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VMAT-based single-isocenter multiple-targets (SIMT) are sensitive to rotational uncertainties and leads to greater possibility of dose coverage loss for off-axis targets. Instead of treating all targets in one plan, it is common practice to separate them into multiple groups based on their distance. It ensures the targets are within reasonable distance and therefore increases robustness, but resulting in increased planning complexity and lower delivery efficiency. Studies have yet to evaluate the impact of separating targets for multiple scattered targets. This work aims to investigate the dosimetric and robustness impact of separating strategy. 10 patients (95 metastases, 4-19 per patient) were retrospectively included. The PTV was 1mm margin expansion to GTV. Two sets of plans were created using HyperArc technique: (1) A single SIMT plan (non-separated) included all PTVs. The PTV to isocenter distance ranged from 2.4cm to 9.5cm (mean 5.7cm). (2) Multiple SIMT plans (separated) so in each plan PTV center to isocenter distance was < = 5cm. 3-4 plans were needed to treat all targets. The mean dose, V6Gy and V12Gy of brain were calculated. The dose coverage against setup uncertainty was evaluated by shifting/rotating dose distribution around the isocenter. 1mm translations in three directions and 1° rotations around three axes (total 12 scenarios) were simulated. The dosimetric changes of GTVs were evaluated using V100% (relative volume covered by prescribed dose) and D99% (relative minimum dose delivered to 99% of the GTV). The non-separated plan increased brain mean dose from separated plan by an average of 22.3cGy (std 15.9cGy). The brain V6Gy V12Gy were increased by 3.3cc and 0.86cc on average in the non-separated plans. Across all 12 1mm/1° translation/rotation uncertainty cases, the average GTV [V100%, D99%] is [99.8%, 103.2%] in non-separated plans, compared with [99.9%, 109.0%] in separated plans. Under worst situation, 6/95 GTVs in nonseparated plans have D99% < 95% (lowest 90.5%), while only one GTV in separated plans have D99% < 95% (lowest 94.4%). Compared with separating targets based on their distances, treating all targets in one plan has similar brain dose sparing with slightly increased low dose distribution. Considering setup uncertainty, non-separated plans maintained GTV dose coverage with average V100% similar to separated plans but degraded D99%. The delivery time of non-separated plans is 3-4 times faster than separated plans. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) integration into the human genome is an established driver event in head and neck squamous cell carcinoma (HNSCC). Functionally, HPV oncogenic proteins inhibit tumor suppressor proteins and activate proliferation of infected epithelial cells. Despite seeming homogeneous, individual epithelial cells among a tumor may have significant cellular and molecular heterogeneity which could affect treatment outcomes. This study aimed to better characterize the single cell molecular heterogeneity within HPV-positive HNSCC. Using a well-characterized HPV-positive HNSCC cell line (UM-SCC-47) we performed integrated single cell multiome RNA (scRNA-seq) and ATAC sequencing using 10x and sequencing platforms. Integration site analysis was done using multiple sequence alignment. Multimodal analysis was then performed using the Seurat package. Molecular profiling from sections of four HPV-positive primary tumors was also performed using the Visium Spatial Platform from 10X Genomics. Cell type identification analysis was performed using SpaceXR. HPV-associated phenotype enrichment was resolved relative to the HPVon signature derived from Puram et al., Nature Genetics, 2023. Using single cell ATAC-seq data we performed multiple sequence alignment with the transitive consistency score to demonstrate the HPV16 integration at a single cell level. This showed that there are two integration sites for HPV (3:189879025, 3:189895061). Using scRNAseq we also demonstrated that TP63 expression is positively correlated with E7 expression (R2 = 0.676). Other HPV-associated genes include KTN1 (R=0.43), ALDH1A3 (0.42), PTMA (0.41), and VMP1 (0.40). Spots containing an enriched HPVon signature also have enrichment of multiple gene sets including, MYC signaling (R=0.77), E2F transcription (R=0.65), and G2-M DNA damage checkpoint (R=0.63). Within the cell type identification analysis, after normalizing by tumor proportion greater than 50%, additional gene set analysis showed enrichment of cytoskeletal regulation and cell adhesion pathways. Typically, HPV-associated HNSCC has better prognosis than other forms of HNSCC. Recently, there has been a trend towards treatment de-escalation for patients with the goal to maintain excellent prognosis and minimize treatment side-effects. However, our study supports the emerging hypothesis that high levels of intra-tumoral heterogeneity may be a critical feature of HPV-associated HNSCC that will fail therapy. Our multi—omics single cell analyses show there is significant heterogeneity in HPV expression which should be considered in future biomarker studies. [ABSTRACT FROM AUTHOR]

