Clinical Cancer Research

PurposeWhile circulating tumor DNA (ctDNA) is a promising biomarker for minimal residual disease (MRD) detection in head and neck squamous cell carcinoma (HNSCC), more sensitive assays are needed for accurate MRD detection at clinically-relevant timepoints. Ultrasensitive MRD detection immediately after surgery could guide adjuvant therapy decisions, but early ctDNA dynamics are poorly understood. Experimental DesignWe applied MAESTRO, a whole-genome, tumor-informed, mutation-enrichment sequencing assay, in a pooled testing format called MAESTRO-Pool, to plasma samples from HNSCC patients collected immediately after surgery and during surveillance. We evaluated whether early MRD detection could predict outcomes. ResultsAmong 24 predominantly HPV-independent (95.8%) HNSCC patients, rapid ctDNA clearance occurred by the first postoperative sample (1-3 days postoperatively) in 9 patients without an event (recurrence or death). 13/15 patients with an event were MRD+ (PPV = 92.9%; NPV = 80%) with a median tumor fraction (TFx) of 54 ppm (range 6-1,177 ppm). In the first and last sample of the immediate postoperative window, 8/13 and 10/13 patients had TFx below 100 ppm, respectively, the detection limit of leading commercial assays. Early MRD detection correlated with worse overall survival (HR = 8.3; 95% CI: 1.1-66.1; P = 0.02) and event-free survival (HR = 27.4; 95% CI: 3.5-214.5; P < 0.0001) independent of high-risk pathology. ConclusionsImmediate postoperative MRD detection by MAESTRO was predictive of recurrence and death. Given the ultralow TFxs observed, ultrasensitive assays will be essential for reliable MRD detection during early postoperative timepoints to enable personalized adjuvant therapy decision-making in HNSCC.

Importance: Recurrence rates for locally advanced HPV-independent (HPV-) head and neck squamous cell carcinoma (HNSCC) are high. Circulating tumor DNA (ctDNA)-based minimal residual disease (MRD) assays have shown promise to improve management and surveillance in several tumor types, but their clinical utility in HPV- HNSCC remains understudied. Objective: To evaluate the performance of a tumor-informed ctDNA-based MRD assay (PredicineBEACON) in patients with newly diagnosed, locally advanced HNSCC (LA-HNSCC). Design: Between 12/2020 and 3/2022 ctDNA was assessed before surgery, before the start of adjuvant treatment (MRD-E), within six weeks of completion of treatment (MRD-TC), and during surveillance (MRD-S). Patients were followed for at least 12 months after treatment completion. We used Kaplan-Meier survival analyses to compare recurrence-free survival (RFS) and overall survival (OS) between patients who were MRD positive those who were MRD negative during each time window. Multivariable Cox hazard regressions were used to assess the association between MRD status and outcomes while controlling for established risk factors. Setting: This was a prospective cohort study of patients treated at a large referral center specializing in treatment of HNSCC. Participants: Forty patients with newly diagnosed, LA-HNSCC treated with surgery followed by risk-adjusted adjuvant treatment Intervention: Tumor-informed ctDNA-based MRD testing Main Outcomes and Measures: Recurrence-free survival (RFS) and overall survival (OS) Results: We processed 142 samples from 40 patients. The median age was 63, 27% were female, 87% were Caucasian, and 95% had HPV- disease. Fifty percent (20/40) of patients experienced recurrence. The pre-surgery ctDNA detection rate was 97% (35/36). MRD-TC positivity was associated with worse OS (HR= 7.15; 95% CI: 1.44-35.3) and RFS (HR= 5.39; 95% CI: 1.98-21.07). MRD-S positivity was associated with worse RFS (HR=8.2; 95% CI: 2.06-33.6). The median time from first MRD detection to clinical detection of recurrence was 5 months (range 0.2-21.6). In multivariable analyses, MRD positivity was associated with worse RFS (HR 13.8; 95% CI 2.92-65.7) and worse OS (HR 18.9; 95% CI 2.27-158). Conclusions and Relevance: Tumor-informed ctDNA MRD positivity was associated with worse RFS and OS in patients with HNSCC. MRD testing could serve as a non-invasive, prognostic biomarker in HPV- HNSCC patients. Key PointsQuestion: Among patients with locally advanced HNSCC (LA-HNSCC) treated with curative-intent surgery, is minimal residual disease (MRD) detection using a tumor-informed ctDNA-based assay prognostic for recurrence free survival (RFS) or overall survival (OS)? Findings: In this prospective cohort study of 40 patients with LA-HNSCC, nearly all of whom had HPV-independent disease, MRD positivity during the first 6 weeks after completing treatment and during surveillance was associated with worse OS and RFS. Meaning: Tumor-informed ctDNA MRD detection after treatment completion could serve as a non-invasive, prognostic biomarker in HPV-independent HNSCC patients. Social Media PostFindings by @FadenLab and colleagues show that detection of MRD using a tumor-informed ctDNA-based after treatment completion is associated with worse recurrence free survival and overall survival in HPV-independent HNSCC.

