BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1.

BACKGROUND: While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized. METHODS: BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition. RESULTS: BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice. CONCLUSION: Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

Personalized Composite Dosimetric Score-Based Machine Learning Model of Severe Radiation-Induced Lymphopenia Among Patients With Esophageal Cancer.

PURPOSE: Radiation-induced lymphopenia (RIL) is common among patients undergoing radiation therapy (RT)' Severe RIL has been linked to adverse outcomes. The severity and risk of RIL can be predicted from baseline clinical characteristics and dosimetric parameters. However, dosimetric parameters, e.g. dose-volume (DV) indices, are highly correlated with one another and are only weakly associated with RIL. Here we introduce the novel concept of "composite dosimetric score" (CDS) as the index that condenses the dose distribution in immune tissues of interest to study the dosimetric dependence of RIL. We derived an improved multivariate classification scheme for risk of grade 4 RIL (G4RIL), based on this novel RT dosimetric feature, for patients receiving chemo RT for esophageal cancer. METHODS AND MATERIALS: DV indices were extracted for 734 patients who received chemo RT for biopsy-proven esophageal cancer. Nonnegative matrix factorization was used to project the DV indices of lung, heart, and spleen into a single CDS; XGBoost was employed to explore significant interactions among predictors; and logistic regression was applied to combine the resultant CDS with baseline clinical factors and interaction terms to facilitate individualized prediction of immunotoxicity. Five-fold cross-validation was applied to evaluate the model performance. RESULTS: The CDS for selected immune organs at risk (ie, heart, lung, and spleen) (OR 1.791; 95 CI [1.350, 2.377]) was a statistically significant risk determinant for G4RIL. Pearson correlation coefficients for CDS versus G4RIL risk for individual immune organs at risk were greater than any single DV indicx. Personalized prediction of G4RIL based on CDS and 4 clinical risk factors yielded an area under the curve value of 0.78. Interaction between age and CDS revealed that G4RIL risk increased more sharply with increasing CDS for patients aged ≥65 years. CONCLUSIONS: Risk of immunotoxicity for patients undergoing chemo RT for esophageal cancer can be predicted by CDS. The CDS concept can be extended to immunotoxicity in other cancer types and in dose-response models currently based on DV indices. Personalized treatment planning should leverage composite dosimetric scoring methods rather than using individual or subsets of DV indices.

Metformin in Conjunction With Stereotactic Radiotherapy for Early-stage Non-small Cell Lung Cancer: Long-term Results of a Prospective Phase II Clinical Trial.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is increasingly detected in early stages and there is interest in improving outcomes with stereotactic body radiotherapy (SBRT). As metformin affects NSCLC signaling pathways, it might alter the metabolism of NSCLC treated with SBRT. This study investigated the long-term outcomes of a phase II clinical trial evaluating metformin in conjunction with SBRT for early-stage NSCLC. PATIENTS AND METHODS: The trial evaluated patients with American Joint Commission on Cancer (AJCC) 7th edition Stage I-II, cT1-T2N0M0 NSCLC who were randomized 6:1 to receive metformin versus placebo in conjunction with SBRT. The outcomes analyzed included local failure (LF), progression-free survival (PFS), overall survival (OS), and Common Terminology Criteria for Adverse Events (CTCAE) version 4 toxicities. RESULTS: There were 14 patients randomized to the metformin arm and one to the placebo. Median follow-up was four years. In the metformin group, the median PFS was 4.65 years [95% confidence interval (CI)=0.31-5.93] and median survival was 4.97 years (95%CI=3.05-4.61). Five year PFS was 27.8% (95%CI=5.3-57.3%) and OS was 46.0% (95%CI=16.0-71.9%). The one patient randomized to placebo was alive and without progression at five years. There were no LFs in the primary SBRT treatment volumes and no CTCAE version 4 Grade ≥3 adverse events. CONCLUSION: Outcomes of SBRT and metformin for early-stage NSCLC were similar to historic controls. These findings along with the results of the NRG-LU001 and OCOG randomized trials do not support the therapeutic use of metformin for NSCLC.

