Head Tumors Research Articles

Nutritional status in advanced pancreatic cancer

Background. Exocrine pancreatic insufficiency (EPI) occurs in most patients with advanced pancreatic cancer (PC) and even more often when a tumor is localized in the head of the pancreas. However, insufficient attention is paid to the diagnosis of EPI and assessment of nutritional status, which negatively affects the results of treatment.
 Materials and methods. One hundred fifty eight patients with primary diagnosed locally advanced and metastatic PC were included in the retrospective study. We used the calculation of the odds ratio with 95% CI with an assessment of the p value using the Pearson ÷2 test to compare the incidence of clinical and laboratory parameters of nutritional status with values below the lower limit of normal (LLN) depending on the location of the tumor in the pancreas.
 Results. Fecal elastase test was performed in only 19 (12%) patients. In this group, the incidence of EPI was 73.6%. Pancreatic enzyme replacement therapy in the minimal sufficient dose was prescribed in 17.1% of the general cohort. The level of hemoglobin, erythrocytes and albumin below the LLN was found in patients with the tumor in the pancreatic head, respectively, 2.742 (95% CI 1.27–5.92), 3.788 (95% CI 1.554–9.232) and 9.767 (95% CI 1.255–76.027) times more often than in patients with cancer in the body-tail of the pancreas. In patients with the tumor in the pancreatic head, the lower levels of hemoglobin (median 125 g/l vs 132 g/l, respectively), erythrocytes (4.19 mil/μl vs 4.51 mil/μl), total protein (69.6 g/l vs 71.5 g/l), and albumin (40.1 g/l vs 42 g/l), as well as the values of nutritional indices: prognostic nutritional index, nutritional risk index, hemoglobin, albumin, lymphocyte and platelet ratio index, and prognostic immune nutritional index were observed.
 Conclusion. Diagnosis and treatment of EPI remains inadequately attended. The values of nutritional status parameters in patients with PC in the head are lower than in patients with a tumor in the body-tail of the pancreas, which reflects the contribution of EPI.

The small extracellular vesicle-mediated intercellular transformation of CXCR1Low to CXCR1High tumour cells promotes the progression of head and neck squamous cell carcinoma.

The heterogeneity of tumour cells enables cancers to dynamically adapt to microenvironmental stresses during progression. However, the mechanism underlying the transformation and intercellular communication between heterogeneous tumour cells has remained elusive. Here, we report a "contagion model" that mediates intercellular transformation between heterogeneous tumour cells which facilitates tumour progression. Initially identifying heterogeneous expression of CXCR1, a receptor for interleukin-8, in head and neck squamous cell carcinoma (HNSCC) tumour cells, we found that CXCR1High tumour cells had higher abilities for migration and invasion. Following interleukin-8-mediated activation, CXCR1High cells transformed CXCR1Low cells into CXCR1High cells through the secretion of small extracellular vesicles (sEVs), which increased the proportion of CXCR1High cells and facilitated tumour progression. Mechanistically, we demonstrate that sEVs derived from interleukin-8-activated CXCR1High cells contain high levels of ATP citrate lyase (ACLY), which acetylates NF-κB p65 and facilitates its nuclear translocation to transcribe CXCR1 in CXCR1Low cells. That process could be inhibited by Bempedoic acid, an FDA-approved ACLY-targeted drug. Taken together, our study reveals an sEV-mediated transformation of CXCR1Low to CXCR1High cells that promotes HNSCC progression. This provides a new paradigm to explain the dynamic changes of heterogeneous tumour cells, and identifies Bempedoic acid as a potential drug for HNSCC treatment.

Clinicopathologic comparison of basal cell carcinoma among a diverse patient population in Los Angeles County.

