:2.0, 95% CI 1.19-3.35, P = 0.008), positive margins (HR:1.52, 95% CI:1.01-2.30, P = 0.047), and advanced age (HR:1.02, 95% CI:1.01-1.04, P = 0.010). Conclusion We found that TP53 nonsense mutations are independently associated with worse OS after controlling for significant confounders. Patients with HPV-negative HNSCC with destructive-type TP53 mutations are more likely to be of advanced age and have adverse pathologic features such as ENE and close or positive margins. This study supports characterizing TP53 mutations in patients with HPV-negative HNSCC as it may further optimize patient stratification, treatment, and clinical trial designs.

Purpose Anatomical changes and patient setup uncertainties during intensity modulated proton therapy (IMPT) of head and neck (HN) cancers demand frequent evaluation of delivered dose. This work investigated a cone-beam computed tomography (CBCT) and deformable image registration based therapy workflow to demonstrate the feasibility of proton dose calculation on synthetic computed tomography (sCT) for adaptive IMPT treatment of HN cancer. Materials and Methods Twenty-one patients with HN cancer were enrolled in this study, a retrospective institutional review board protocol. They had previously been treated with volumetric modulated arc therapy and had daily iterative CBCT. For each patient, robust optimization (RO) IMPT plans were generated using ±3 mm patient setup and ±3% proton range uncertainties. The sCTs were created and the weekly delivered dose was recalculated using an adaptive dose accumulation workflow in which the planning computed tomography (CT) was deformably registered to CBCTs and Hounsfield units transferred from the planning CT. Accumulated doses from ±3 mm/±3% RO-IMPT plans were evaluated using clinical dose-volume constraints for targets (clinical target volume, or CTV) and organs at risk. Results Evaluation of weekly recalculated dose on sCTs showed that most of the patient plans maintained target dose coverage. The primary CTV remained covered by the V95 > 95% (95% of the volume receiving more than 95% of the prescription dose) worst-case scenario for 84.5% of the weekly fractions. The oral cavity accumulated mean dose remained lower than the worst-case scenario for all patients. Parotid accumulated mean dose remained within the uncertainty bands for 18 of the 21 patients, and all were kept lower than RO-IMPT worst-case scenario for 88.7% and 84.5% for left and right parotids, respectively. Conclusion This study demonstrated that RO-IMPT plans account for most setup and anatomical uncertainties, except for large weight-loss changes that need to be tracked throughout the treatment course. We showed that sCTs could be a powerful decision tool for adaptation of these cases in order to reduce workload when using repeat CTs.

Background Radiation recall dermatitis (RRD) is an acute inflammatory skin reaction occurring in a skin area previously exposed to radiotherapy and triggered by subsequent intake of a drug, most commonly a chemotherapeutic agent. RRD secondary to antibiotics has also been reported but is a rare phenomenon overall and there are no reports of RRD in association with ceftriaxone exposure. Methods We report on a 59-year-old patient who had received radiotherapy to the neck bilaterally and who developed RRD 6 months later after a single dose of intramuscular ceftriaxone. Results The patient's rash resolved without further intervention over the ensuing 2 days following administration of a single dose of ceftriaxone. Conclusion This case illustrates that while RRD secondary to antibiotic exposure is rare, it is part of the differential diagnosis to be considered for acute dermatitis when there is a past history of radiotherapy to the same skin area.