Plasmonic nano-aperture label-free imaging of single small extracellular vesicles for cancer detection.

BACKGROUND: Small extracellular vesicle (sEV) analysis can potentially improve cancer detection and diagnostics. However, this potential has been constrained by insufficient sensitivity, dynamic range, and the need for complex labeling. METHODS: In this study, we demonstrate the combination of PANORAMA and fluorescence imaging for single sEV analysis. The co-acquisition of PANORAMA and fluorescence images enables label-free visualization, enumeration, size determination, and enables detection of cargo microRNAs (miRs). RESULTS: An increased sEV count is observed in human plasma samples from patients with cancer, regardless of cancer type. The cargo miR-21 provides molecular specificity within the same sEV population at the single unit level, which pinpoints the sEVs subset of cancer origin. Using cancer cells-implanted animals, cancer-specific sEVs from 20 µl of plasma can be detected before tumors were palpable. The level plateaus between 5-15 absolute sEV count (ASC) per µl with tumors ≥8 mm3. In healthy human individuals (N = 106), the levels are on average 1.5 ASC/µl (+/- 0.95) without miR-21 expression. However, for stage I-III cancer patients (N = 205), nearly all (204 out of 205) have levels exceeding 3.5 ASC/µl with an average of 12.2 ASC/µl (±9.6), and a variable proportion of miR-21 labeling among different tumor types with 100% cancer specificity. Using a threshold of 3.5 ASC/µl to test a separate sample set in a blinded fashion yields accurate classification of healthy individuals from cancer patients. CONCLUSIONS: Our techniques and findings can impact the understanding of cancer biology and the development of new cancer detection and diagnostic technologies.

Small extracellular vesicles (sEVs) are tiny particles derived from cells that can be detected in bodily fluids such as blood. Detecting sEVs and analyzing their contents may potentially help us to diagnose disease, for example by observing differences in sEV numbers or contents in the blood of patients with cancer versus healthy people. Here, we combine two imaging methods ­ our previously developed method PANORAMA and imaging of fluorescence emitted by sEVs­to visualize and count sEVs, determine their size, and analyze their cargo. We observe differences in sEV numbers and cargo in samples taken from healthy people versus people with cancer and are able to differentiate these two populations based on our analysis of sEVs. With further testing, our approach may be a useful tool for cancer diagnosis and provide insights into the biology of cancer and sEVs.

Redefining FLASH RT: the impact of mean dose rate and dose per pulse in the gastrointestinal tract.

Background: The understanding of how varying radiation beam parameter settings affect the induction and magnitude of the FLASH effect remains limited. Purpose: We sought to evaluate how the magnitude of radiation-induced gastrointestinal (GI) toxicity (RIGIT) depends on the interplay between mean dose rate (MDR) and dose per pulse (DPP). Methods: C57BL/6J mice were subjected to total abdominal irradiation (11-14 Gy single fraction) under conventional irradiation (low DPP and low MDR, CONV) and various combinations of DPP and MDR up to ultra-high-dose-rate (UHDR) beam conditions. The effects of DPP were evaluated for DPPs of 1-6 Gy while the total dose and MDR were kept constant; the effects of MDR were evaluated for the range 0.3- 1440 Gy/s while the total dose and DPP were kept constant. RIGIT was quantified in non-tumor-bearing mice through the regenerating crypt assay and survival assessment. Tumor response was evaluated through tumor growth delay. Results: Within each tested total dose using a constant MDR (>100 Gy/s), increasing DPP led to better sparing of regenerating crypts, with a more prominent effect seen at 12 and 14 Gy TAI. However, at fixed DPPs >4 Gy, similar sparing of crypts was demonstrated irrespective of MDR (from 0.3 to 1440 Gy/s). At a fixed high DPP of 4.7 Gy, survival was equivalently improved relative to CONV for all MDRs from 0.3 Gy/s to 104 Gy/s, but at a lower DPP of 0.93 Gy, increasing MDR produced a greater survival effect. We also confirmed that high DPP, regardless of MDR, produced the same magnitude of tumor growth delay relative to CONV using a clinically relevant melanoma mouse model. Conclusions: This study demonstrates the strong influence that the beam parameter settings have on the magnitude of the FLASH effect. Both high DPP and UHDR appeared independently sufficient to produce FLASH sparing of GI toxicity, while isoeffective tumor response was maintained across all conditions.

