Background The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing, and human papillomavirus (HPV)-associated OPSCC is contributing substantially to this trend. Salivary HPV testing enables early detection of HPV-driven OPSCC. The GeneXpert system (Cepheid) is an automated polymerase chain reaction (PCR)-based diagnostic platform designed primarily for cervical cancer screening using vaginal swabs. Our goal was to adapt this platform for HPV testing using salivary oral rinse (SOR) samples to enable early detection of HPV-driven OPSCC. Methods Patients suspected of having any type of head and neck cancers and people at higher risk of acquiring HPV infections were recruited (n = 67). A volume of 5 to 10 mL of SOR samples was tested using the GeneXpert system. Genomic DNA was extracted from the same SOR samples and analyzed using quantitative real-time PCR for human papillomavirus type 16 (HPV-16). The detection status of HPV-16 was used to evaluate the efficiency of HPV detection using the GeneXpert system. Results The GeneXpert system had excellent specificity of 100% and sensitivity of 72.09%. There was strong agreement with quantitative real-time PCR results (κ = .649). This confirms the suitability of SOR as a sample medium on the GeneXpert system to detect oral HPV with minimal bias. Conclusions The GeneXpert system can effectively detect HPV in SOR samples. It offers rapid HPV detection, helps identify people at high risk of developing HPV-associated OPSCC, thereby holding promise for the early detection of HPV-driven OPSCC. Practical Implications The results of this study showed the feasibility of using SOR samples on the GeneXpert platform as a scalable, noninvasive screening approach for the early detection of patients at higher risk of developing HPV-associated OPSCC.

9585 Background: Cosibelimab, a high-affinity programmed death ligand-1 (PD-L1)–blocking antibody, is approved by the US Food and Drug Administration (FDA) at a recommended dose of 1200 mg every 3 weeks (Q3W) for the treatment of adults with locally advanced or metastatic cutaneous squamous cell carcinoma (laCSCC or mCSCC) who are not candidates for curative radiation or surgery. The CSCC expansion cohort of the pivotal open-label Phase 1 study (NCT03212404) evaluated cosibelimab 800 mg every 2 weeks (Q2W) in patients with laCSCC or mCSCC and 1200 mg Q3W in patients with mCSCC. Results through March 2023 demonstrated robust and sustained responses with cosibelimab 800 mg Q2W. The 800-mg Q2W and 1200-mg Q3W regimens are considered similar based on pharmacokinetic criteria outlined by the FDA for alternative PD-L1–blocking antibody dosing regimens. We present new follow-up data for the 800-mg Q2W laCSCC cohort. Methods: Eligible patients were ≥18 years old and had an Eastern Cooperative Oncology Group performance status of 0 or 1 and unresectable laCSCC not amenable to local therapy. Cosibelimab 800 mg was administered intravenously on Day 1 of each 14-day cycle until complete response, worsening disease progression, toxicity, or clinical deterioration. The primary endpoint was objective response rate (ORR; best overall response: complete or partial) by independent central review per RECIST V1.1; duration of response (DOR) was the key secondary endpoint. Follow-up data presented are from investigator assessment. Other secondary endpoints included safety. Results: By the January 31, 2025, data cutoff, 64 patients with laCSCC received ≥1 dose of cosibelimab 800 mg Q2W; their median (range) age was 77 (51–95) years, and 66% were male. Patients received a median of 29 (2–89) doses over a median duration of 60 (4–184) weeks. The ORR (95% confidence interval) was 50% (37%, 63%), including 17 complete and 15 partial responses; over a median follow-up duration of 31 (1–59) months the median DOR was not reached. Treatment-emergent adverse events (TEAEs) were reported in 61 (95%) patients and considered treatment-related in 50 (78%) patients; none were fatal. The most common AEs were anemia and diarrhea, recorded in 17 (27%) patients each. Grade ≥3 TEAEs were reported in 26 (41%) patients and considered treatment-related in 7 (11%). Hypertransaminasemia, in 2 (3%) patients, was the only serious treatment-related TEAE reported. Immune-related AEs (irAEs) were recorded in 22 (34%) patients; Grade ≥3 irAEs occurred in 1 (2%) patient and were dermatologic in nature (maculo-papular and pruritic rashes) and considered treatment-related. Conclusions: Responses to treatment with cosibelimab 800 mg Q2W remained robust and durable with stable ORR after continued follow-up in a larger cohort of patients with laCSCC, with no new safety signals identified. Clinical trial information: NCT03212404 .