To examine the impact of ethnicity, Spanish language preference, socioeconomic status, and treatment setting on utilization of supportive services before radiotherapy (RT) among head and neck cancer patients and to determine whether a lack of these services is associated with an increased rate of adverse events.Demographic, staging, and treatment details were retrospectively collected for patients treated at a safety-net hospital (n = 56) or adjacent private academic hospital (n = 183) from January 1, 2014, to June 30, 2016. Supportive care services evaluated were limited to speech/swallowing therapy and nutrition therapy. Adverse events and performance measures examined included weight loss during RT, gastric tube placement, emergency department visits, hospital admissions, and missed RT days.On multivariable analysis, patients receiving treatment at the safety-net hospital were less likely to receive speech/swallowing services. Receiving speech/swallowing therapy before treatment was associated with less weight loss during treatment, and in conjunction with nutrition therapy, was associated with fewer missed RT days.Safety-net hospital treatment was associated with a lack of utilization of pre-RT speech/swallowing therapy which in turn was associated with increased weight loss. Interventions aimed at improving utilization of these services would improve treatment tolerance and patient outcomes.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to universal therapeutic resistance. Here, we integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes, and bulk multi-omic profiles across 11 adult IDH-mutant or IDH-wild-type gliomas to delineate sources of intratumoral heterogeneity. We found that local DNA methylation instability, or epimutation burden, was elevated in more aggressive tumors, reflected intratumoral variability, linked with transcriptional disruption, and associated with environmental stress response. We show that the activation of cell-state specific transcription factors is impacted by epimutations and that loosened epigenetic control may facilitate cellular plasticity. We validated the impact that the common environmental stressor hypoxia has on the epigenetic stability and shifts in cell states through perturbation-based in vitro experiments coupled with single-cell genomics. Our analyses support that glioma cells under stress hijack epigenetic mechanisms that regulate cell state transitions to overcome challenges posed by nutrient-poor microenvironments and therapeutic insults. We confirmed the link between cellular stress and epigenetic instability by analyzing larger cohorts of bulk longitudinally collected and spatially separated DNA methylation data. Increased DNA methylation instability was associated with accelerated disease progression, and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work provides an integrative framework to better understand glioma evolution and highlights the importance of epigenetic heterogeneity in shaping therapeutic response. Citation Format: Kevin C. Johnson, Kevin J. Anderson, Elise T. Courtois, Amit D. Gujar, Floris P. Barthel, Frederick S. Varn, Diane Luo, Martine Seignon, Eunhee Yi, Hoon Kim, Marcos R. Estecio, Ming Tang, Nicholas E. Navin, Rahul Maurya, Chew Yee Ngan, Niels Verburg, Philip C. De Witt Hamer, Ketan Bulsara, Michael L. Samuels, Sunit Das, Paul Robson, Roel G. Verhaak. Single-cell multimodal glioma analyses reveal epigenetic regulators of cellular plasticity and environmental stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2084.

Background: Adaptive radiotherapy is being used in few institutions in patients with head and neck cancer having bulky disease using periodic computed tomography imaging accounting for volumetric changes in tumor volume and/or weight loss. Limited data are available on ART in the postoperative setting. We aim to identify parameters that would predict the need for ART in patients with head and neck cancer and whether ART should be applied in postoperative setting. Materials and Methods: Twenty patients with stage III–IV head and neck cancer were prospectively accrued. A computed tomography simulation was done prior to treatment and repeated at weeks 3 and 6 of concurrent intensity-modulated radiotherapy and chemotherapy. The final plan was coregistered with the subsequent computed tomography images, and dosimetric/volumetric changes at weeks 1 (baseline), 3, and 6 were quantified in high-risk clinical target volumes, low-risk clinical target volumes , right parotid , left parotid , and spinal cord . An event to trigger ART was defined as spinal cord maximum dose >45 Gy, parotid mean dose >26 Gy, and clinical target volume coverage

Objective: MRI provides clear visualization of spinal cord, tumor, and bone for patient positioning and verification during MRI-guided radiotherapy (MRI-RT). Therefore, we wished to evaluate spine stereotactic ablative radiotherapy (SABR) feasibility with MRI-RT. Given dosimetric limitations of first generation Co-60 MRI-RT, we then evaluated improvements by newer linear accelerator (linac) MRI-RT. Methods: Nine spinal metastases were treated with Co-60 MRI-RT. Seven received a single 16 Gy fraction, and two received three fractions totaling 24 or 30 Gy. After replanning with linac MRI-RT software, comparisons of organ at risk and dose spillage objectives between Co-60 and linac plans were performed. Results: Spinal cord and cauda equina dose constraints were met in all Co-60 cases. Treatments were delivered successfully with real-time imaging during treatment and no treatment-related toxicities. While limits for dose spillage into surrounding soft tissues were not achieved due to the limitations of the Co-60 system, this could be corrected with linac MRI-RT delivery. Conclusions: MRI-RT SABR of spinal metastases is feasible with Co-60 MRI-RT. Dose delivery is improved by linac MRI-RT. Advances in knowledge: This is the first report of MRI-RT for SABR of spinal metastases. The enhanced visualization of anatomy by MRI may facilitate RT dose escalation for spine SABR.