Basal cell carcinoma (BCC) is the most common malignancy in the United States. The majority of cases are identified in Non-Hispanic Whites (NHW) and are far less demonstrated in patients of colour (POC). However, the Hispanic population represents a large and growing proportion of the US population, and skin cancer diagnoses in Hispanics are rising. Thus, the goal of this study is to examine clinicopathologic differences between BCC in Hispanics versus NHW. A retrospective chart review of Hispanic and NHW patients with BCC at Los Angeles County+USC Medical Center from January 2018 to March 2020 was performed. In total, 101 BCC samples from the first 100 patients identified of Hispanic ancestry, as well as 50 BCC samples identified from the first 50 patients identifying as NHW, were included for analysis. Patient characteristics (age, sex, medical history, and ethnicity), as well as tumour characteristics (location, subtype, tumour depth, and perineural invasion), were collected. We used between subjects t-tests for continuous variables, and chi-square tests for categorical variables. In total, 151 specimens were collected amongst 122 subjects (79 Hispanics and 43 NHW patients). Among NHW, the majority of patients (74.4%) were men, but among the Hispanic population, the majority (68.4%) were female (p<0.001). Prior history of other skin cancer was more common among NHW (67.4%) than Hispanics (31.6%) (p=<0.001). The Hispanic population had a significantly higher proportion of head and neck tumours (p=0.0004) but a lower proportion of extremity tumours (p=0.001) compared to NHW. Pigmented BCC was significantly more common among Hispanic patients (p<0.01). Finally, within the Hispanic group, there was a significant association between sex and histology (p=0.004), with Hispanic men demonstrating more aggressive mix histology compared to Hispanic women. Our study supports the notion that BCC disparities occur among POC compared to NHW. This includes variations in epidemiologic factors such as sex and past medical history, primary tumour location, and pathologic characteristics. Further research should be conducted to identify additional differences in skin cancer presentation in POC to reduce the gaps in skin cancer knowledge and care.

Abstract 4352: A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing

Abstract Persistent Human Papillomavirus (HPV) infection causes an increased risk of Head and Neck Squamous Cell Carcinoma (HNSCC). However, a comprehensive understanding of the intricate molecular events within virus-positive tumors remains a critical knowledge gap. Therefore, we used long-read sequencing to study the intricacies of HPV integration events within the genomic landscape of HPV+ HNSCC tumors. A total of 16 tissue biopsies, including 10 tumors and 6 matched adjacent normal tissues from histopathologically confirmed HNSCC subjects were sequenced by LSK114 chemistry of Oxford Nanopore Technology with an average coverage of 30X and 10X, respectively. The predicted mitochondrial haplotype from Mitomaster confirmed that the tumor/normal pair was from the same individual. The HPV integration status was determined by aligning fastq_pass files at the Cancer Genomics Cloud and viewing the breakpoints in the Integrated Genome Viewer. HPV lineage and sub-lineage classification was determined using ClustalOmega. We detected HPV16 in 8/10 tumors, with sublineages A1 and A2 accounting for 38% (3/8) and D3 for 25% (2/8). One tumor sample had a very low HPV load with only one HPV16 sequence read and low detection by PCR. Interestingly, we found that 5/8 HPV positive tumors had episomal HPV16 with different states. So, two tumors had intact HPV episomes, two more had either a small deletion or an insertion in the episomal HPV, and the last one had a rearranged episomal multimer (Table 1). The remaining three tumors had integrated HPV16: one with type 1 integrations at 11q12.13 and two others with type 2 integrations at chr1q42.12 and chr14q21.1. Our results indicate that episomal HPV16 is more prevalent than integrated HPV16 in primary HPV+ HNSCC samples. These findings provide new insights into the molecular mechanisms and consequences of HPV infection in HNSCC. Table 1: HPV characterization in HNSCC studied tissue biopsies. Patient ID Race HPV status HPV sublineages Predicted Haplotype HPV pattern #PID_1 Caucasian HPV16 + A2 T2b (T2b) Type 2 Integration (chr1q42.12) #PID_2 - HPV- - I1a (I1a1b) - #PID_3 Caucasian HPV16 + A1 H1q (H1q) Episomal (rearranged multimer) PID_4 African American HPV16 + A1 - Episomal (deletion of 636bp from 4892 to 5528) #PID_5 - HPV16 + A2 H1 (H1+16189) Type 1 Integration (complex chr11q12.13) *#PID_6 Caucasian HPV16 + - T2b (T2b) - PID_7 Caucasian HPV16 + D3 - Episomal #PID_8 African American HPV16 + D3 L1c (L1c3a) Type 2 Integration (chr14q21.1) PID_9 African American HPV16 + A2 - Episomal (insertion of 257bp from 7310-7567) PID_10 Caucasian HPV16 + A1 - Episomal *Very weakly HPV16 positive; #Tumor-normal pair Citation Format: Sonam Tulsyan, Vera Mukhina, Isabel Rodriguez, Subhendu R. Choudhury, Erin Allor, Kyle Hatte, Michael Dean, Daria A. Gaykalova. A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4352.

