Introduction:Head and neck cancer (HNC) treatment on the Unity MR-Linac (MRL) (Elekta AB, Stockholm, Sweden) has been developed using the novel adapt-to-shape Lite (ATS-lite) method to create clinically acceptable adaptive treatments clinician-free. Here we investigate patient experience and acceptability of this technique. Methods:Ten HNC patients treated to 65 Gy in 30 fractions with MRI-guided adaptive radiotherapy (MRIgART) within the PERMIT trial (NCT03727698), were included. Data collected comprised patient demographics, treatment time, and patient experience, using an established MRL questionnaire.Back-up plans were created for use on the conventional linac with CT guidance, to prevent missed fractions. The frequency of use was collected and categorised to reflect the cause. Results:The median total treatment time for ATS-lite method was 39 min. The percentage of treatments under 60 mins was 98.8 %.Questionnaire response rate was 85% and individual question response rate was 99%. Ninety-six percent of responses scored 2 or 3 on the Likert scale, a positive answer. The lowest scoring question was "I forced myself to manage the situation," with a mean (SD) of 2.4 (0.9).The MRL delivered 84.7 % of treatments. The back-up plan was used for 46 fractions, 7 attributed to patient tolerance (n = 2 patients). Conclusion:Average treatment times for the ATS-lite HNC MRIgART are acceptable and faster than reported ATP treatment times. Patient-reported experience was extremely positive. Use of back-up plans attributable to lack of patient tolerance was low. This technique can used with the confidence that patient experience is not negatively impacted.

Head and neck cancer and its treatment poses significant physiologic and psychologic burdens for patients. Involvement of the whole multidisciplinary team is crucial in addressing the broad functional deficits those patients experience. In addition, cancer recurrence or development of a new second malignancy is common. Current strategies are unrefined and could be criticized for being a 'one-size fits all'. Clinical trials are examining new strategies of surveillance in head and neck cancer, which may provide evidence for more risk-adapted, technology-driven, and ultimately more personalized follow-up paradigms.

Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient prognosis. To gain insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MAPK axis represents a hub of tumor-stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together, these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.

Abstract Genomic analysis of the T-cell receptor (TCR) reveals the strength, breadth, and clonal dynamics of the adaptive immune response to pathogens or cancer. The diversity of the TCR repertoire, however, means that sequencing is technically challenging, particularly for samples with low-quality, degraded nucleic acids. Here, we developed and validated FUME-TCRseq, a robust and sensitive RNA-based TCR sequencing methodology that is suitable for formalin-fixed paraffin-embedded samples and low amounts of input material. FUME-TCRseq incorporates unique molecular identifiers into each molecule of cDNA, allowing correction for sequencing errors and PCR bias. Using RNA extracted from colorectal and head and neck cancers to benchmark the accuracy and sensitivity of FUME-TCRseq against existing methods demonstrated excellent concordance between the datasets. Furthermore, FUME-TCRseq detected more clonotypes than a commercial RNA-based alternative, with shorter library preparation time and significantly lower cost. The high sensitivity and the ability to sequence RNA of poor quality and limited amount enabled quantitative analysis of small numbers of cells from archival tissue sections, which is not possible with other methods. Spatially resolved FUME-TCRseq analysis of colorectal cancers using macrodissected archival samples revealed the shifting T-cell landscapes at the transition to an invasive phenotype and between tumor subclones containing distinct driver alterations. In summary, FUME-TCRseq represents an accurate, sensitive, and low-cost tool for the characterization of T-cell repertoires, particularly in samples with low-quality RNA that have not been accessible using existing methodology. Significance: FUME-TCRseq is a TCR sequencing methodology that supports sensitive and spatially resolved detection of TCR clones in archival clinical specimens, which can facilitate longitudinal tracking of immune responses through disease course and treatment.

IntroductionThere is a need to improve the outcomes of patients with head and neck squamous cell carcinoma (HNSCC) and nasopharyngeal carcinoma (NPC), especially in recurrent unresectable and metastatic (R/M) setting. Antibody-drug conjugates (ADC) and bispecific antibodies (BsAb) may deliver promising results.MethodsWe conducted a systematic literature review to identify ADC and BsAb clinical trials, involving patients with HNSCC and NPC, from database creation to December 2023. We reported trial characteristics, overall response rate (ORR), overall survival (OS), and grade ≥ 3 treatment-related adverse events (trAEs).Results23 trials (65 % phase I) were found, involving 540 R/M patients (355 [20trials] HNSCC and 185 [5trials] NPC). There were 13 ADC (n = 343) and 10 BsAb (n = 197) trials. 96 % patients were refractory to standard of care treatments. ORR ranged from 0 to 100 %, with the highest ORR for GEN1042 plus chemoimmunotherapy. ORRs for monotherapies were 47 % for ADC, and 0–37 % for BsAb. MRG003 reached in HNSCC 43 % and NPC 47 %. BL-B01D1 54 % in NPC. Longest median OS was seen with MRG003 and KN046. Grade ≥ 3 trAEs were 28–60 % in ADC trials, and 3–33 % BsAb. Grade ≥ 3 myelosuppressive trAEs were typically seen in 8 ADC trials, while 4 BsAb showed infusion-related reactions (IRR). Four treatment-related deaths were reported (1 pneumonitis), all ADC trials.ConclusionADC and BsAb antibodies show promise in R/M HNSCC and NPC. Results are premature by small sample sizes and lack of control arm. ADC mainly caused myelosuppression and a pneumonitis case, and BsAb IRR. Further research is warranted in this setting.