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the GD2 disialoganglioside and chimeric antigen receptor modified T-cells targeting GD2 (GD2-CART) eradicate DMGs in preclinical models. Arm A of the Phase I trial NCT04196413 administered one IV dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline glioma at two dose levels (DL1=1e6/kg; DL2=3e6/kg) following lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) GD2-CART infusions (10-30e6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability, and identification of a maximally tolerated dose of IV GD2-CART. Secondary objectives included preliminary assessments of benefit. Thirteen patients enrolled and 11 received IV GD2-CART on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CART manufacturing was successful for all patients. No dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric tumor reductions (52%, 54%, 91% and 100%). One patient exhibited a complete response ongoing for >30 months since enrollment. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. Sequential IV followed by ICV GD2-CART induced tumor regressions and neurological improvements in patients with DIPG and sDMG. DL1 was established as the maximally tolerated IV GD2-CART dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

PurposePatients with metastatic castration-resistant prostate cancer (mCRPC) with tumors that display disease heterogeneity, including features of androgen receptor (AR)-independence and/or lineage plasticity, have particularly poor outcomes. Non-invasive methods to prognosticate on such poor-risk features continue to pose a major challenge. We previously showed that imaging could be used to quantitatively identify patients by virtue of AR-expression and glycolysis, and that those with AR-negative and glycolytic-positive disease were associated with a poorer prognosis. Here, we extend our analysis by creating a model integrating imaging parameters with clinical determinants to comprehensively prognosticate progression free (PFS) and overall survival (OS). Patients and Methods124 CRPC patients underwent dual FDG/FDHT PET scans in a clinical trial spanning 7-years. For each patient, clinical markers and five index lesions showing abnormal tracer uptake and anatomical tumor spread were analyzed. Univariate analysis was performed on PET and clinical variables for OS and PFS. Cox proportional-hazard models were developed for OS and PFS with an analysis of covariance determining association between FDG/FDHT and OS/PFS, after adjustment for serum markers. Concordance probability estimates (CPE) quantified the discriminatory power of our model. ResultsFDG SUVmaxavg uptake was associated with OS (HR=1.55, P=0.027) with the 4th quartile of FDG SUVmaxavg ([&ge;]7.8) showing a 14-month reduction in OS relative to the 1st quartile ([&le;]4.22). Moreover, through multivariate modeling, the incorporation of FDG PET with clinical markers yielded a model with moderate discriminatory power (CPE=0.74). FDHT SUVmaxavg uptake was however not associated with OS (HR=1.14, P=0.5). Furthermore, FDHT SUVmax as a covariate lacked robust association with PFS (HR=0.78, P=0.08). ConclusionsIn this prognostic model that integrates clinical and molecular imaging data, FDG PET is an important element of identifying poor-risk patients by OS. FDHT, however, does not prognosticate for OS, and further lacks robust association with PFS, likely due to the complexities of imaging AR in mCRPC patients.

Background and ObjectiveMRI-driven active surveillance (AS) is increasingly used for prostate cancer (PCa) management. To determine the oncological safety of contemporary, MRI-driven AS and identify patients at higher risk of AS failure. MethodsThis retrospective cohort study included AS patients with MRI-localised PCa from three US and UK centres. The primary outcome was AS failure, a composite of PCa-specific mortality, metastasis, progression to [&ge;]GG4, or post-treatment biochemical recurrence. The secondary outcome was disease progression, defined as histological progression to GG3 or progression to locally advanced disease. Hazard ratios (HRs) were estimated using multivariable Cox models, with multiplicity-adjusted log-rank tests comparing event-free survival across subgroups. Key Findings and Limitations719 patients (median follow-up 5.2 years) were included. Of those, 629 (87%) had stable disease; 36 (5%) experienced AS failure, including 8 (1%) cases of metastasis and no PCa-related deaths; 54 (8%) had disease progression. Cribriform GG2 histology was the strongest predictor of AS failure (HR 12.7 95% CI, 4.8-33.6; P < 0.001), followed by tumor MRI-visibility (HR 5.0; 95% CI, 1.5-16.5; P = 0.009) and non-cribriform GG2 histology (HR 3.4; 95% CI, 1.6-7.0; P = 0.001). MRI-invisible, non-cribriform GG2 and all GG1 tumors had comparable event-free survival (Padj > 0.05 for both outcomes). The study is limited by retrospective design. Conclusions and clinical implicationsContemporary MRI-driven AS is safe, including for patients with non-cribriform GG2 tumours, particularly those that are MRI-invisible. Conversely, patients with cribriform GG2 disease are at increased risk of AS failure and therefore warrant upfront treatment.

BackgroundRecurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is an aggressive cancer with a median overall survival of only 12 months. Existing biomarkers have limited ability to predict treatment response or survival, exposing many patients to the potential toxicity of treatment without certain clinical benefit. Circulating tumor DNA (ctDNA) has emerged as a non-invasive, real-time biomarker that could address these challenges. MethodsWe analyzed 137 plasma samples from 16 patients with R/M HNSCC undergoing immune checkpoint blockade (ICB)-based therapy. A tumor-informed, highly sensitive next-generation sequencing liquid biopsy assay (RaDaR, NeoGenomics Laboratories, Inc.) was applied to track ctDNA changes at baseline and throughout treatment. Univariable and multivariable analyses were used to assess the association between ctDNA negativity and key clinical outcomes: disease control (best objective response of stable disease, partial response, or complete response), three-year overall survival (OS), and three-year progression-free survival (PFS). We also assessed a machine learning model to predict disease progression based on ctDNA dynamics. ResultsMultivariable analysis revealed that ctDNA negativity during treatment was significantly associated with improved disease control (OR 21.7, 95% CI 1.86-754.88, p=0.0317), three-year OS (HR 0.04, 95% CI 0.00-0.47, p=0.0103), and three-year PFS (HR 0.03, 95% CI 0.00-0.37, p=0.0057). The machine learning model predicted disease progression with 88% accuracy (AUC 0.89). ConclusionSerial ctDNA monitoring predicted disease control, survival, and progression in patients with R/M HNSCC receiving treatment with ICB, suggesting that incorporation of ctDNA into clinical practice could enhance treatment decision-making for clinicians and improve patient outcomes.

Translational RelevanceDespite definitive local therapy, some men with prostate cancer develop biochemical recurrence (BCR-PCa) and progress to metastatic disease. Current standard-of-care, androgen receptor pathway inhibitors (ARPIs) with or without androgen deprivation therapy (ADT) carry substantial long-term morbidity. This trial tested a novel, non-hormonal approach of 5-azacitidine (AZA) plus all-trans retinoic acid (ATRA) to induce tumor cell dormancy via epigenetic reprogramming. The regimen was well tolerated, with manageable toxicities, and showed preliminary signals of delayed PSA progression and prolonged PSA doubling time in some patients, suggesting dormancy induction. One patient achieved durable disease stabilization with ATRA maintenance. These first-in-human findings indicate that epigenetic reprogramming may modulate dormancy in BCR-PCa, offering a potential strategy to delay or minimize ADT use and its toxicities. This approach highlights the translational potential of preventing or delaying overt metastases by activating dormancy pathways in early recurrent prostate cancer. PurposeBiochemical recurrence (BCR) after definitive local therapy remains a major clinical challenge in prostate cancer (PCa), with heterogeneous disease trajectories and few established strategies to delay further progression without prolonged androgen deprivation. This pilot study evaluated the combination of 5-azacitidine (AZA) and all-trans retinoic acid (ATRA) to induce tumor dormancy and delay clinical progression in patients with BCR. Experimental DesignIn a prospective, open-label, randomized, single-institution pilot trial, patients with BCR of PCa and no recent hormonal or definitive therapy received low-dose AZA and sequential ATRA. The co-primary endpoints were changes in prostate-specific antigen doubling time (PSADT) and time to next treatment (TTNT). Safety and biomarker analyses, including bone morphogenetic protein (BMP) signaling and dormancy marker NR2F1 in circulating tumor cells (CTCs), were evaluated to investigate treatment effects on minimal residual disease dormancy. ResultsFourteen patients were enrolled. Treatment resulted in an increase in median PSADT from 2.45 to 4.56 months. The median TTNT was 9.6 months, with 28.6% of patients experiencing TTNT over 12 months. No new safety signals were identified; adverse events were consistent with those expected for AZA and ATRA. Analysis of circulating BMP4 and BMP7 suggested that higher BMP4 levels may correlate with treatment response. Notably, all patients achieved testosterone recovery post-treatment, likely reflecting the avoidance of ongoing androgen deprivation. Across the cohort, treatment with AZA+ATRA led to a reduction in total CTC numbers and an apparent increase in the fraction of NR2F1-positive CTCs in responders, although the small cohort size limited statistical testing. ConclusionsThe combination of AZA and ATRA was feasible and prolonged PSA kinetics in a subset of patients with BCR of PCa, with a favorable safety profile. This epigenetic approach promoting tumor dormancy presents a potential strategy to defer progression and delay the need for continuous hormonal suppression. Larger studies are warranted to validate these findings and further explore biomarkers predictive of clinical benefit.

PurposeRelapse is a major cause of failure in human papillomavirus (HPV)-independent head and neck squamous cell carcinoma (HNSCC). Clinicopathologic criteria for adjuvant treatment remain imprecise and have not changed for decades. We investigated whether circulating tumor DNA (ctDNA) in lymphatic exudate collected via surgical drains ("lymph") 24-hours after surgery identified molecular residual disease (MRD) and compared its performance to time-matched plasma. Experimental DesignUsing an ultra-sensitive tumor-informed sequencing approach, tumor variants were called in lymph and plasma to classify patients as ctDNA-positive or ctDNA-negative, trained in an initial cohort of 36 patients and replicated in an independent cohort of 37 patients. Progression-free survival (PFS) was compared in ctDNA+ vs. ctDNA-patients. ResultsLymph identified MRD in two independent multi-site cohorts (initial cohort sensitivity = 76%, specificity = 63%, P = 0.01; replication cohort sensitivity = 65%, specificity = 70%, P = 0.04). Lymph performance was enhanced in locoregional relapse (sensitivity = 78%, specificity = 67%, P = 0.0004) and generalized to early-stage patients. Analysis of matched plasma collected at this early timepoint was not predictive of recurrence (sensitivity = 35%, specificity = 72%, P = 0.7). In patients with intermediate-risk pathology, lymph ctDNA was associated with recurrence (sensitivity = 88%, specificity = 67%, P = 0.0008), suggesting an opportunity for improved stratification of patients who may benefit from additional adjuvant treatment. ConclusionPostoperative lymph is a novel, proximal, and early source of MRD with the potential to introduce more precision into adjuvant therapy decision-making and improve outcomes, especially for intermediate-risk HPV-independent HNSCC patients. Translational RelevancePostoperative lymphatic exudate represents a proximal analyte for MRD detection in HPV-independent HNSCC designed specifically for use in the immediate post-surgical window when adjuvant therapy decisions must be made. Accurate MRD identification at this early timepoint has the potential to augment traditional pathology and personalize adjuvant treatment paradigms in HPV-independent HNSCC.