Abstract 5415: Fibroblast mediated head and neck tumor cell invasion in 3D culture is independent of partial epithelial to mesenchymal transition

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with a five-year-survival of 61-64%. Recent studies have demonstrated tumor keratinocytes with features of a partial transition from epithelial to a mesenchymal phenotype, also known as partial-EMT (pEMT), at the edge of invading tumor islands associates with metastatic disease. As such, pEMT likely results from direct interactions between tumor cells and their surrounding microenvironment. To test if specific secreted factors or cell interactions drive the acquisition of pEMT, we first analyzed a series of primary and established HNSCC cell populations to determine TGF-β1 responsiveness since TGF-β1 is a known driver of pEMT. Of the eight populations tested, six displayed an increased level of pSMAD3 upon incubation with 5 ng/mL of TGF-β1 (p &amp;lt; 0.0001). To determine if this TGF-β1 response leads to an increase in pEMT phenotype, a pEMT-score (derived from mRNA expression of 9 pEMT markers) for each HNSCC population was determined following TGF-β1 treatment. As expected, the two non-responsive HNSCC populations showed no increase in pEMT-score, whilst five of the six TGF-β1-responsive HNSCC populations demonstrated a significant increase in pEMT-score (p = 0.006). To assess if TGF-β induced pEMT phenotype correlates with increased invasive potential, we produced 3D skin equivalent models using the eight HNSCC keratinocyte populations, in the presence or absence of primary fibroblasts and/or TGF-β1 treatment. TGF-β1 treatment alone caused a significant increase in invasion of all five TGF-β1-induced pEMT HNSCC populations (r2 = 0.449, p = 0.0045), whilst the three populations previously shown to be pEMT unresponsive to TGF-β1 treatment showed no increase. Interestingly, addition of primary fibroblasts without TGF-β1 treatment caused a universal dramatic increase in invasion in 3D organ culture (p = 0.0064, 34-fold increase), regardless of the previously determined TGF-β1-induced pEMT status. Unexpectedly, TGF-β1 treatment in the presence of fibroblasts caused no significant increase in invasion in 3D cultures (r2 = 0.00001, p = 0.9906). We next compared levels of pEMT markers in 3D cultures using immunofluorescence and observed a significant increase in PDPN in six of the eight HNSCC populations (p = 0.0136) with the addition of fibroblasts suggesting that factors independent of TGF-β1 contributes to pEMT marker expression and invasion in 3D culture. Overall, these data demonstrate fibroblasts induce HNSCC keratinocyte invasion regardless of the population’s ability to respond to TGF-β1-induced pEMT and that pEMT may be driven by factors independent of TGF-β1 in 3D culture. Citation Format: Pyung Hun Park, Michael M. Alexander, Joseph M. Curry, Adam J. Luginbuhl, Andrew P. South. Fibroblast mediated head and neck tumor cell invasion in 3D culture is independent of partial epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5415.