Pembrolizumab has received approval in the UK as first-line monotherapy for recurrent and/or metastatic HNSCC (R/M HNSCC) following the results of the KEYNOTE-048 trial, which demonstrated a longer overall survival (OS) in comparison to the EXTREME chemotherapy regimen in patients with a combined positive score (CPS) ≥1. In this article, we provide retrospective real-world data on the role of pembrolizumab monotherapy as first-line systemic therapy for HNSCC across 18 centers in the UK from March 20, 2020 to May 31, 2021. 211 patients were included, and in the efficacy analysis, the objective response rate (ORR) was 24.7%, the median progression-free survival (PFS) was 4.8 months (95% confidence interval [CI]: 3.6-6.1), and the median OS was 10.8 months (95% CI 9.0-12.5). Pembrolizumab monotherapy was well tolerated, with 18 patients having to stop treatment owing to immune-related adverse events (irAEs). 53 patients proceeded to second-line treatment with a median PFS2 of 10.2 months (95% CI: 8.8-11.5). Moreover, patients with documented irAEs had a statistically significant longer median PFS (11.3 vs. 3.3 months; log-rank p value = <.001) and median OS (18.8 vs. 8.9 months; log-rank p value <.001). The efficacy and safety of pembrolizumab first-line monotherapy for HNSCC has been validated using real-world data.

Purpose: Swallow-related motion of the larynx is most significant in the cranio-caudal directions and of` short duration. Conventional target definition for radical radiation therapy includes coverage of the whole larynx. This study longitudinally examined respiration- and swallow-related laryngeal motions using cine-magnetic resonance imaging. We further analyzed the dosimetry to organs at risk by comparing 3D-conformal radiation therapy (3D-CRT), volumetric modulated arc therapy (VMAT), and intensity modulated radiation therapy (IMRT) techniques. Methods: Fifteen patients with T1-2 N0 glottic squamous cell carcinomas were prospectively recruited for up to 3 cine-MRI scans on the Elekta Unity MR-Linear accelerator, at the beginning, middle, and end of a course of radical radiation therapy. Swallow frequency and motion of the hyoid bone, cricoid and thyroid cartilages, and vocal cords were recorded during swallow and rest. Adapted treatment volumes consisted of gross tumor volume + 0.5-1 cm to a clinical target volume with an additional internal target volume (ITV) for personalized resting-motion. Swallow-related motion was deemed infrequent and was not accounted for in the ITV. We compared radiation therapy plans for 3D-CRT (whole larynx), VMAT (whole larynx), and VMAT and IMRT (ITV for resting motion). Results: Resting- and swallow-related motions were most prominent in the cranio-caudal plane. There were no significant changes in the magnitude of motion over the course of radiation therapy. There was a trend of a progressive reduction in the frequency of swallow. Treatment of partial larynx volumes with intensity modulated methods significantly reduced the dose to carotid arteries, compared with treatment of whole larynx volumes. Robustness analysis demonstrated that when accounting for intrafraction swallow, the total dose delivered to the ITV/planning target volume was maintained at above 95%. Conclusions: Swallow-related motions are infrequent and accounting for resting motion in an ITV is sufficient. VMAT/IMRT techniques that treat more conformal targets can significantly spare critical organs at risk such as the carotid arteries and thyroid gland, potentially reducing the risk of carotid artery stenosis-related complications and other long-term complications.

Supplementary Tables 1 and 2, and Supplementary Figure 1

A, Bar plots showing the proportion of each V gene detected by FUME-TCRseq (right column) and immunoSEQ (left column) in three representative FFPE head and neck cancer samples (HN1, HN2, HN3). B, Bar chart showing the numbers of unique TCR clonotypes detected in the three samples with FUME-TCRseq and immunoSEQ methodologies. C, Heat map showing the Morisita overlap between each sample. D, Plots showing the frequencies of the 10 most common clonotypes detected in each sample by immunoSEQ and the frequencies at which they appear in the FUME-TCRseq data.