BackgroundSurgery is a common treatment for early-stage HPV-associated head and neck squamous cell carcinoma (HPV+HNC). Selection of patients who require adjuvant treatment is based on clinicopathologic risk factors, which have poor individualized prognostic capacity. Circulating tumor HPV DNA (ctHPVDNA) is a highly sensitive and specific biomarker for HPV+HNC at diagnosis, but current clinically available assays lack the necessary sensitivity for accurate minimal residual disease (MRD) detection after surgery. Here, we applied a significantly more sensitive HPV whole genome sequencing (WGS) assay to determine the prognostic value of ctHPVDNA-based MRD detection and compare this head-to-head with existing approaches and clinical standard of care. Patients and methods103 patients with AJCC 8 Stage I-IV HPV+HNC treated with definitive surgery were prospectively enrolled. Blood was collected before surgery, after surgery, and in surveillance and analyzed by clinically validated HPV WGS and droplet digital (dd)PCR assays. The primary hypothesis tested was that patients with MRD detection after surgery would have inferior disease-free survival (DFS) and overall survival (OS). ResultsWith a median follow up of 27 months, patients with ctHPVDNA detected after surgery had significantly worse 2-year DFS and OS compared to those without ctHPVDNA (DFS 60%, 95% CI:31-80% vs 100%, p<0.001; OS 73%, 95% CI:43-89% vs 98%, 95% CI:88-100, p=0.002). Patients with ctHPVDNA detected following multi-modality treatment completion also had significantly worse 2-year DFS and OS compared to patients without ctHPVDNA (DFS 0% vs 100%, p<0.001) (OS 50%, 95% CI:11-80% vs 100%, p<0.001). MRD status was a stronger predictor of DFS than standard clinicopathologic criteria (HR 25.2; p = 0.003). Lead time from molecular detection of recurrence to clinical detection of recurrence was up to 17.5 months and nearly twice as long compared to ddPCR (7.1 vs 4.1 months). ConclusionApplying an ultrasensitive HPV WGS liquid biopsy, HPV+HNC patients with ctHPVDNA detected after surgery and following treatment completion had significantly worse DFS and OS, highlighting the potential for MRD status for personalized adjuvant treatment decision-making. HighlightsO_LIApplying an ultrasensitive HPV WGS liquid biopsy, HPV+HNC patients with MRD after surgery and following treatment completion had significantly worse DFS and OS C_LIO_LIPatients with MRD had a strong benefit from adjuvant treatment in decreasing recurrence and death while patients without MRD showed no benefit C_LIO_LIMRD status after surgery outperformed conventional clinicopathologic features in predicting recurrence C_LIO_LIMedian lead time from molecular detection of recurrence to clinical detection was >7 months, and up to 17.5 months, double existing liquid biopsy approaches C_LI

PURPOSEA non-surgical alternative for treating cervical intraepithelial neoplasia (CIN) 2/3 is an unmet need due to a risk of cervical incompetency. METHODSPepCan consists of four human papillomavirus (HPV) type 16 E6 peptides and a Candida skin testing reagent (adjuvant). In this randomized, double-blind Phase 2 study, women with biopsy-confirmed CIN2/3 were treated with PepCan or Candida at one to one ratio. Four intradermal injections were given every 3 weeks, with observation visits at 6 and 12 months post-vaccination. Quadrant biopsies were performed at the 12-month visit, and those whose lesions regressed to no CIN were considered to be complete responders. Regression rates of each treatment group were compared to that of a historical placebo group. RESULTSWith the intention-to-treat analysis, PepCan (n=39) showed 30.8% efficacy (95% confidence interval [CI], 17 to 47.6; p=0.25) while Candida (n=42) demonstrated 47.6% efficacy (95% CI, 32 to 63.6; p<0.001). Likewise, with the per-protocol analysis, PepCan (n=24) showed 45.8% efficacy (95% CI, 25.6 to 67.2; p=0.08) and Candida (n=29) showed 62.1% efficacy (95% CI, 42.3 to 79.3; p<0.001). There was no difference between efficacy of PepCan and Candida. No dose-limiting toxicity was observed. HPV-specific T cell responses were elicited in both groups. Vaccine-induced HPV-specific CD4 and CD8 T cells were present in cervix regardless of histological response. Single-cell RNA-seq revealed increased expression of granzymes, CCR5, and EOMES in HPV-specific CD8-positive T cells of a histological responder, compared to non-responders. Six cytokines (CCL4, CCL5, interleukin-9, lymphotoxin-, platelet-derived growth factor-{beta}{beta}, tumor growth factor-{beta}1) were significantly decreased in both Candida recipients and histological responders suggesting that Candida may possibly exert its anti-tumor effects through these systemic mediators. CONCLUSIONSCandida may be effective in inducing histological regression. PepCan and Candida treatments are safe. Candida should be evaluated in a Phase 3 trial as a potential new treatment for CIN2/3.