Abstract 6036: Flash proton radiotherapy mitigates radiation-induced salivary gland dysfunction and oral mucositis in mice

Abstract Background- Recent studies have reported that ultra-high dose rate “FLASH” Proton radiation therapy (F-PRT) decreases normal tissue toxicity while maintaining tumor-controlling efficacy compared to Standard Proton RT (S-PRT), that are used for patient treatments. However, although highly efficacious in eliminating tumors, damage to the otherwise healthy salivary glands and oral mucosa is often unavoidable in patients with head and neck cancer, leaving patients with lifelong xerostomia and other comorbidities. Aim- In this study, we aimed to investigate the effect of F-PRT on radiation-induced oral mucositis and salivary gland dysfunction and in controlling orthotopic tumor growth. Methods- The head and neck area of C57Bl/6 mice was irradiated with a single dose of 16 Gy or a fractionated dose of 8 Gy x 3 of F-PRT (128Gy/s) or S-PRT (0.95 Gy/s). Oral mucositis was analyzed by histopathological examination. Radiation-induced xerostomia was studied by measuring the saliva flow rate of mice. To examine the ability of F-PRT to control orthotopic head and neck tumors, tongue tumors were generated in mice and then irradiated with F-PRT/S-PRT. Results- Following irradiation with a single dose or a fractionated dose, saliva flow was reduced by both treatments. However, the F-PRT-treated mice showed a significant improvement in salivary flow at 14 days (p&amp;lt;0.05) and 28 days (p&amp;lt;0.005) post irradiation. Expression of AQP5 was found to be significantly down-regulated at 2, 5, 10, and 14 days post irradiation with S-PRT. However, mice irradiated with F-PRT showed a significant restoration of the AQP5 expression post-RT. Oral mucositis started to appear on day 14, which showed more severity on day 28 post-irradiation with S-PRT. Histopathological analysis showed the presence of lingual gland atrophy only in the tongue of mice treated with S-PRT 28 and 60 days post-irradiation. F-PRT ameliorates radiation-induced jawbone loss compared to S-PRT as observed in mice irradiated either with a single dose or in a fractionated regime. The surviving fraction of F-PRT-treated mice with orthotopic tongue tumors was significantly ameliorated compared to S-PRT-treated mice. Conclusion- This study demonstrates that F-PRT minimizes radiation-induced normal tissue toxicity after irradiation in mice’s head and neck region. Moreover, the ability of F-PRT to control orthotopic head and neck tumors further determines the efficacy of this modality for clinical applications. Citation Format: Priyanka Chowdhury, Anastasia Velalopoulou, Ioannis Verginadis, George Morcos, Michele Kim, James Metz, Lei Dong, Alexander Lin, Constantinos Koumenis. Flash proton radiotherapy mitigates radiation-induced salivary gland dysfunction and oral mucositis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6036.