In Merkel cell carcinoma (MCC), clinical factors, including advanced age and immunosuppression, are variably prognostic and poorly predictive of treatment response. There is a clear need for robust genetic markers of aggressive disease to inform treatment selection and prognostication. In a cohort of 54 MCC patients, we assessed the impact of tumor mutational burden (TMB), single nucleotide (SNV) and copy number variations (CNV), and Merkel Cell Polyomavirus (MCPyV) integration on MCC-specific survival outcomes. Genomic DNA was harvested from paraffin-embedded tissue blocks obtained from patients with biopsy-proven MCC of any anatomic subsite. A total of 54 patients with MCC were included from 2016 – 2019. We performed targeted exome sequencing of a 226-gene panel on a sequencing platform and used our established bioinformatics pipelines for targeted TMB, SNV, CNV, and MCPyV integration site calls. We then assessed the prognostic impact of specific genetic alterations on MCC-specific survival using Kaplan-Meier curves and Cox regression. Demographic and clinical characteristics of our cohort are shown in the Table. Median (range) follow-up duration after treatment completion was 43 (1 – 78) months. Five-year MCC-specific survival was 75 % (95 % CI: 56.5 – 85.9%). The median (range) targeted TMB was 1 (0 – 69). Frequently altered genes included LRP1B (n = 10, 18.5 %), FAT1 (n = 9, 16.7 %), KMT2D (n = 9, 16.7 %), RB1 (n = 7, 13.0 %), and ABL1 (n = 6, 11.1 %). In 36 of 44 (81.8 %) MCPyV-positive cases, virus was integrated into the host genome with a median (range) of 2 (1 – 30) distinct integration events. In six tumors, MCPyV integrated into COSMIC Tier 1 or Tier 2 cancer-related host genes, thus possibly altering their native function, including ABL2, BLM, HIF1A, KMT2A, MYCL , and TET2. A receiver operating characteristic curve was used to identify a targeted TMB = 13 as a cut-point to maximize differences in MCC-specific survival. Using this cut-point, a higher targeted TMB strongly predicted MCC-specific survival (p = 0.001), even when controlling for clinical nodal status. Tumor MCPyV status, virus state (i.e., integrated vs episomal), and specific SNV/CNVs did not predict MCC-specific survival. Higher tumor mutational burden portends worse survival in MCC and may be a clinically useful biomarker of treatment response and for prognostication for this challenging disease. [ABSTRACT FROM AUTHOR]

Radiotherapy is the main treatment for esophageal cancer. Previous studies have shown that radiotherapy not only kills tumor cells directly, but also reshapes the immune microenvironment of the tumor. It has been reported an increase in the recruitment of myeloid-derived suppressor cells (MDSC) can occur in tumor tissue after ionizing radiation. Exosomes are mediators of intercellular information exchange and are also involved in the regulation of the tumor microenvironment. In this study, we wanted to understand whether MDSC in esophageal cancer tissue are involved in the regulation of tumor cell response to ionizing radiation via exosomes. KYSE-150 was used to construct a subcutaneous transplantation tumor model in nude mice. And then mice irradiated with 5 Gy×5fx and 0 Gy×5fx respectively. After irradiation, the spleens of the mice were used to isolate MDSC, and collect the cell supernatants to extract the exosomes. Based on the exosomes, we divided the experiment into three groups (control, exosomes, exosomes+radiation). Exosomes were injected into a nude mouse model of esophageal cancer via the tail vein or co-cultured with KYSE-150 cells. Mice were irradiated with a 5 Gy×5fx after completion of injection, and KYSE-150 cells were irradiated with a single dose 4 Gy. After radiation, KYSE-150 cells were used to detect cell cloning, apoptosis and cell cycle by flow cytometry, cell proliferation by CCK 8. XRCC4,XRCC5,XRCC6,γH2AX,ATM expression in cells and tumor tissue were measured by Western blot and RT-PCR. The tumor volume was significantly reduced after 5 Gy x 5fx radiation. When exosomes co-cultured with KYSE-150 cells, decrease in apoptosis and increase in cell cloning and cell proliferation were found in the exosomes+radiation group and exosomes group after radiation when compared with the control group, with this change being more pronounced in the exosome+radiation group. The results of the cell cycle assay showed that after ionizing radiation, the proportion of cells in the G0/G1 phase was significantly lower, and the proportion of cells in the S and G2/M phases were significantly higher in the exosomes+radiation group and exosomes group when compared to the Control group. The protein and mRNA expression of XRCC4,XRCC5,XRCC6,γH2AX,ATM in cells were increased in exosomes+radiation group and exosomes group after radiation when compared with the control group, with this change being more obvious in the exosome+radiation group. After irradiation, tumor volumes were measured in nude mice and the results showed that exosomes+radiation group tumors were the largest in volume, while the control group regressed most significantly after irradiation. MDSC-derived exosomes have a tumor growth-promoting effect in esophageal squamous carcinoma, which is enhanced by ionizing radiation, and this may be related to the accelerated repair of damage in tumor tissue after radiation. [ABSTRACT FROM AUTHOR]