Esophageal Cancer Outcomes After Definitive Chemotherapy With Intensity Modulated Proton Therapy.

Purpose: The effectiveness of intensity-modulated proton therapy (IMPT) for esophageal cancer treated with definitive concurrent chemoradiation therapy remains inadequately explored. We investigated long-term outcomes and toxicity experienced by patients who received IMPT as part of definitive esophageal cancer treatment. Patients and Methods: We retrospectively identified and analyzed 34 patients with locally advanced esophageal cancer who received IMPT with concurrent chemotherapy as a definitive treatment regimen at The University of Texas MD Anderson Cancer Center from 2011 to 2021. The median IMPT dose was 50.4 GyRBE in 28 fractions; concurrent chemotherapy consisted of fluorouracil and/or taxane and/or platinum. Survival outcomes were determined by the Kaplan-Meier method, and toxicity was scored according to the Common Terminology Criteria for Adverse Events version 4.0. Results: The median age of all patients was 71.5 years. Most patients had stage III (cT3 cM0) adenocarcinoma of the lower esophagus. At a median follow-up time of 39 months, the 5-year overall survival rate was 41.1%; progression-free survival, 34.6%; local regional recurrence-free survival, 78.1%; and distant metastasis-free survival, 65.0%. Common acute chemoradiation therapy-related toxicities included hematologic toxicity, esophagitis (and late-onset), fatigue, weight loss, and nausea (and late-onset); grade 3 toxicity rates were 26.0% for hematologic, 18.0% for esophagitis and 9.0% for nausea. No patient had grade ≥3 wt loss or radiation pneumonitis, and no patients had pulmonary fibrosis or esophageal fistula. No grade ≥4 events were observed except for hematologic toxicity (lymphopenia) in 2 patients. Conclusion: Long-term survival and toxicity were excellent after IMPT for locally advanced esophageal cancer treated definitively with concurrent chemoradiation therapy. When available, IMPT should be offered to such patients to minimize treatment-related cardiopulmonary toxicity without sacrificing outcomes.

Comparing 90-Day Postoperative Mortality After Neoadjuvant Proton-Based Versus Photon-Based Chemoradiotherapy for Esophageal Cancer.

Purpose: Evidence suggests that proton-beam therapy (PBT) results in less toxicity and postoperative complications compared to photon-based radiotherapy in patients who receive chemoradiotherapy followed by esophagectomy for cancer. Ninety-day mortality (90DM) is an important measure of the postoperative (nononcologic) outcome as proxy of quality-of-care. We hypothesize that PBT could reduce 90DM compared to photon-based radiotherapy. Materials and Methods: From a single-center retrospective database patients treated with chemoradiotherapy before esophagectomy for cancer were selected (1998-2022). Univariable logistic regression was used to study the association of radiotherapy modality with 90DM. Three separate methods were applied to adjust for confounding bias, including multivariable logistic regression, propensity score matching, and inverse probability of treatment weighting. Stratified analysis for the age threshold that maximized the difference in 90DM (ie, ≥67 vs <67 years) was performed. Results: A total of 894 eligible patients were included and 90DM was 5/202 (2.5%) in the PBT versus 29/692 (4.2%) in the photon-based radiotherapy group (P = .262). After adjustment for age and tumor location, PBT versus photon-based radiotherapy was not significantly associated with 90DM (P = .491). The 90DM was not significantly different for PBT versus photon-based radiotherapy in the propensity score matching (P = .379) and inverse probability of treatment weighting cohort (P = .426). The stratified analysis revealed that in patients aged ≥67 years, PBT was associated with decreased 90DM (1.3% vs 8.8%; P = .026). Higher age significantly increased 90DM risk within the photon-based radiotherapy (8.8% vs 2.7%; P = .001), but not within the PBT group (1.3% vs 3.2%; P = .651). Conclusion: No statistically significant difference was observed in postoperative 90DM after esophagectomy for cancer between PBT and photon-based neoadjuvant chemoradiotherapy. However, among older patients a signal was observed that PBT may reduce 90DM risk.