Abstract 4206: Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression

Abstract Purpose: A substantial contribution to the poor prognosis for head and neck squamous cell carcinoma (HNSCC) patients is the progression to distant metastasis. This occurs in 30% of patients over the course of their disease, and radically reduces the 5-year survival rate to under 50%. Investigations in recent years have begun to unveil the intricate interplay of the extracellular matrix (ECM) in both tumor-intrinsic signaling and immunoregulation. However, studies conducted in the context of metastatic have been limited. Here, we employ spatial transcriptomics on HNSCC patient biopsies to identify ECM components that may contribute to cancer progression and immune suppression. Experimental design: Comprehensive transcriptome profiling of HNSCC patient biopsies was conducted using the GeoMx Digital Spatial Profiler (DSP). This approach allowed us to characterize regions of the tumor microenvironment (TME) by immunofluorescent staining for subsequent bioinformatic analysis. To investigate the biological consequences of associations observed in our dataset, we performed in vivo murine studies. We have produced a series of SCC C57BL/6 murine cell lines derived from Smad4-/-KrasG12D Keratin15+ stem cells, which faithfully mimic the clinical progression of HNSCC tumors, including metastasis. Using these murine models, we applied relevant pharmacologic inhibitors and genetic knockdown of targets identified with our GeoMx DSP analysis. In vitro immune-based and functional assays were conducted to elucidate the underlying mechanisms contributing to SCC progression. Results: GeoMx DSP analysis revealed that laminins were enriched in stage IV HNSCC patient stroma, relative to lower stage patients. This coincided with TGFβ enrichment and inversely associated with signatures of anti-tumor immune infiltration. Analysis of The Cancer Genome Atlas data revealed similar inverse correlations in SCCs, distinct from breast cancer and lung adenocarcinoma. Our metastatic SCC murine lines likewise exhibited elevated levels of these laminin proteins, accompanied by high levels of laminin-binding integrins and TGFβ-Smad signaling. Independent of impacting cell proliferation, shRNA knockdown of laminin decreased tumor volume in an immune-competent setting. Targeting laminin-integrin signaling with the small molecule inhibitors Galunisertnib and Buparlisib also reduced the metastatic capacity of these cells in C57BL/6 mice. Conclusions: Spatial transcriptomic profiling of the HNSCC TME uncovers intricate associations and promising targets not readily discernible in bulk RNAseq analyses. Validation through genetic and pharmacological approaches in immune-competent models underscores their potential as strategies to reduce HNSCC burden. Citation Format: John D. Aleman, Khoa A. Nguyen, Erica Wong, Yao Ke, Hanne T. Lind, Clifford G. Tepper, Andrew C. Birkeland, Christian D. Young, Sana D. Karam, Xiao-Jing Wang. Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4206.

Abstract 5188: Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer globally with a poor 5-year survival rate of ~50%. With the emerging successes with immune checkpoint blockade (ICB) in the metastatic and recurrent setting, there is a need for predictive biomarkers to identify patients likely to achieve clinical benefit. Whilst PD-L1 expression on tumour cells and PD-1 on immune cells have been investigated, these remain inconclusive biomarkers. To better understand and map the PD-L1/PD-1 receptor-ligand interactions, we evaluated a novel in situ proximity ligation assay (is PLA) across whole tissue sections of pre-treatment HNSCC patients to received ICB therapy. Pre-treatment FFPE whole tissue samples were collected from n=25 advanced stage HNSCC patients across two major Queensland Hospitals (Royal Brisbane and Women’s Hospital and the Princess Alexandra Hospital). Tissues were stained with multiplex immunofluorescence and the Navinci Diagnostics fluorescent is PLA assay for PD-1/PD-L1. Tissues were incubated with the PD1/PD-L1 antibodies and subsequent incubation with oligonucleotide-tagged secondary probes enabled PLA, which highlights PD1 and PD-L1 interactions. Following the application of secondary probes, the sections underwent a ligation step, then a post-blocking, and an amplification process to enhance the detection of protein interactions. Next, the sections were treated with a detection solution to visualize the amplification product. The PLA assay findings were measured against clinical endpoints (PFS/OS criteria). In n=5/25 HNSCC tissues, positive PD-1/PD-L1 interactions were measured across the tumour microenvironment (TME), specifically in the immune rivers on the immediate tumour periphery. These cells showed highly specific binding of the PLA product. These areas positive by the PLA assay were in stark contrast to the tumour bulk/nests which were absent in PD-1/PD-L1 interactions. Notably, the PLA PD-1/PD-L1 do not correlate with tumour PD-L1 expression or tumour proportion scores (TPS). Rather, our study highlighted the difference in spatially mapping PD-1/PD-L1 interactions which may better recapitulate the tumour-immune recognition and activity in the TME. Taken together, these data provide a novel approach for determining clinically relevant receptor/ligand interactions in the HNSCC TME. Citation Format: Habib Sadeghirad, Vahid Yaghoubi Naei, James Monkman, Subham Basu, Agata Wicher, Rahul Ladwa, Brett GM Hughes, Arutha Kulasinghe. Defining PD-1/PD-L1 receptor-ligand interactions in the head and neck cancer tumor microenvironment identifies unique ‘immune cell rivers’ of cellular interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5188.