9588 Background: Immune checkpoint inhibitors (ICI) are standard first-line therapy for advanced cutaneous squamous cell carcinoma (cSCC); however, 35% of patients experience disease progression. Evidence guiding optimal second-line management remains limited. We evaluated real-world outcomes of second-line treatment strategies in patients with cSCC resistant to ICI. Methods: A retrospective analysis of patients with unresectable locally advanced or metastatic cSCC treated with ICI monotherapy at two tertiary centres in Brisbane, Australia, was conducted. Patients with progressive disease (PD) were identified and classified as having primary resistance (PD < 4 months) or acquired resistance (PD ≥4 months). Second-line strategies included local therapy (LT - surgery and/or radiotherapy), systemic therapy +/- LT or best supportive care (BSC). Outcomes included progression-free survival from second-line initiation (PFS2), and overall survival (OS) using Kaplan–Meier estimates. Results: Seventy-four patients were identified. Median age was 74 years. Most patients had locoregionally advanced disease (77%), head and neck primary tumours (70%), ECOG performance status 0–1 (76%), and were immunocompetent (70%). Primary resistance occurred in 54% and acquired resistance in 46%, of which 21% progressed after ICI cessation. Locoregional progression was observed in 70% of patients, while 30% progressed with distant disease. Management of ICI resistance included LT (N = 32), systemic therapy +/- LT (N = 16), and BSC (N = 26). An objective response rate of 65% was observed in patients undergoing systemic therapy +/- LT with responses to both ICI rechallenge and/or EGFR targeted therapy (7 of 11 patients). With a median follow up of 46.6m the Median PFS2 was 10.7m. PFS2 was numerically higher in patients undergoing systemic therapy vs LT alone (11.9m vs 8.4m). Of those that underwent LT alone, further PD was seen in 16 patients (50%), with half requiring systemic therapy. Median OS for the overall cohort was 19.0m. OS was numerically reduced in immunocompromised patients (11.6m) and in those with primary ICI resistance (6.9m) vs those who had acquired resistance (22.5m) and progression after ICI cessation (47.1m). Conclusions: Selected patients with advanced cSCC, particularly those with acquired resistance, derive meaningful benefit from second-line therapy following ICI progression. Outcomes remain poor for immunosuppressed patients or those with primary resistance, highlighting a major unmet clinical need and the importance of prospective studies in the post-immunotherapy setting.

Aim Anti-programmed death-1/ligand-1 (anti-PD-(L)1) therapies are effective for advanced cSCC. This international study examines response patterns and subsequent management strategies following anti-PD-(L)1 therapy. Method This retrospective cohort study involved 10 centres across 6 countries. Key outcomes included objective response rate (ORR), progression-free survival (PFS), duration of response (DOR), and overall survival (OS) for anti-PD-(L)1 and subsequent therapy. Results One-hundred and fifty-nine patients with advanced cSCC who received anti-PD-(L)1 were evaluated. At median follow-up of 19.3 months (95% CI; 17.5 – 22.2), ORR was 67%. The 12-month and 24-month PFS rates were 62% (95% CI: 55 - 70) and 55% (95% CI: 47 - 64), while 12-month and 24-month OS rates were 81% (95% CI: 75- 88) and 71% (95% CI: 63 – 80). Disease progression occurred in 47 patients (30%); 11% who progressed within 6 weeks of receiving anti-PD-(L)1 therapy, 55% with primary resistance, 28% with secondary resistance, 6% with late progression. Among 21 patients who received subsequent systemic therapies; 8 received cetuximab monotherapy (ORR 50%, DOR 8.5 months [95% CI; 6.9 - NR]) and 5 received anti-PD-(L)1 rechallenge (ORR 20%, one responder with response duration of 3.2 months). In multivariate analysis, a head and neck primary tumour site was associated with higher ORR, and prolonged PFS. Conclusion Our real-world findings reinforce the efficacy of anti-PD-(L)1 therapy in patients with advanced cSCC, with outcomes influenced by primary tumour location. The heterogeneity of subsequent treatments highlights an unmet need in the management of patients who progress on these therapies.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most prevalent cancer diagnosed in Australia, accounting for significant morbidity and mortality. Locoregional recurrence occurs in approximately half of HNSCC patients. Immunotherapy response rates in HNSCC remain modest at 20-35%. Therefore, it is essential to review and build on the evidence around how immunotherapy response is shaped by the tumour microenvironment (TME). Tertiary lymphoid structures (TLS) are emerging as predictors of immunotherapy response. This review summarises recent studies on TLS in HNSCC and investigates whether these have potential as predictive biomarkers.