BackgroundCancers that do not respond to immunotherapy typically harbor a non-T cell-inflamed tumor microenvironment (TME), characterized by the absence of type I/II interferon (IFN) signaling and CD8+ T cell infiltration. We previously reported IDH1 somatic mutations were enriched in non-T cell-inflamed tumors across tumor types. Consistent with this, mutant IDH1 (mIDH1) has been demonstrated to drive immune exclusion through metabolic reprogramming of the TME, and IDH inhibition enhanced anti-tumor immunity in preclinical models. Based on these pan-cancer observations, we conducted a Phase II study assessing the preliminary activity of ivosidenib, an IDH1 inhibitor, in combination with nivolumab, an anti-PD1 antibody, in patients with mIDH1 advanced solid tumors (NCT04056910). MethodsPatients with an advanced or refractory solid tumor harboring an IDH1 mutation, but no prior exposure to IDH1 inhibitor, were enrolled. Participants were administered ivosidenib 500 mg by mouth daily with nivolumab 480 mg intravenously every 4 weeks. Given heterogeneity in tumor types, including some where RECIST response is uncommon (i.e. sarcoma), a composite primary endpoint was utilized including either six-month progression free survival (PFS6) or overall response rate (ORR). Translational analyses included pharmacodynamic, proteomic, and spatial transcriptomic investigations. Results15 patients were enrolled (median age, 54 years; female, 53.3%; ECOG 1, 60%; glioma, 46.7%; R132H, 40%). One patient had a partial response (ORR 6.7%) and was without progression at 6 months (PFS6) whereas two other patients achieved PFS6 alone. In total, 3 out of 15 patients met the primary endpoint (3/15; 20%). The median PFS was 1.94 months. The most common treatment adverse events were leukopenia (67%), rash (67%), diarrhea (33%), nausea (27%), and QTC prolongation (27%). Pharmacodynamic studies demonstrated combining ivosidenib and nivolumab significantly decreased the plasma (R)-2HG concentration and correlated with clinical benefit. Serum proteomic and spatial omic analysis suggested immune-modulatory effects of mIDH1 inhibition plus anti-PD1. ConclusionsIn treatment refractory mIDH1 solid tumors, the combination of ivosidenib and nivolumab was safe however demonstrated similar anti-tumor activity (predominantly as disease stabilization) compared with that previously described for ivosidenib monotherapy. Translational investigation suggests further evaluation of IDH1 inhibition as a combination partner with immune-checkpoint inhibition may be justified. Implications for practiceThis study demonstrates that ivosidenib combined with nivolumab was safe in patients with advanced IDH1-mutant solid tumors. The clinical activity was modest and comparable to ivosidenib monotherapy. Pharmacodynamic and exploratory translational analyses revealed immune-related changes in the tumor microenvironment, suggesting that mutant IDH1 inhibition may modulate immune signaling. These findings support further investigation of IDH1 inhibitors as immunotherapy partners and highlight the importance of integrated translational analyses to inform therapeutic development.

IntroductionCerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring neoplastic processes of the central nervous system. We hypothesize that analysis of CSF-tDNA in patients with advanced lung cancer improves the sensitivity of leptomeningeal disease (LMD) diagnosis and enables central nervous system response monitoring. MethodsWe applied CAPP-Seq using a lung cancer-specific sequencing panel to 81 CSF, blood, and tissue samples from 24 patients with advanced lung cancer who underwent lumbar puncture (LP) for suspected LMD. A subset of the cohort (N = 12) participated in a prospective clinical trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment with. ResultsCSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearmans {rho}, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). ConclusionsDetection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA may improve the sensitivity of LMD diagnosis, enable improved prognostication, and drive therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

Despite responses of chimeric antigen receptor (CAR)-T cells in relapsed/refractory (R/R) large B cell lymphoma (LBCL) patients, over half of patients eventually relapse. Methods to detect early disease persistence are needed to identify patients at high-risk of treatment failure. We recently developed MAESTRO, an ultrasensitive, tumor-informed measurable residual disease (MRD) assay, which can detect parts-per-million (ppm) levels of circulating tumor DNA (ctDNA) using minimal sequencing. We applied MAESTRO to 140 samples from 28 patients (15 durable responders at 12 months, 13 nonresponders) to identify treatment failure following axicabtagene ciloleucel (axi-cel) administered at our institution between 2018 and 2022. Responder and nonresponder patients had similar baseline tumor burden. By 1 week after infusion, responders had marked ctDNA reduction compared to nonresponders, p<0.001. At weeks 2 and 4, responders had ctDNA levels approaching 0 ppm, while nonresponders had persistence of ctDNA, each p<0.001. At day 0, 21% of patients had ctDNA fractions below 0.01%, hence these individuals would not have qualified for ctDNA monitoring with a less sensitive test. Our results confirm feasibility of highly sensitive MRD detection by ctDNA for early identification of patients at high risk of disease progression from axi-cel.