Background No study has determined the incidence of long-term opioid use, or risk factors for long-term use, ≥1 year after radiotherapy. Methods Medical records of 276 head/neck cancer patients were retrospectively assessed for persistent opioid use 1-year after curative-intent radiotherapy. Numerous potential risk factors were assessed and the physicians' documented reasons for continued use were qualitatively categorized as suspected opioid use disorder (OUD) or as medically indicated for control of ongoing pain. Results Of note, 20 of 276 patients continued using opioids long-term. High maximum opioid dose and the use of opioids and/or psychotropics/non-opioid analgesics at the radiation oncology intake visit were associated with this outcome. Three patients continued due to suspected OUD and 17 due to medical indications. Conclusion Of note, 7.2% of patients developed long-term opioid use, which was associated with high maximum opioid dose and early initiation of opioids and/or psychotropics/non-opioid analgesics. Physicians cited medical indications as the primary reason for continued use.

SummaryCancer progression depends on both cell-intrinsic processes and interactions between different cell types. However, large-scale assessment of cell type composition and molecular profiles of individual cell types within tumors remains challenging. To address this, we developed epigenomic deconvolution (EDec), an in silico method that infers cell type composition of complex tissues as well as DNA methylation and gene transcription profiles of constituent cell types. By applying EDec to The Cancer Genome Atlas (TCGA) breast tumors, we detect changes in immune cell infiltration related to patient prognosis, and a striking change in stromal fibroblast-to-adipocyte ratio across breast cancer subtypes. Furthermore, we show that a less adipose stroma tends to display lower levels of mitochondrial activity and to be associated with cancerous cells with higher levels of oxidative metabolism. These findings highlight the role of stromal composition in the metabolic coupling between distinct cell types within tumors.

For early-stage glottic cancers, intensity-modulated radiation therapy (IMRT) has been shown to have comparable local control to 3D-conformal radiotherapy with the advantage of decreased dose to the carotid arteries. The planning target volume (PTV) for early glottic cancers typically includes the entire larynx, plus a 3 to 5 mm uniform margin. The air cavity within the larynx creates a challenge for the inverse optimization process as the software attempts to “build up” dose within the air. This unnecessary attempt at dose build-up in air can lead to hot spots within the rest of the PTV and surrounding soft tissue. We hypothesized that removal of the air from the PTV would decrease hot spots and allow for a more homogeneous plan while still maintaining adequate coverage of the PTV. We analyzed 20 consecutive patients with early-stage glottic cancer, T1-2N0, who received IMRT at our institution from April 2015 to December 2016. Each patient received 63 to 65.25 Gy in 2.25 Gy per fraction. Two plans were created for each case: one in which the PTV included the laryngeal air cavity and one in which the air cavity was subtracted from the PTV to create a new PTV-air structure. Dosimetric variables were collected for PTV-air structure from both IMRT plans, including V100%, D98% D2%, and D0.2%. Dosimetric variables for spinal cord and the carotid arteries were also recorded. Homogeneity index (HI) defined as D98/D2 was calculated. Two-sided t-tests were used to compare dosimetric variables. The median PTV volume was 69.9 cc (standard deviation [SD] ± 28.7 cc) and the median air cavity volume removed was 11.0 cc (SD ± 3.4 cc). A 2-sided t-test revealed a statistically significant decrease in max dose (112.7% vs 108.8%, p value = 0.0002) and improvement of HI (0.93 vs 0.91, p value = 0.0023) for the PTV air in the IMRT plan optimized for PTV air, which had air excluded, compared to the IMRT plan optimized for PTV with air included. There was no significant worsening of PTV-air coverage or significant increase in doses to the organs at risk (OARs). The removal of the air cavity from the PTV for early-stage glottic cancers does not compromise PTV coverage or sparing of OARs and can result in a more homogeneous IMRT plan. A more homogeneous plan has the potential to reduce treatment morbidity, although further study is warranted to investigate the clinical impact of air cavity removal from the PTV.