Background C-POST is a phase 3, double-blind, multicenter, placebo-controlled trial (NCT03969004) of adjuvant cemiplimab for the treatment of CSCC with high risk of recurrence after surgery and radiation therapy. We present the primary analysis including efficacy, safety, and PROs. Methods C-POST included patients with local and/or regional CSCC after surgery and post-operative radiation therapy, at high risk of recurrence based on nodal and/or non-nodal criteria. Patients were randomized 1:1 to cemiplimab 350 mg or placebo Q3W for 12 weeks, then cemiplimab 700 mg or placebo Q6W up to 36 weeks (up to 48 weeks total). Crossover was allowed after disease recurrence. The primary endpoint was disease-free survival (DFS). Secondary endpoints included freedom from local-regional recurrence (FFLRR), freedom from distant recurrence (FFDR), overall survival (OS), and safety. The pre-specified PRO analysis focused on QLQ-C30 global health status/quality of life (GHS/QoL), functional (physical, role, emotional), and symptom (fatigue, pain) scales. Overall PRO changes from baseline across treatment cycles were analyzed using mixed-effects models for repeated measures. The PRO responder analysis used a 10-point threshold for clinically meaningful change. The data cutoff (~50% of final DFS events; pre-specified threshold) was October 4, 2024. Results From June 2019 to August 2024, 415 patients (209/206 cemiplimab/placebo) were randomized: median age, 71 years (range 33–95); 83.9% male; 82.7% head and neck primary; 58.3%/41.7% high-risk nodal/non-nodal categories. Median (range) follow-up was 24 (2–64) months. DFS was superior with cemiplimab versus placebo (24 vs 65 events). Median DFS was not reached in the cemiplimab arm and was 49.4 months in the placebo arm; HR 0.32 (95% CI: 0.20–0.51; P<0.0001). Kaplan–Meier estimated 24-month DFS was 87.1% (95% CI: 80.3–91.6) for cemiplimab and 64.1% (95% CI: 55.9–71.1) for placebo. Cemiplimab improved FFLRR (HR 0.20; 95% CI: 0.09–0.40) and FFDR (HR 0.35; 95% CI: 0.17–0.72) versus placebo. Kaplan–Meier estimated 24-month FFLRR/FFDR was 94.6% (95% CI: 89.1–97.3)/94.3% (95% CI: 89.0–97.1) for cemiplimab and 76.7% (95% CI: 69.1–82.6)/83.8% (95% CI: 76.3–89.0) for placebo. OS HR (April 2025 data cutoff; 33 deaths) for cemiplimab versus placebo was 0.78 (95% CI: 0.39–1.56). Grade ≥3 treatment-emergent adverse events (TEAEs) occurred in 23.9% and 14.2%, and discontinuations due to TEAEs occurred in 9.8% and 1.5% of patients receiving cemiplimab and placebo, respectively. Overall changes from baseline on QLQ-C30 GHS/QoL, functioning, and symptom scores were small and similar between arms. Most patients in both arms reported maintenance or clinically meaningful improvement in QoL in all scales across all cycles (cemiplimab: 55.9–86.8%; placebo: 55.5–88.2%). Kaplan–Meier estimated median time to first deterioration in QoL was similar between arms in all scales (cemiplimab: 5.6–25.6 months; placebo: 8.3–22.2 months). Conclusion Cemiplimab is the first systemic adjuvant immunotherapy to demonstrate statistically significant and clinically meaningful reduction in disease recurrence for high-risk CSCC, and was consistent with the known safety profile of cemiplimab monotherapy in advanced or metastatic disease. QoL was maintained on cemiplimab, with no clinically meaningful differences versus placebo.