Effective treatments for recurrent, radiation-relapsed meningiomas (RR-meningiomas) following surgery and radiation therapy (RT) are limited. Inhibitors of the programmed-death-1 (PD-1) or programmed-death ligand-1 (PD-L1) pathway have shown modest activity in single-arm phase II studies of RR-meningiomas. This study aimed to evaluate the immunological effects of combining avelumab, a PD-L1 inhibitory antibody, with proton beam therapy (PBT) in RR-meningiomas. Patients with grade 1-3 RR-meningiomas were treated with neoadjuvant avelumab plus hypofractionated PBT, followed by surgery and adjuvant avelumab. Correlative analyses included RNA-sequencing (RNA-seq), whole exome sequencing (WES), multiplex immunofluorescence (MxIF), single-nucleus RNA-seq (snRNA-seq) of pre- and post-treatment tumor tissues, and flow cytometry (FC) of serial blood samples. Nine patients were enrolled: three achieved an immunologic response and prolonged progression-free survival (PFS > 36 months). At a median follow-up of 47.2 months, the median PFS was 19.1 months (95% CI: 15.2-23.0). RNA-seq showed a dynamic change of tumor microenvironment (TME) signatures. MxIF revealed marked infiltration of T cells and CD68+CD206- (M1-phenotype) macrophages in the post-treatment tissues of responders, a pattern absent in non-responders, whose pre- and post-treatment tissues predominantly featured CD206+ (M2-phenotype) macrophages. These findings were supported by snRNA-seq, which identified FN1-associated immunosuppressive macrophage subtype enriched in non-responders. Additionally, FC revealed elevated peripheral primed T-cell signatures one-month after treatment initiation in responders, suggesting a potential predictive biomarker. Avelumab combined with RT may elicit an immune response in a subset of RR-meningiomas, leading to prolonged remission. Further investigations are warranted to validate these findings and to develop predictive biomarkers in larger prospective studies. SummaryThis phase I window-of-opportunity trial demonstrates that neoadjuvant PD-L1 blockade combined with hypofractionated proton therapy can induce robust immunologic responses and prolonged progression-free survival in a subset of patients with radiation-relapsed meningioma, supported by multi-omic profiling of the tumor microenvironment and peripheral blood.

Outcomes for high-grade pediatric brain tumor patients remain poor, but there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. We present the results from a phase I clinical trial of IL13BBz-CAR T cells infused weekly into the cerebral ventricles in pediatric and young adult patients with recurrent or refractory brain tumors. The trial met its primary objectives of feasibility, safety, and tolerability, with one dose-limiting toxicity. 8 of 16 patients evaluable for response experienced radiographic size decreases consistent with biologic activity and with an anti-tumor response. Two patients met protocol criteria for response. Median survival for patients receiving lymphodepletion was 20.5 months from diagnosis and 6.9 months from treatment for patients with midline glioma, and 187 months from diagnosis and 7.5 months from treatment for patients with ependymoma. Importantly, patients who did not receive lymphodepletion developed anti-CAR humoral and cellular immune responses detectable in the CSF and peripheral blood, whereas patients receiving lymphodepletion had no evidence of CSF anti-CAR immunity. Taken together, these findings demonstrate the safety, tolerability, and biological activity of locoregionally-delivered IL13BBz-CAR T cells for children and young adults with CNS tumors. Moreover, we show that anti-CAR immune responses arise in patients not receiving lymphodepletion, but not in the CSF of patients receiving systemic lymphodepletion. Further investigation of adoptive cellular therapies combined with immunosuppression is warranted in this patient population. ClinicalTrials.gov registration: NCT04510051.