OBJECTIVES In this study, we aim to determine the frequency of adherence to National Comprehensive Cancer Network follow-up guidelines in a population of head and neck cancer patients who received curative treatment. We will also assess the impact of race, ethnicity, socioeconomic status, and treatment setting on utilization of follow-up care. METHODS This study included patients with biopsy-proven, nonmetastatic oropharyngeal or laryngeal cancer treated with radiotherapy between January 1, 2014, and June 30, 2016, at a safety-net hospital or adjacent private academic hospital. Components of follow-up care analyzed included an appointment with a surgeon or radiation oncologist within 3 months and posttreatment imaging of the primary site within 6 months. Univariable and multivariable analyses were conducted using a logistic regression model to estimate odds ratios and corresponding 95% confidence intervals. RESULTS Two hundred and thirty-four patients were included in this study. Of those, 88.8% received posttreatment imaging of the primary site within 6 months; 88.5% attended a follow-up appointment with a radiation oncologist within 3 months; and 71.1% of patients attended a follow-up appointment with a surgeon within 3 months. On multivariable analysis, private academic hospital treatment versus safety-net hospital treatment was associated with increased utilization of both surgical and radiation oncology follow-up. Non-Hispanic black (NHB) patients, Hispanic patients, and those with a low socioeconomic status were also less likely to receive follow-up. CONCLUSION Safety-net hospital treatment, socioeconomic status, Hispanic ethnicity, and NHB race were associated with decreased follow-up service utilization. Quality improvement initiatives are needed to reduce these disparities. LEVEL OF EVIDENCE 2b Laryngoscope, 129:2303-2308, 2019.

Objective To examine the impact of treatment setting and demographic factors on oropharyngeal and laryngeal cancer time to treatment initiation (TTI). Study Design Retrospective case series. Setting Safety net hospital and adjacent private academic hospital. Subjects and Methods Demographic, staging, and treatment details were retrospectively collected for 239 patients treated from January 1, 2014, to June 30, 2016. TTI was defined as days between diagnostic biopsy and initiation of curative treatment (defined as first day of radiotherapy [RT], surgery, or chemotherapy). Results On multivariable analysis, safety net hospital treatment (vs private academic hospital treatment), initial diagnosis at outside hospital, and oropharyngeal cancer (vs laryngeal cancer) were all associated with increased TTI. Surgical treatment, severe comorbidity, and both N1 and N2 status were associated with decreased TTI. Conclusion Safety net hospital treatment was associated with increased TTI. No differences in TTI were found when language spoken and socioeconomic status were examined in the overall cohort.

Online adaptive radiotherapy (ART) with frequent imaging has the potential to improve dosimetric accuracy by accounting for anatomical and functional changes during the course of radiotherapy. Presented are three interesting cases that provide an assessment of online adaptive magnetic resonance-guided radiotherapy (MRgRT) for lung stereotactic body radiotherapy (SBRT). The study includes three lung SBRT cases, treated on an MRgRT system where MR images were acquired for planning and prior to each treatment fraction. Prescription dose ranged from 48 to 50 Gy in four to five fractions, normalized to where 95% of the planning target volume (PTV) was covered by 100% of the prescription dose. The process begins with the gross tumor volume (GTV), PTV, spinal cord, lungs, heart, and esophagus being delineated on the planning MRI. The treatment plan was then generated using a step-and-shoot intensity modulated radiotherapy (IMRT) technique, which utilized a Monte Carlo dose calculation. Next, the target and organs at risk (OAR) contours from the planning MRI were deformably propagated to the daily setup MRIs. These deformed contours were reviewed and modified by the physician. To determine the efficacy of ART, two different strategies were explored: 1) Calculating the plan created for the planning MR on each fraction setup MR dataset (Non-Adapt) and 2) creating a new optimized IMRT plan on the fraction setup MR dataset (FxAdapt). The treatment plans from both strategies were compared using the clinical dose-volume constraints. PTV coverage constraints were not met for 33% Non-Adapt fractions; all FxAdapt fractions met this constraint. Eighty-eight percent of all OAR constraints studied were better on FxAdapt plans, while 12% of OAR constraints were superior on Non-Adapt fractions. The OAR that garnered the largest benefit would be the uninvolved lung, with superior sparing in 92% of the FxAdapt studied. Similar, but less pronounced, benefits from adaptive planning were experienced for the spinal cord, chest wall, and esophagus. Online adaptive MR-guided lung SBRT can provide better target conformality and homogeneity and OAR sparing compared with non-adaptive SBRT in selected cases. Conversely, if the PTV isn't adjacent to multiple OARs, then the benefit from ART may be limited. Further studies, which incorporate a larger cohort of patients with uniform prescriptions, are needed to thoroughly evaluate the benefits of daily online ART during MRgRT.