6083 Background: C-POST, a phase 3 trial (NCT03969004) in patients (pts) with high risk CSCC after surgery and radiotherapy (RT), demonstrated superior disease-free survival (DFS) for adjuvant cemiplimab (cemi) vs placebo (pbo) (HR, 0.32; P <0.0001); treatment discontinuation due to adverse events occurred in 9.8% vs 1.5% of pts (Rischin, et al. N Engl J Med . 2025). With approximately 6 months of additional follow-up, we present exploratory analyses of DFS per high-risk criteria and per the interval between completion of prior RT and study randomization (2-6 wk vs >6 wk). These additional analyses provide key data to understand the importance of risk factors and the impact of time post RT. Methods: Pts were randomized 1:1 (N=415) to adjuvant cemi or pbo (350 mg cemi or pbo Q3W for 12 wk, then 700 mg cemi or pbo Q6W for 36 wk). All pts had nodal high-risk disease (with extracapsular extension [ECE] and ≥1 node ≥20 mm, or ≥3 nodes regardless of ECE) and/or non-nodal high-risk disease (in-transit metastases, perineural invasion, T4 lesions, or recurrent CSCC with ≥1 other feature). Tumors could meet ≥1 high-risk criteria. Randomization occurred within 2-11 wk after completion of RT. Data cutoff was April 7, 2025. Results: Among 415 pts (209/206 cemi/pbo) randomized, the DFS HR was 0.35 (95% CI, 0.23-0.55) at a median follow-up of 30 months. The most common high-risk criterion was nodal ECE, present in 48.4% of pts (201/415). DFS was improved with cemi vs pbo across all high-risk features, including both nodal and non-nodal criteria (Table 1). A consistent benefit was observed regardless of whether the interval between prior RT completion and study randomization was 2-6 wk (DFS events, 17/117 [14.5%] vs 37/112 [33.0%]; HR, 0.39; 95% CI, 0.22-0.70) or >6 wk (DFS events, 12/91 [13.2%] vs 31/93 [33.3%]; HR, 0.36; 95% CI, 0.19-0.71). Conclusions: In this phase 3 study, adjuvant cemi demonstrated a DFS benefit vs pbo across all high-risk features and regardless of the interval between prior RT completion and randomization. Clinical trial information: NCT03969004 . DFS with cemi vs pbo: Analyses per high-risk criteria. Criteria a Met high risk criteria, n/N (%) DFS events in cemi arm, n/N (%) DFS events in pbo arm, n/N (%) DFS HR (95% CI) Nodal high risk ECE with ≥1 node ≥20 mm 201/415 (48.4) 14/105 (13.3) 27/96 (28.1) 0.44 (0.23-0.84) ≥3 nodes 71/415 (17.1) 7/34 (20.6) 18/37 (48.6) 0.34 (0.13-0.92) Non-nodal high risk In-transit metastases 41/415 (9.9) 2/20 (10.0) 9/21 (42.9) 0.07 (0.01-0.58) T4 lesion 33/415 (8.0) 6/17 (35.3) 5/16 (31.3) 0.58 (0.16-2.11) Perineural invasion 64/415 (15.4) 3/32 (9.4) 8/32 (25.0) 0.27 (0.07-1.04) Recurrent CSCC with ≥1 additional high-risk criteria 105/415 (25.3) 9/55 (16.4) 22/50 (44.0) 0.19 (0.08-0.45) a Tumors could meet ≥1 high-risk criteria.