PurposeLimited CNS bioavailability and pharmacodynamics are obstacles to effective systemic therapies for glioblastoma. One strategy to overcome these challenges is drug combinations enhancing CNS penetration and/or tumor chemosensitivity. LP-184, a synthetic acylfulvene class alkylator, induces DNA damage and inhibits glioblastoma cell viability in pre-clinical models. LP-184 is a prodrug converted to active metabolites by intracellular prostaglandin reductase 1 (PTGR1) that is over-expressed in >70% of glioblastoma. DNA damage induced by LP-184 is MGMT agnostic and reversed by transcription-dependent NER. PatientsLP-184 was evaluated in a Phase 1a study (NCT05933265) in 63 adult patients with advanced malignancies including 16 patients with recurrent glioblastoma. All patients with glioblastoma received prior standard-of-care therapy and most had received 1 or more additional therapies before enrollment. ResultsPatients with glioblastoma experienced more frequent transaminitis, Grade 1-2 nausea and a trend towards more frequent and severe thrombocytopenia compared to the non-glioblastoma cohort. Otherwise, overall toxicity profiles were similar. Clinical pharmacokinetic analysis combined with published pre-clinical intra-tumoral bioavailability data ([~]20% penetration) predicted that LP-184 at the recommended dose for expansion (RDE) would achieve cytotoxic levels if combined with spironolactone, a BBB permeable ERCC3 degrader and TC-NER inhibitor that sensitizes glioblastoma cells to LP-184 3-6-fold. We show that three daily doses of spironolactone deplete orthotopic glioblastoma PDX ERCC3 protein by [~] 80% and increases tumor LP-184 cytotoxicity 2-fold. ConclusionsLP-184 is well tolerated at the RDE, and we establish a clinically translatable scheme for dosing spironolactone in combination with LP-184 for a future Phase 1b clinical trial. Statement of translational relevanceTreatment failure in glioblastoma reflects inadequate drug brain exposure and DNA repair- mediated resistance. LP-184, a novel acylfulvene alkylator, generates MGMT-independent DNA lesions predominantly repaired by transcription-coupled NER. In a Phase 1a dose finding trial, LP-184 was well-tolerated at the recommended dose for expansion (RDE) in participants with advanced cancers, including recurrent glioblastoma. Plasma drug levels achieved predicted effective systemic exposures but not brain concentrations based on projected 20% brain penetrance. Pharmacokinetic modeling indicates that NER inhibition could increase tumor chemosensitivity with the addition of spironolactone. The optimal dosing regimen for spironolactone combined with LP-184 was identified in orthotopic PDX models, facilitating advancement to Phase 1b/2a testing of LP-184 plus spironolactone.

Immune-checkpoint blockade (ICB) has shown significant efficacy across various tumor types. However, tumors with low intraepithelial T-cell infiltration, often referred to as "cold" tumors, are expected to yield poor responsiveness to ICB. We investigated the potential of low-dose radiotherapy (LDRT) to enhance ICB responses in 25 patients with multimetastatic immune-excluded solid tumors through a multi-cohort phase I clinical trial (RACIN). Primary endpoint was to determine the safety and tolerability of the combination of a backbone treatment, comprising nivolumab, ipilimumab, aspirin/celecoxib, and low-dose cyclophosphamide (Cy) in association with escalated LDRT. Secondary endpoints included among others disease control rate (DCR) and overall survival (OS). Exploratory endpoints included biomarkers and molecular correlates of response. The combination treatment showed a manageable safety profile, with Grade 3 or higher adverse events in 12% to 21% of patients across cohorts. The overall DCR was 42%. Progression free survival (PFS) across all cohorts was 2.1 months (95% C.I.: 1.8 - 4.2 months), with the highest PFS observed in cohort 1 which received 0.5 Gy (5.7 months, (95% C.I.: 1.9 - 11.3 months). Median OS was 14.0 months (95% CI: 8.5-24.6 months), with one patient with ovarian cancer still maintaining a complete response at three years follow-up. Site-paired tumor biopsies collected for each patient at baseline and after LDRT +/- Cy enabled the comprehensive characterization of the dynamics of excluded tumor microenvironments (TME) at the single cell level. Response to LDRT and ICB was associated with DNA damage and repair responsiveness and the presence of detectable intratumoral PD1+CD8+ tumor infiltrating lymphocytes (TILs) at baseline. Our data revealed that LDRT amplified CD8+ TIL functionality in responding patients offering mechanistic insights on how LDRT improves ICB effectiveness. In contrast, we observed a radiosensitivity of TILs in tumors of non-responders. Detailed single cell immune profiling before LDRT also highlighted a lack of key immune stimulatory myeloid cells that can therefore limit ICB efficacy in excluded tumors. Collectively, this study represents the most comprehensive profiling of longitudinal samples of cancer patients treated with LDRT. Our findings highlight several genetic, transcriptomic and TME parameters associated with response to combinatorial LDRT and ICB in advanced immune-excluded solid cancers, generating rationale for their validation in larger cohorts. One sentence summaryCombinatorial treatment with low-dose radiotherapy and immune checkpoint blockade in patients with immune-excluded tumors enhances CD8 T cell functionality, particularly in those with DNA repair deficiencies, offering new biomarkers for patient selection.

Immune checkpoint blockade impedes the negative regulatory signals for T-cell response and permits more effective immune detection and eradication of cancer cells. This single-arm phase II clinical trial (ACTRN12616001019493) within the Molecular Screening and Therapeutics (MoST) program evaluates the clinical activity and safety of combination immunotherapy with durvalumab and tremelimumab in patients with advanced cancers, prioritsing rare cancers (<6 per 100,000 annual incidence) and patients having failed standard treatments for their cancer type. MethodsEligible patients were determined by the molecular tumour board based on the absence of actionable genomic findings (n=64) and biomarker enriched (n=48) at screening. Patients received durvalumab 1500 mg and tremelimumab 75 mg every four weeks for 4 cycles, followed by durvalumab alone for another 9 cycles. The primary endpoint was progression-free survival at 6 months (PFS6) and secondary endpoints included objective response, time to progression (TTP) on trial to TTP on prior therapy (TTP2/TTP1>1.3), overall survival and treatment tolerability. ResultsBetween December 2016 and 2019, 112 patients were enrolled on the study. There was a female predominance (55%), most had an ECOG performance status of 0 (66%), aged <65 years (75%), with rare cancers (84%). The PFS6 rate was 32% (95% CI 23 to 40%); 16 of 112(14%) achieved an objective response; TTP2/TTP1>1.3 for 22 of 63 (35%) patients with an evaluable ratio; median overall survival 11.9 months (95% CI 11.0 to 14.8), and there were no new safety concerns. High tumour cell PD-L1 correlated with improved PFS and OS and TMB with PFS alone. More PD-1+CD4+ T-cells and circulating follicular T-helper (cTfh) cells at baseline were strongly associated with better PFS and OS. ConclusionDurvalumab plus tremelimumab demonstrated a signal of clinical activity in treatment-refractory patients with rare cancers. A PFS6 of 32% and 35% of patients achieving a TTP2/TTP1>1.3 suggests an improved disease trajectory on trial. Translational correlates provided insights into biological associations with clinical outcomes across tumour types.