Background Patients with head and neck cancer are at high risk for second primary malignancies. Human papillomavirus (HPV)-driven tumors are generally high-grade oropharyngeal cancers. We analyzed the incidence of second primary malignancy of the head and neck in patients with primary squamous cell carcinoma (SCC) of the head and neck and temporal trends in the HPV era. Methods The Surveillance, Epidemiology, and End Results (SEER) database was queried for patients with SCC of the head and neck (range, 1973–2008). Cumulative incidence rates of second primary malignancy of the head and neck were compared based on competing risk analysis. Results A total of 104,639 cases were included in this study, of which 4616 patients had second primary malignancy of the head and neck. Oropharyngeal cancer incidence increased over time. Estimated incidence rate/10,000 person-years (105.5, 80.6, and 50.2 for 1973–1989, 1990–1999, and 2000–2008, respectively) and cumulative incidence rates (10-year rates of 6.68%, 5.72%, and 4.59% for 1973–1989, 1990–1999, and 2000–2008, respectively) of second primary malignancies of the head and neck for patients with oropharyngeal cancer decreased over time (p

Introduction Patients with stage III non-small-cell lung cancer and poor performance status and/or weight loss do not seem to benefit from standard therapy. Based on the preclinical interaction between epidermal growth factor receptor inhibitors and radiation, we designed a trial of induction chemotherapy followed by thoracic radiotherapy and concurrent erlotinib. Methods Patients with poor-risk unresectable stage III non-small-cell lung cancer received two cycles of carboplatin at an AUC of 5 and nab-paclitaxel at 100 mg/m on days 1 and 8 every 21 days, followed by erlotinib administered concurrently with thoracic radiotherapy. Maintenance was not permitted. Molecular analysis was performed in available specimens. Seventy-two eligible patients were required to test whether the 1-year survival rate was less than 50% or greater than or equal to 65% with approximately 90% power at a significance level of 0.10. Results From March 2008 to October 2011, 78 patients were enrolled, three of whom were ineligible. The median age was 68 (range, 39-88) and 32% were aged greater than or equal to 75 years. Patients were evenly distributed between stages IIIA and IIIB and the majority had performance status 2. The overall response rate was 67% and the disease control rate was 93%. Treatment was well tolerated. The median PFS and OS were 11 and 17 months, respectively. The overall 12-month OS was 57%, which narrowly missed the prespecified target for significance. Conclusions Patients with poor-risk stage III non-small-cell lung cancer had better than expected outcomes with a regimen of induction carboplatin/nab-paclitaxel followed by thoracic radiotherapy and erlotinib. However, as per the statistical design, the 12-month OS was not sufficiently high to warrant further studies.

Background The emergence of transoral robotic surgery (TORS) ignited the debate between surgical and nonsurgical strategies on oropharyngeal squamous cell carcinoma (SCC) management; a question further complicated by human papillomavirus (HPV). We evaluated the survival by treatment strategy independently in HPV-related and HPV-nonrelated oropharyngeal SCC. Methods The National Cancer Database was queried for patients with oropharyngeal SCC with known HPV status who underwent primary TORS or primary radiotherapy. The overall survival (OS) was compared by treatment strategy, including propensity matching to control for confounders. Results Of 1873 patients, 73% were HPV-positive and 30% were treated with TORS. The propensity-matched patients with HPV-positive disease displayed no significant difference in 3-year survival; 95% versus 91% (P = .116) for the TORS versus primary radiotherapy. In the HPV-negative cohort, TORS was associated with superior survival; 84% versus 66% (P = .01). Conclusion The TORS-based approach was associated with superior survival in patients with HPV-negative oropharyngeal SCC; similar difference was not observed in patients with HPV-positive disease.