Abstract Circulating tumor cells (CTCs) are clinically significant biomarkers for cancer detection and monitoring due to their role in metastasis. However, their extreme rarity in peripheral blood and phenotypic heterogeneity, particularly following epithelial-to-mesenchymal transition (EMT), pose challenges for reliable isolation. Conventional epitope-dependent capture methods often fail to detect mesenchymal-like CTCs. Label-free microfluidic platforms offer a promising alternative by exploiting biophysical properties such as size and deformability. Building on our prior work, we developed a high-throughput multi-flow microfluidic (MFM) device to isolate diverse CTCs from clinically relevant blood volumes, with a focus on oropharyngeal cancer (OPC), where CTCs exhibit favorable size characteristics for inertial separation. A parallel microfluidic device comprising six inertial separation channels was fabricated using dry film photolithography and PDMS molding. Two versions were constructed: Chip A (20 mm in length) and Chip B (25 mm in length), both with a 150 µm x 50 µm cross-section. Device performance was evaluated using fluorescent microparticles (10.3, 15.5, and 18.7 µm) and head and neck cancer cell lines CAL27, SCC9, and HPV16-positive SCC2. Samples were processed at 600 µL/min with a buffer flow of 1.2 mL/min. Cell recovery and purity were assessed by high-content imaging and immunofluorescence. SCC2 DNA enrichment was quantified by qPCR. Clinical validation involved 20 p16+ oropharyngeal cancer patients. The MFM device achieved a 5-fold increase in throughput compared to the earlier single-channel design, while maintaining high separation efficiency. Chip A (∼14 µm cutoff) recovered 90-94% of CAL27 and SCC9 cells with <17% WBC contamination. Chip B (∼12 µm cutoff) improved recovery to 92%-97%, but with slightly higher WBC carryover. PCR analysis of HPV16-positive SCC2 cells confirmed >35-fold enrichment of tumor DNA in the CTC fraction. In clinical samples, CTCs were detected in 60% of OPC patients, including epithelial, mesenchymal-like, and dual CK/CSV-positive phenotypes. Notably, dual CK/CSV-positive CTCs, indicative of partial EMT, were observed in 25% of patients. p16 expression was observed in CTCs from 4 of 14 patients, suggesting potential concordance with tumor HPV status. The device preserved cell integrity, enabling downstream molecular analysis. The parallel MFM platform enables robust, label-free isolation of phenotypically diverse CTCs from clinically relevant blood volumes. Its ability to detect EMT-associated phenotypes and HPV16/p16 expression supports its utility in head and neck oncology. Compatibility with molecular workflows such as qPCR and successful clinical validation in OPC patients highlight its translational potential for liquid biopsy applications, including disease monitoring and precision oncology. Citation Format: Chameera E. Weeramange, Jian Zhou, Sreedevi Damodaran, Chiara F. Ghera, Lizbeth Kenny, Brett Hughes, Sarju Vasani, Omar Breik, Rahul Ladwa, Ian Papautsky, Chamindie Punyadeera. Parallel microfluidic platform enables efficient label-free capture of p16+ circulating tumor cells in oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1072.

Perineural spread (PNS) from cutaneous squamous cell carcinoma (cSCC) to the skull base is increasingly recognised as a route of cancer spread. Management historically involved definitive radiotherapy to treat PNS at the skull base. In the endemic region with modern magnetic resonance neurogram (MRN) and skull base surgical expertise, the outcome has improved over the years. With the advent of immunotherapy, the outcome may be maintained while preserving critical organs in the head and neck region. We conducted a critical review of the literature to establish the treatment outcomes and pattern of failure as practice evolved. Furthermore, we described our skull base surgical and radiotherapy management guideline and outlined the emerging paradigm shift in the immunotherapy era.