Impact of Cancer Therapy on Clonal Hematopoiesis Mutations and Subsequent Clinical Outcomes.

Exposure to cancer therapies is associated with an increased risk of clonal hematopoiesis (CH). The objective of our study was to investigate the genesis and evolution of CH following cancer therapy. In this prospective study, we undertook error-corrected duplex DNA sequencing in blood samples collected prior to and at two timepoints following chemoradiation in patients with esophageal or lung cancer recruited from 2013-2018. We applied a customized workflow to identify the earliest changes in CH mutation count and clone size and determine their association with clinical outcomes. Our study included 29 patients (87 samples). Their median age was 67 years, 76% (n = 22) were male; the median follow-up period was 3.9 years. The most mutated genes were DNMT3A, TET2, TP53, and ASXL1. We observed a two-fold increase in the number of mutations from before to after treatment in TP53, which differed from all other genes examined (P < .001). Among mutations detected before and after treatment, we observed an increased clone size in 38% and a decreased clone size in 5% of TP53 mutations (odds ratio = 3.7; 95% CI = 1.75-7.84; P < .001). Changes in mutation count and clone size were not observed in other genes. Individuals with an increase in the number of TP53 mutations following chemoradiation experienced shorter overall survival (hazard ratio = 7.07; 95% CI = 1.50-33.46; P = .014). In summary, we found an increase in the number and size of TP53 CH clones following chemoradiation that were associated with clinical outcomes.

On the acceptance, commissioning, and quality assurance of electron FLASH units.

Background & Purpose: FLASH or ultra-high dose rate (UHDR) radiation therapy (RT) has gained attention in recent years for its ability to spare normal tissues relative to conventional dose rate (CDR) RT in various preclinical trials. However, clinical implementation of this promising treatment option has been limited because of the lack of availability of accelerators capable of delivering UHDR RT. Commercial options are finally reaching the market that produce electron beams with average dose rates of up to 1000 Gy/s. We established a framework for the acceptance, commissioning, and periodic quality assurance (QA) of electron FLASH units and present an example of commissioning. Methods: A protocol for acceptance, commissioning, and QA of UHDR linear accelerators was established by combining and adapting standards and professional recommendations for standard linear accelerators based on the experience with UHDR at four clinical centers that use different UHDR devices. Non-standard dosimetric beam parameters considered included pulse width, pulse repetition frequency, dose per pulse, and instantaneous dose rate, together with recommendations on how to acquire these measurements. Results: The 6- and 9-MeV beams of an UHDR electron device were commissioned by using this developed protocol. Measurements were acquired with a combination of ion chambers, beam current transformers (BCTs), and dose-rate-independent passive dosimeters. The unit was calibrated according to the concept of redundant dosimetry using a reference setup. Conclusions: This study provides detailed recommendations for the acceptance testing, commissioning, and routine QA of low-energy electron UHDR linear accelerators. The proposed framework is not limited to any specific unit, making it applicable to all existing eFLASH units in the market. Through practical insights and theoretical discourse, this document establishes a benchmark for the commissioning of UHDR devices for clinical use.

Survival outcomes and toxicity of adjuvant immunotherapy after definitive concurrent chemotherapy with proton beam radiation therapy for patients with inoperable locally advanced non-small cell lung carcinoma.