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

Background This study was a retrospective analysis of elderly patients treated with stereotactic body radiotherapy (SBRT) in the setting of a multi-institutional consortium. Patients and Methods Three institutions pooled data on patients aged ≥ 75 years who received SBRT for stage I non–small-cell lung cancer (NSCLC). Forty-seven tumors in 46 patients were analyzed in patients aged 75 to 92 years (median, 82 years). Treatment was delivered during 2007 to 2009, with a median follow-up of 12.4 months. All patients underwent staging positron emission tomography–computed tomography (PET-CT), and 87% of tumors were confirmed by biopsy results. Total doses were 35 to 60 Gy, mainly in 3 to 5 fractions. All tumors were treated using a linear accelerator, with 96% of patients receiving 3-dimensional (3D) conformal RT and 4% undergoing intensity modulated RT (IMRT). Results At the time of analysis, the local failure rate was 2% (1 of 47). The regional failure rate was 9% (4 of 47). The distant failure rate was 6% (3 of 47). The combined failure rate was 15% (7 of 47) because 1 patient experienced both regional and distant failure. Among 20 tumors with any acute toxicity, there were no ≥ grade 3 toxicities. Pneumonitis (n = 10) grades 1 (n = 3) and 2 (n = 2) was seen in 15% and 10% of patients, respectively; these data were missing for 25% of patients. Conclusion SBRT in patients aged ≥ 75 years with stage I NSCLC proved tolerable, with toxicity rates comparable to those in younger patients. Excellent rates of local, regional, and distant control were achieved at a median follow-up of 12.4 months. This patient population represents a rapidly growing segment of the early lung cancer population, and SBRT appears to be a safe and effective treatment option for patients who are not optimal candidates for surgery.

Head and neck soft tissue sarcoma (HNSTS) is a distinct entity within the soft tissue sarcoma group and the evidence supporting treatment options for this disease is poorly defined. Using the National Cancer Database, this study aims to reveal adjuvant therapy utilization patterns and their outcomes.HNSTS patients constituted 6.5% of all sarcoma patients and the analysis was limited to non-metastatic patients who underwent resection between 2004 and 2012. Chi-square test assessed the distribution of demographic, tumor and treatment variables in the treatment subgroups. Kaplan-Meier method estimated overall survival and proportional hazards model estimated survival hazard ratios for prognostic factors including age, comorbidity, gender, race, size, grade, margin status, histology, chemotherapy administration and radiotherapy technique/dose.The cohort included 2493 patients with a median age of 62years. Adjuvant therapies were more frequently delivered in high-grade, large tumors and with positive surgical margins. Limiting the analysis to 788 high-grade HNSTS patients, adjunctive radiotherapy improved 5-year survival from 44% (95% confidence interval (CI): 38-50) to 49% (CI: 43-55); reducing mortality hazards ratio by 24% (p0.001). On multivariate analysis, older age, positive surgical margins and large tumor size were significant predictors of poor survival. These findings were consistent after propensity score analysis.Our analysis suggests that adjuvant radiotherapy is associated with improved survival in high-grade HNSTS. Higher dose and precise technique did not accentuate this benefit; however, further studies are needed to refine the treatment strategies, including the role of chemotherapy in the management of this rare disease.

For decades, the standard of care for radiation treatment of early larynx cancers has been conventional treatment using opposed lateral fields encompassing the larynx and overlying neck structures, including the adjacent carotid arteries. While intensity-modulated radiotherapy (IMRT) has replaced conventional radiotherapy for all other head/neck cancer situations, the use of IMRT to treat early glottic cancers remains controversial. The article reviews the published experience with IMRT for this clinical situation and provides a detailed review of the literature on radiation-induced carotid toxicity and how it might apply to the controversy. Finally, we discuss whether the radiation oncology community should transition to IMRT as a new standard of care for the treatment of early glottic cancers.

Extensive-stage small cell lung cancer (ESCLC) includes metastatic disease and locally advanced disease confined to the thorax that cannot be encompassed in a typical radiation portal. We assessed and then compared the benefits of thoracic radiotherapy (TRT) and/or brain radiotherapy (BRT) on overall survival (OS) between the intrathoracic (T-ESCLC) and metastatic (M-ESCLC) groups using the Surveillance Epidemiology and End Results database. TRT and BRT data were available for 10150 patients treated from 1988−1997. The T-ESCLC group included 1774 patients. The Kaplan–Meier method was used to estimate OS and the proportional hazards model was used to estimate OS hazard ratios for prognostic factors including age, gender, race, tumor size, T/N stage, TRT, and BRT. The 2-year OS for T-ESCLC was 7.8 % compared to 3 % in the M-ESCLC group (p

Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.

2518Background: DNA-dependent protein kinase (DNA-PK), regulates one of the major pathways responsible for repair of DNA double-strand breaks. The combination of RT and DNA-PK inhibition (DNA-PKi) ...