BackgroundThe effect of chemoradiation on the anti-cancer immune response is being increasingly acknowledged; however, its clinical implications in treatment responses are yet to be fully understood. Human papillomavirus (HPV)-driven malignancies express viral oncogenic proteins which may serve as tumor-specific antigens and represent ideal candidates for monitoring the peripheral T-cell receptor (TCR) changes secondary to chemoradiotherapy (CRT).MethodsWe performed intra-tumoral and pre- and post-treatment peripheral TCR sequencing in a cohort of patients with locally-advanced HPV16-positive cancers treated with CRT. An in silico computational pipeline was used to cluster TCR repertoire based on epitope-specificity and to predict affinity between these clusters and HPV16-derived epitopes.ResultsIntra-tumoral repertoire diversity, intra-tumoral and post-treatment peripheral CDR3β similarity clustering were predictive of response. In responders, CRT triggered an increase peripheral TCR clonality and clonal relatedness. Post-treatment expansion of baseline peripheral dominant TCRs was associated with response. Responders showed more baseline clustered structures of TCRs maintained post-treatment and displayed significantly more maintained clustered structures. When applying clustering by TCR-specificity methods, responders displayed a higher proportion of intra-tumoral TCRs predicted to recognise HPV16 peptides.ConclusionsBaseline TCR characteristics and changes in the peripheral T-cell clones triggered by CRT are associated with treatment outcome. Maintenance and boosting of pre-existing clonotypes are key elements of an effective anti-cancer immune response driven by CRT, supporting a paradigm in which the immune system plays a central role in the success of CRT in current standard-of-care protocols.

A, Hematoxylin and eosin showing the regions of FFPE adenoma and carcinoma that were macrodissected for RNA extraction and FUME-TCRseq. Scale bar, 5 mm. B, Bar chart showing the numbers of unique TCR clonotypes detected in carcinoma and adenoma regions. C, Bar chart showing the diversity of the TCR repertoire in carcinoma and adenoma regions. D, Plot showing the frequencies of the 10 most common clonotypes detected in the adenoma region and the frequencies at which they appear in the carcinoma.

A, Hematoxylin and eosin images and annotated BaseScope staining for point mutations in PIK3CA (Case C537, top) or KRAS (Case 539, bottom). Scale bars, 5 mm (top) and 1 mm (bottom). Regions circled in green were macrodissected for FUME-TCRseq. B, Bar chart showing the numbers of unique TCR clonotypes detected in the PIK3CA WT and mutant (E545K) subclones of Case C537. C, Bar chart showing the diversity of the TCR repertoire in PIK3CA WT and mutant subclones of Case C537. D, Plot showing the frequencies of the 10 most common clonotypes detected in the PIK3CA E545K mutant subclone and the frequencies at which they appear in the WT subclone. Arrow, TCR sequence CASSYGHPNTEAFF. E, Bar chart showing the numbers of unique TCR clonotypes detected in the KRAS WT and mutant (G12C) subclones of Case C539. F, Bar chart showing the diversity of the TCR repertoire in KRAS WT and mutant subclones of Case C539. G, Plot showing the frequencies of the 10 most common clonotypes detected in the KRAS G12C mutant subclone and the frequencies at which they appear in the WT subclone.

Schematic representation of the FUME-TCRseq methodology, whereby RNA is reverse-transcribed using a UMI-containing primer, followed by multiplex PCR amplification of the CDR3 region using a panel of 38 primers and a second PCR to add sample-specific indexes. SP1 (33bp) and SP2 (24bp) are binding sites for universal Illumina sequencing primers and are used for paired-end sequencing of the amplified TCR and sequencing of the 8bp index 1 (i1) and the 8bp index 2 (i2). P7 (24bp) and P5 (29bp) are flow cell binding sequences for Illumina-sequencing platforms.

e18009 Background: Clinical trials drive improvements in patient care with head and neck squamous cell carcinoma (HNSCC), presenting opportunities for optimizing clinical outcomes. However, a significant number of patients (pts) miss out on new therapeutic opportunities due to screen failure (SF) after signing the informed consent form. Understanding the circumstances leading to SF might improve enrollment processes. Methods: Retrospective cohort analysis of pts with HNSCC intended for treatment within a clinical trial at the Royal Marsden Cancer Centre but who experienced SF between Jan 2016 and Jan 2024. Demographic and cancer-related characteristics, comorbidities, consent and SF dates, SF causes, and subsequent outcomes after SF were analyzed. Results: 76 pts who experienced SF were identified across 33 different trials. Cohort characteristics and type of trials offered are described in the table. The largest number of SFs was due to exclusion criteria being met in screening tests in 37 pts (49%): Pathology sample did not meet the quality standards in 11 pts (14%); abnormal laboratory tests in 5pts (6%); abnormal echocardiogram/reduced ejection fraction in 5 pts (6%); vessel invasion/ encasement in 4 pts (5%); non-measurable disease in 2 pts (3%); abnormal audiometry in 1pt (1%) among other reasons. The next commonest cause of SF was rapid progression during screening in 28 pts (37%): 11% compromised airway; 11% active bleeding; 5% central nervous system progression and 7% other rapid progression causes. Other causes of SF included: withdrawn consent in 7 pts (9%), comorbidity-related complication in 3 pts (5%) and study termination by sponsor in 2 pts (3%). The median time between consenting and SF was 20 days (range 4-42). After SF, 8% reconsented for the same trial, 56% received standard therapy, and 36% received best supportive care. Conclusions: This study reveals abnormal screening tests and rapid progression as primary causes for SF in patients with HNSCC. Timely referral , airway assessments and improved tissue acquisition could improve enrollment and minimize SF. [Table: see text]