Prognostic role of p16 overexpression in sinonasal squamous cell carcinoma: A retrospective analysis of Alberta patients

AbstractObjectiveSinonasal squamous cell carcinoma (SNSCC) is rare in the general population. No clear and consistent etiologic correlation between human papillomavirus (HPV) and SNSCC has yet been delineated in the literature. p16 is a tumor suppressor protein used as a surrogate marker for HPV. This study aims to evaluate the relationship between p16 overexpression in SNSCC and its role in prognosis and survival.MethodsA population‐based retrospective analysis was performed using prospectively collected data from the Northern Alberta Head and Neck Tumour Board, the Alberta Cancer Registry, and the Alberta Cancer Research Biobank. p16 overexpression was analyzed from pathologic samples of patients meeting study criteria, and participants were dichotomized by status. Subsequently, nonparametric analysis of demographics, initial staging, and initial treatment were performed, and a Kapan–Meier curve was developed to assess differences in survival.ResultsSixteen patients were included in the analysis. p16 overexpression was seen in 68.8% of patients. p16 positive and negative groups were comparable for age, gender, smoking status, stage, and treatment. A statistically significant 5‐year survival advantage was observed in patients with p16 positive SNSCC (P = 0.013).ConclusionsThis is the first Canadian study to demonstrate a high prevalence of p16 positivity in SNSCC and its presence denoting a statistically significant survival advantage. Results demonstrate a previously unconfirmed role of oncogenic HPV in SNSCC.

Abstract 224: Combining functional precision medicine and radiotherapy with bio-printed organoids in head and neck squamous cell carcinoma

Abstract The study aims to establish a high throughput ex vivo organoid-based drug screening platform for head and neck squamous cell carcinoma (HNSCC) to investigate interactions between radiotherapy and different chemotherapies or targeted therapy agents and ultimately provide functional precision medicine therapeutic suggestions. HNSCC is the sixth most common cancer worldwide. Both HPV+ and HPV- cancers can be treated with surgery, radiotherapy, chemotherapy, or a combination of these. Cetuximab is currently the only FDA-approved targeted agent for HNSCC. Few clinical trials to date have shown an improved clinical outcome when combining targeted agents with radiotherapy, thus there is a need to identify new therapeutic combinations to improve outcomes. In order to characterize HNSCC tumor behavior and sensitivity to therapy, we have leveraged our high throughput 3D tumor organoid screening platform. Our approach entails seeding tumor cells in extracellular matrix around the perimeter of tissue culture wells in ring-like structures, which we have successfully deployed in other cancer types (Phan et al, 2019; Al Shihabi et al, 2022; Nguyen et al, 2022; Al Shihabi et al 2023). To optimize a chemoradiation platform, we used the HN30, HN31, and SCC154 cell lines representing HPV- primary, HPV- metastatic, and HPV+ head and neck tumors. The cell-line-derived 3D models are exposed to both radiotherapy and various chemotherapy or targeted therapy drugs. We measured cell responses by ATP-release assays and by applying our machine learning-based brightfield image analysis pipeline (Al Shihabi et al, 2022). Our results showed that the HPV+ SCC154 was the most sensitive line to radiotherapy while HPV- metastatic HN31 was the most resistant. HN31 was resistant to most drug treatments except for gemcitabine, docetaxel, and mitomycin c, while HN30 and SCC154 were sensitive to a broader set of agents. We have investigated combinatorial efficacy of radiation and chemotherapy as well as targeted agents and extended this platform to HNSCC patient-derived tumor organoids. Our proof-of-concept study highlights how our organoid-based drug screening platform can be used to test chemoradiation approaches that are more effective and personalized. Citation Format: Luda Lin, Krishna K. Bommakanti, Andreanne V. Sannajust, Yazeed Alhiyari, Brandon Mo, Lauran Evans, Peyton Tebon, Maie St. John, Alice Soragni. Combining functional precision medicine and radiotherapy with bio-printed organoids in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 224.