Head and neck squamous cell carcinoma (HNSCC) represents a biologically diverse group of malignancies within the upper aerodigestive tract and oral cavity and remains a significant cause of global morbidity and mortality. Increasing evidence highlights that the tumour microenvironment (TME) plays a central role in disease progression and therapeutic response. Distinct immune and stromal profiles have been observed between HPV positive (HPV+) and HPV negative (HPV-) HNSCC, underpinning their differing natural history and responses to immunotherapy. Despite advances, only 15-20% of patients with recurrent or metastatic HNSCC (RMHNSCC) respond to immunotherapy, with no difference in response being seen between HPV+ and HPV- disease. This may reflect the complexity and heterogeneity of the TME. This review explores how emerging spatial omics technologies, combining molecular, and spatial context, are reshaping our understanding of the HNSCC microenvironment, with emphasis on recurrent and/or metastatic disease. By delineating the spatial organisation of immune, stromal, and metabolic features, these approaches provide new insights into mechanisms of treatment resistance, prognostic biomarkers, and therapeutic vulnerabilities. Understanding spatial TME dynamics across HPV-related subtypes may ultimately guide more precise and effective treatment strategies for HNSCC.

9582 Background: Patients with advanced cutaneous squamous cell carcinoma (cSCC) or basal cell carcinoma (BCC) frequently develop new primary keratinocyte cancers (KCs) due to field cancerisation. Emerging data suggest immune checkpoint inhibitors (ICIs) may reduce this burden. We evaluated the incidence of new KCs in a prospective immunotherapy cohort. Methods: De-Squamate was a prospective phase II trial evaluating neoadjuvant pembrolizumab using a response-adapted management strategy in resectable cSCC. As part of protocol-mandated safety monitoring, all new primary KCs were prospectively recorded as adverse events. Events included histologically confirmed new cSCCs, BCCs, or other keratinocyte malignancies diagnosed and resected during or after treatment. Person-time was calculated for periods on pembrolizumab and off treatment. Incidence rates were calculated per person-year. Rate ratios were estimated using crude analyses and adjusted models accounting for within-patient clustering. Results: Among the 27 patients, 54 new KCs were recorded. Patients contributed 14.9 person-years on pembrolizumab and 33.4 person-years off treatment. KC incidence was lower during active therapy than after discontinuation (73.7 vs 143.6 per 100 person-years). The unadjusted rate ratio for new KC development off treatment versus on treatment was 1.95 (95% CI, 1.01–3.75). Adjusted analyses using negative-binomial modelling produced similar estimates (adjusted rate ratio 1.94; 95% CI, 0.64–5.89). Reductions were most pronounced for BCCs, with more modest effects observed for new primary cSCCs (table 1). Conclusions: In this prospective cohort, new KCs occurred at approximately twice the rate after discontinuation of pembrolizumab compared with during active therapy. These findings support the hypothesis that ICIs may suppress carcinogenesis within chronically damaged skin fields. Larger studies are needed to clarify the potential immunopreventative effect of ICIs in patients with field-driven KCs. Clinical trial information: NCT05025813 . Incidence of new keratinocyte cancers by treatment exposure. Outcome On Pembrolizumab Off Pembrolizumab (Follow-up) Unadjusted Rate Ratio† (95% CI) Adjusted Rate Ratio‡ (95% CI) Person-years 14.9 33.4 – – All keratinocyte cancers 11 events0.74 / PY 43 events1.29 / PY 1.95 (1.01–3.75) 1.94 (0.64–5.89) Cutaneous SCC (new primaries) 6 events0.40 / PY 10 events0.30 / PY 0.75* – Basal cell carcinoma 5 events0.34 / PY 38 events1.14 / PY 3.35* – †Off-treatment vs on-treatment. ‡Adjusted for patient-level clustering using negative-binomial model. *Crude comparison shown; formal adjusted estimates limited by small event numbers.