INTRODUCTION: Adjuvant immunotherapy (IO) following concurrent chemotherapy and photon radiation therapy confers an overall survival (OS) benefit for patients with inoperable locally advanced non-small cell lung carcinoma (LA-NSCLC); however, outcomes of adjuvant IO after concurrent chemotherapy with proton beam therapy (CPBT) are unknown. We investigated OS and toxicity after CPBT with adjuvant IO versus CPBT alone for inoperable LA-NSCLC. MATERIALS AND METHODS: We analyzed 354 patients with LA-NSCLC who were prospectively treated with CPBT with or without adjuvant IO from 2009 to 2021. Optimal variable ratio propensity score matching (PSM) matched CPBT with CPBT + IO patients. Survival was estimated with the Kaplan-Meier method and compared with log-rank tests. Multivariable Cox proportional hazards regression evaluated the effect of IO on disease outcomes. RESULTS: Median age was 70 years; 71 (20%) received CPBT + IO and 283 (80%) received CPBT only. After PSM, 71 CPBT patients were matched with 71 CPBT + IO patients. Three-year survival rates for CPBT + IO vs CPBT were: OS 67% vs 30% (P < 0.001) and PFS 59% vs 35% (P = 0.017). Three-year LRFS (P = 0.137) and DMFS (P = 0.086) did not differ. Receipt of adjuvant IO was a strong predictor of OS (HR 0.40, P = 0.001) and PFS (HR 0.56, P = 0.030), but not LRFS (HR 0.61, P = 0.121) or DMFS (HR 0.61, P = 0.136). There was an increased incidence of grade ≥3 esophagitis in the CPBT-only group (6% CPBT + IO vs 17% CPBT, P = 0.037). CONCLUSION: This study, one of the first to investigate CPBT followed by IO for inoperable LA-NSCLC, showed that IO conferred survival benefits with no increased rates of toxicity.

Consolidation Osimertinib Versus Durvalumab Versus Observation After Concurrent Chemoradiation in Unresectable EGFR-Mutant NSCLC: A Multicenter Retrospective Cohort Study.

INTRODUCTION: Durvalumab improves survival when used as consolidation therapy after chemoradiation (CRT) in patients with stage III NSCLC. The optimal consolidation therapy for patients with EGFR-mutant (EGFRmut) stage III NSCLC remains unknown. METHODS: In this multi-institutional, international retrospective analysis across 24 institutions, we evaluated outcomes in patients with stage III EGFRmut NSCLC treated with concurrent CRT followed by consolidation therapy with osimertinib, durvalumab, or observation between 2015 and 2022. Kaplan-Meier method was used to estimate real-world progression-free survival (rwPFS, primary end point) and overall survival (secondary end point). Treatment-related adverse events (trAEs) during consolidation treatment were defined using Common Terminology Criteria for Adverse Events version 5.0. Multivariable Cox regression analysis was used. RESULTS: Of 136 patients with stage III EGFRmut NSCLC treated with definitive concurrent CRT, 56 received consolidation durvalumab, 33 received consolidation osimertinib, and 47 was on observation alone. Baseline characteristics were similar across the three cohorts. With a median follow-up of 46 months for the entire cohort, the median duration of treatment was not reached (NR) for osimertinib (interquartile range: NR-NR) and was 5.5 (interquartile range: 2.4-10.8) months with durvalumab. After adjusting for nodal status, stage III A/B/C, and age, patients treated with consolidation osimertinib had significantly longer 24-month rwPFS compared to those treated with durvalumab or in the observation cohorts (osimertinib: 86%, durvalumab: 30%, observation: 27%, p < 0.001 for both comparisons). There was no difference in rwPFS between the durvalumab and the observation cohorts. No significant difference in overall survival across the three cohorts was detected, likely due to the limited follow-up. Any-grade trAE occurred in 52% (2 [6.1%] grade ≥3) and 48% (10 [18%] grade ≥3) of patients treated with osimertinib and durvalumab, respectively. Of 45 patients who progressed on consolidation durvalumab, 37 (82%) subsequently received EGFR tyrosine kinase inhibitors. Of these, 14 (38%) patients developed trAEs including five patients with pneumonitis (14%; 2 [5.4%] grade ≥3) and five patients with diarrhea (14%; 1 [2.7%] grade ≥3). CONCLUSIONS: This study suggests that among patients with stage III unresectable NSCLC with a sensitizing EGFR mutation, consolidation osimertinib was associated with a significantly longer rwPFS compared to durvalumab or observation. No unanticipated safety signals were observed with consolidation osimertinib.