PurposeNovel therapies to prevent lethal castration resistant prostate cancer in response to standard-of-care androgen deprivation therapy (ADT) are required. Unfortunately, most prostate cancers are "immune cold" and fail to respond to checkpoint inhibitors (CPIs). To assess whether ADT induces changes that enable more effective CPI therapy, we examined the tumor immune micro-environment (TiME) following neoadjuvant ADT (nADT). DesignRadical prostatectomy specimens from 43 nADT-treated patients were stratified into three duration groups and compared to each other and matched controls. RNA sequencing and quantitative multispectral immunofluorescence (qmIF) staining were performed to analyze transcriptomic and TiME abundance and cellular spatial relationship differences after nADT. ResultsImmune and inflammatory pathways, particularly of antigen presentation and adaptive immune response, were increased, most notably in tumors receiving 3-5 months nADT. qmIF revealed a complex temporal response in the TiME, with a dramatic influx of CTLs and T-helper cells after 3-5 months of nADT. However, after 6 months nADT, M2-like tumor associated macrophages (TAMs) and Tregs were strikingly increased while CTLs decreased. Spatially, CTLs and T-helper cells, clustered near tumor cells at 3-5 months nADT, were replaced by M2-TAMs in tumors receiving [&ge;] 6 months of nADT. ConclusionThese data reveal the induction of a bi-phasic response in the TiME: robust CTL activation 3-5 months after nADT is initiated, followed by myeloid immunosuppression in tumors receiving prolonged nADT. This ADT-induced reprogramming of the TiME suggests a critical window of opportunity where short-duration ADT might augment CPI efficacy, converting cold into immunologically responsive tumors. Translational RelevanceImmune Checkpoint inhibitors (CPIs) have not been effective in treating most human prostate cancers. This study describes the temporal dynamics of the immune response of primary prostate cancers to neoadjuvant androgen deprivation therapy (nADT), and suggests a strategic approach to improve the efficacy of CPIs in prostate cancer. After several months of nADT, inflammation and immune-related pathways were activated, accompanied by a robust infiltration of both CD8+ and CD4+ T cell into prostate tumors, indicating effector T cell education and activation. In contrast, six or more months nADT leads to an immunosuppressive shift, evidenced by increased M2-like tumor associated macrophages and regulatory T cells. Thus, our findings suggest a critical window of opportunity following nADT for initiating CPIs. This provides a rationale for the precise sequencing of nADT and CPI regimens to maximize therapeutic benefit.

PurposeBuparlisib, a pan-class I PI3K inhibitor, combined with paclitaxel, demonstrated improved survival in the BERIL-1 trial for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). However, predictive biomarkers of benefit remain undefined. We evaluated spatial biomarkers derived from hematoxylin and eosin (H&E) images using artificial intelligence (AI)-based digital pathology. Materials and MethodsWhole-slide H&E images (n=144) from BERIL-1 were analyzed using a deep learning model trained to segment tissue compartments and classify individual cell phenotypes. Three prospectively defined spatial features were evaluated: (1) tumor-infiltrating lymphocyte (TIL) density in the tumor area; (2) tumor microenvironment (TME) heterogeneity; and (3) granulocyte fraction in the tumor invasive margin (TIM). Cox proportional hazards model was used to evaluate biomarker-treatment interactions, with patients stratified by biomarker status. ResultsHigh TIL density (>10%) defined by deep learning-derived analysis of H&E was associated with a significantly improved overall survival with buparlisib versus placebo (HR{square}={square}0.25; 95% CI, 0.01-0.64; p = 0.002), as were high TME heterogeneity (HR{square}={square}0.47; 95% CI, 0.27-0.80; p = 0.005) and granulocyte enrichment in the TIM (HR = 0.51; p = 0.014); in a within-arm proximity analysis, higher granulocyte-tumor cell proximity correlated with improved OS on buparlisib (HR = 0.32; p < 0.001). AI-derived spatial metrics outperformed CD3 immunohistochemistry staining in stratifying survival outcomes. In patients with oropharyngeal tumors, human papillomavirus-positive cases were more frequent among those with high TILs. ConclusionsSpatial features extracted from standard H&E slides using AI-driven digital pathology can predict OS benefit from buparlisib in R/M HNSCC. These cost-effective and scalable biomarkers support image-based patient selection strategies and are being prospectively evaluated in the ongoing BURAN phase 3 trial.