Despite the advancements made in the diagnosis and treatment of head and neck squamous cell carcinoma (HNSCC), metastasis remains the primary cause of cancer-related deaths. Circulating tumour cells (CTCs) may offer a valuable platform for personalised treatment selection. Towards this goal, the establishment of short-term CTC cultures would allow ex vivo characterisation and drug sensitivity testing. A negative enrichment technique (RosetteSep (TM), Human CD45 depletion cocktail) was used to capture CTCs for downstream culturing. Whole-exome sequencing was applied to compare the genomic features in primary tumour and CTC cultures. In vitro functional assays and in vivo mouse models were used to confirm the origin and biological properties of cultured CTCs. Cytotoxicity assays were used to measure CTCs' responsiveness to chemotherapeutic drugs. We successfully derived 16 CTC cell lines from HNSCC patients' blood samples (16/107). Tumoural p16-positivity notably increased the successful culture rate. CTC cultures displayed similar migratory and invasive capabilities compared to the American Type Culture Collection HNSCC cell lines. In addition, CTC cultures generated a subcutaneous tumour in immunocompromised NSG mice, which was confirmed by a pathologist to be of HNSCC. CTCs derived from post-treatment blood samples showed a trend towards reduced sensitivity to Cisplatin and 5-fluorouracil, although this did not reach statistical significance (Mann-Whitney test, p > .05). HNSCC-derived CTC cultures closely recapitulate the features of primary tumours, providing a model system for in vitro drug testing and may pave the way towards a personalized medicine approach for HNSCC.

ABSTRACT:Although human papillomavirus (HPV)–positive oropharyngeal squamous cell carcinoma (OPSCC) is associated with better prognosis than HPV-negative disease, ∼30% of cases relapse despite curative-intent radiotherapy (±chemotherapy). We aimed to develop a proteomic signature associated with risk of recurrence within HPV+OPSCC. We analyzed tumor specimens from 124 patients with T1–4N0–3M0 HPV+OPSCC: 50 patients with residual or recurrent disease within 5 years of treatment and 74 age and performance status–matched patients with no recurrence. Proteomic analysis was performed on archival formalin-fixed, paraffin-embedded primary tumor core biopsy specimens and matched normal adjacent tissues using quantitative data-independent acquisition mass spectrometry. Recurrence-free survival (RFS), both locoregional and distant, was the primary endpoint. Univariate Cox regression analysis identified peptides associated with RFS, from which a risk score was established to generate a peptide-based signature. A total of 7,597 protein groups were identified across the cohort, 1,565 of which were differentially abundant between tumor and normal adjacent tissues, with 1,218 being significantly increased in tumors. Improved 5-year RFS (q-value < 0.5) was associated with 405 differentially abundant peptides (from 233 unique proteins) within the 124 tumors. Among them a 26-peptide signature encompassing 26 protein groups was associated with RFS and was able to stratify patients into low, intermediate, or high risk of recurrence (concordance index = 0.941, P < 0.0001). Data available via ProteomeXchange PXD036891. Overall, a 26-peptide signature can be used to risk-stratify HPV+OPSCC. Validation of this proteomic prognostic signature in an independent cohort is required to assess its potential use in future clinical trials to better tailor initial therapy. SIGNIFICANCE:HPV+OPSCC incidence is increasing, with heterogeneous treatment outcomes despite favorable prognosis. Current de-escalation strategies show inferior results, highlighting the need for precise risk stratification. Using data-independent acquisition mass spectrometry proteomics, we identified a 26-peptide signature that stratifies patients into risk categories, potentially enabling personalized treatment decisions and optimal patient selection for de-escalation trials.