Postoperative radiotherapy (PORT) is necessary in patients with locally advanced esophageal squamous cell carcinoma (LA-ESCC) who have not received neoadjuvant chemoradiotherapy (NCRT). However, the optimal clinical target volume has under hot debate for decades. This clinical trial aims to estimate the optimal radiation volume of PORT for patients with LA-ESCC. Patients confirmed LA-ESCC (pT3-4N0-3M0) after esophagectomy without NCRT were randomly assigned to either large-field irradiation (LFI, primary lesion and lymph node tumor bed plus elective nodal irradiation) arm or small-field irradiation (SFI, primary lesion and lymph node tumor bed alone) arm with a ratio of 1:1, stratified by T stage and number of lymph node metastasis (LN<3 vs LN≥3). The primary endpoint was disease-free survival (DFS); secondary endpoints included overall survival (OS), adverse events and first failure patterns. The log-rank test was used to estimate the survival differences. The Chi-square test was used to compare adverse events and failure patterns between groups. In the intention-to-treat analysis, a total of 401 patients with LA-ESCC were randomly assigned to the LFI arm (n=210) and the SFI arm (n=191). With a median follow-up of 38.9 months, the DFS and OS rates of the whole population at 1-year and 3-year were 72.9% and 51.8%, 88.4% and 61.7%, respectively, which were close to patients who received NCRT. Patients in the two irradiation arms had similar DFS (LFI vs SFI, 46.6 vs 48.1 months; HR=0.92, 95% CI, 0.68-1.25; p=0.60). The difference of OS between the two arms did not reach significant (NR vs 73.5 months, HR=0.82, 95% CI, 0.59-1.14; p=0.26). The locoregional recurrence-free survival (HR=0.57, 95% CI 0.35-0.92; p=0.022) and the failure patterns (p=0.021) between two arms were significantly different. Locoregional failure occurred in 12.9% and 20.4% of patients in the LFI arm and SFI arm, respectively; The differences of distant diseases and distant metastasis-free survival of patients between the two arms had no significance. The most frequent toxicity was grade 2 esophagitis (LFI vs SFI, 22.9% vs 16.8%). Grade 3 acute adverse events occurred in 6.7% and 2.6% of patients in the LFI and SFI arms, respectively. No toxicity of grade 4 or 5 was recorded in this study. Both postoperative irradiation fields are alternatives for LA-ESCC patients without NCRT with the promising survival outcomes comparable to CROSS study. Patients in the LFI arm achieved better locoregional control with a safety profile consistent with the SFI arm. [ABSTRACT FROM AUTHOR]

Purpose/objective(s): A novel O-ring Linac (ORL) offers a fully enclosed gantry with a single energy (6 MV) flattening filter-free photon beam, improved kV CBCT, a maximum treatment length of 36 cm (dual-isocenter setup), and a translation-only 3DOF couch for IGRT. The purpose of this study was to evaluate the viability of this ORL for use in a single-room clinic setting and to assess implementation challenges.Materials/methods: Treatment statistics of the single-energy ORL over a 1-year period from our main campus (5-linac lineup) were used to develop a ORL machine utilization model; 2 years of treatment statistics from 3 single-room locations with multi-energy C-arm Linac were analyzed to generate a population-based disease distribution model for a typical single-room center. A percentage frequency distribution of key anatomical sites is summarized in Table 1. The ORL utilization model was then extended to the network disease distribution model to assess the percentage of patients in network that can be treated using the ORL.Results: Table 1 shows the 9 anatomical sites accounting for ∼96% of network cases, all of which have corresponding active treatment protocols using the ORL at our main campus. Some 2D/3D treatments required modifications to planning workflow based on technical limitations of the ORL. Less than 1% of treatments at network locations required field sizes exceeding 36 cm, which is the ORL's limit. Electron treatments constitute ∼10% of network cases, of which 74% were for breast boost treatments. Considering 80% of breast boost treatments at our main campus were delivered on ORLs, over half of network electron cases can be safely treated on an ORL with photons. kV-CBCT is required for all ORL daily treatments. Despite the lack of 6DOF couch, ∼80% of ORL treatment fractions did not require manual repositioning for rotational corrections. Averaged appointment durations were reduced from 20 min on C-Arm Linac to 14 min on ORL.Conclusion: Our model indicated that approximately 95% of the patient population at a single-Linac clinic can be treated using the ORL photon-only system. Streamlined IGRT workflow improved the ORL's overall efficiency compared to C-Arm Linac. Although some modifications to planning workflows are required, the ORL improves overall efficiency and is in general suitable for a single-room clinic. [ABSTRACT FROM AUTHOR]