INTRODUCTION:Immune checkpoint inhibitors (ICIs) are increasingly used in older adults, but tolerability among frail patients remains uncertain. This study examined immune-related adverse events (irAEs) and treatment outcomes in frail and non-frail adults aged ≥65 years. MATERIALS AND METHODS:A retrospective single-centre review was conducted at Princess Alexandra Hospital (Queensland, Australia) of patients aged ≥65 who commenced at least one cycle of ICI between December 2021 and July 2023. Frailty was assessed using the Australian 58-item Frailty Index (FI) (non-frail <0.25, frail ≥0.25). The primary outcome was any irAE as defined by Common Terminology Criteria for Adverse Events (CTCAE v5). Secondary outcomes included treatment discontinuation, cycles, interruptions, and corticosteroid use. Univariable and multivariable logistic regression modelling was performed, adjusting for age, sex, cycles, tumour type, stage, concurrent therapy, and Eastern Cooperative Oncology Group Performance Status (ECOG-PS). RESULTS:Of 122 adults (mean age 75.8 years, 66.4 % male), 51 (41.8 %) were frail. Twenty patients were still receiving ICIs and had not yet completed treatment at last census, leaving 102 records with evaluable outcomes for the final modelling. Any irAE occurred in 53 non-frail adults (74.7 %) and 28 frail adults (54.9 %) (adjusted OR 0.41, 95 % CI 0.19-0.89, p = 0.02). Non-frail adults had more grade 1 events (62.0 % vs 43.1 %, p = 0.045) and skin-related events (48 % vs 27 %, p = 0.04). Grade ≥ 3 events occurred in 11.3 % non-frail vs 11.8 % frail (OR 1.10, 95 % CI 0.38-3.17). Early discontinuation was 60.6 % in non-frail vs 74.5 % in frail (OR 1.90, 95 % CI 0.86-4.19, p = 0.12). Median cycles were 10 (IQR 4-16) in non-frail vs 6 (IQR 4-11) in frail (p = 0.04). DISCUSSION:Frailty (FI ≥0.25) was associated with fewer irAEs, but frail adults received fewer cycles. These findings are exploratory and should be interpreted cautiously as differences in exposure likely confound toxicity comparisons; time-to-event analyses are warranted.

With nearly a million new cases expected, the global death rate from mucosal head and neck cancer is projected to reach around 50% in 2024. In the U.S., patients with localized tumors have a five-year survival rate of 50-60%. While immune checkpoint inhibitors (ICIs) have shown promise in extending survival, significant challenges remain, particularly the limited effectiveness of PD-1/PD-L1 blockade therapies. Since many studies have shown that PD-L1 protein expression alone may not be a reliable predictor of response to ICI therapy, understanding the spatial context of PD-1/PD-L1 interactions might be crucial for identifying immune evasion mechanisms and predicting responses to ICIs in head and neck cancer. To address this, we aimed to take a more comprehensive approach by mapping PD-1/PD-L1 interactions across n=35 mucosal head and neck squamous cell carcinoma (HNSCC) tissue samples collected before ICI treatment, using a combination of high-plex spatial proteomics and the in situ Navinci Proximity Ligation Assay (isPLA). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained using multiplex immunofluorescence and the Navinci Diagnostics isPLA assay to detect PD-1/PD-L1 interactions. After incubation with primary anti-PD1 and anti-PD-L1 antibodies, secondary probes tagged with oligonucleotides were applied, enabling PLA to highlight the interactions through a ligation step and an amplification process to enhance the visibility of the protein interactions. Finally, a detection solution was applied to visualize the amplification product at the sites of PD1/PD-L1 interaction.We identified distinct spatial patterns of PD-1/PD-L1 interactions at the tumor-stroma interface, particularly in progressive disease (PD) patients, who exhibited dense layers of isPLA+ macrophages, CD3e T cells, and tumor cells, suggesting an immunosuppressive barrier in this region. In contrast, complete response (CR) patients showed rare or absent barriers, with enriched immune cell aggregates, including B cells and T cells, near the tumor boundary. These findings were corroborated through neighborhood clustering analysis, revealing significant associations between isPLA+ regions and poor prognosis.The spatially resolved nature of our analysis underscores the critical role of PD-1/PD-L1 interactions in shaping the tumor microenvironment (TME) and highlights their potential as predictive biomarkers for ICI therapy efficacy. Unlike methods that detect mere colocalization, isPLA technology enables us to detect true PD-1/PD-L1 interactions, which are more likely to affect immune suppression directly. Our study also emphasizes the need for further exploration of macrophage and T cell subtypes, as well as PD-1/PD-L1 interactions across tumor-stroma boundaries in various cancers to improve immunotherapy strategies. Vahid Yaghoubi Naei, Rafael Tubelleza, James Monkman, Habib Sadeghirad, Meg L. Donovan, Tony Blick, Agata Wicher, Sara Bodbin, Subham Basu, Robert Stad, Catherine Barnett, Ken O’Byrne, Rahul Ladwa, Caroline Cooper, Majid Ebrahimi Warkiani, Brett GM Hughes, Arutha Kulasinghe. Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 750.