Abstract 6658: TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy

Abstract Background: Oncolytic viruses, such as the chimeric vesicular stomatitis virus variant VSV-GP have dual modes of action, direct tumor-cell killing, and activation of tumor-specific immunity. While tumor cells deficient in type I interferon (IFN) response are permissive to VSV-GP replication and killing, IFN competent tumor cells are protected from VSV-GP-mediated oncolysis. Our previous data demonstrated that VSV-GP therapy turns immunologically ‘cold’ tumors ‘hot’ by increasing infiltration of T cells into the tumor. The aim of this study was to use the VSV-GP platform to locally express a T cell engager (TcE) molecule to redirect these T cells to destroy non-permissive IFN competent tumor cells expressing the target of the TcE on their cell surface. Methods: We selected EpCAM as the target antigen and used a bispecific EpCAM-TcE molecule consisting of two variable single chain fragments against EpCAM and CD3, respectively. VSV-GP encoding EpCAM-TcE (VSV-GP-EpCAM-TcE) or an invariant control TcE (VSV-GP-TNP-TcE) were generated as previously described. T cells were co-cultured with human target cells expressing EpCAM on their cell surface, either infected with the VSV-GP-EpCAM-TcE virus or with supernatant from VSV-GP-EpCAM-TcE infected cells. T cell activation, proliferation, degranulation and cytokine secretion were analyzed by flow cytometry and multiplex cytokine assays as a measure of TcE mediated cross-linking of T cells to their target on cancer cells. Patient-derived head and neck squamous cell carcinoma (HNSCC) tumors were sectioned into approximately 200 µM slices and infected with the virus. T cell activation was measured by cytokine secretion. The therapeutic potential of VSV-GP-EpCAM-TcE was tested in vivo in cell-line derived xenograft (CDX) models implanted in immunodeficient mice humanized with donor-derived PBMCs or CD34+ cells. Results: Virus fitness was maintained for VSV-GP encoding the TcE molecule. Using co-culture assays, we demonstrated that EpCAM-TcE molecule is successfully expressed by VSV-GP and can activate both CD4+ and CD8+ T cells resulting in proliferation, degranulation, and cytokine secretion. Furthermore, we validated the concept using patient-derived HNSCC tumor slices which are representative of complex human tumor ecosystems. We could confirm that cancer cells infected by VSV-GP-EpCAM-TcE virus express functional TcE molecule and activates T cells to secrete IFNγ and TNFα. Finally, we tested the concept in two different CDX models where VSV-GP-EpCAM-TcE showed improved tumor growth inhibition due to a combined effect of oncolysis and T cell activation by EpCAM-CD3 TcE. Summary: The oncolytic virus VSV-GP can be successfully armed with a TcE to redirect tumor-infiltrating T cells to destroy tumor cells which are not permissive to viral replication. This resulted in an enhanced therapeutic efficacy compared to just the oncolytic effect. Citation Format: Aparna Ponnurangam, Theresa Schwaiger, Sophie Walter, Simone Bartl, Melissa Mayr, Monika Petersson, Tanja Schoenberger, Fabian Heinemann, Peter Gross, Wolfgang Rist, Srinath Kasturirangan, Charlotte Lempp, Jutta Petschenka, Krishna Das, Nolden Tobias. TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6658.

Abstract 2414: Feasibility and clinical utility of blood based NGS in head and neck carcinomas: A single center experience from precision medicine program

Abstract Background: While blood-based next-generation sequencing is increasingly integral to clinical practice across various malignancies, the routine assessment of circulating tumor DNA (ctDNA) in head and neck cancer remains underexplored, with limited supportive data. Methods: This study focused on head and neck cancer patients enrolled in the Gustave Roussy precision medicine program (STING, NCT04932525), undergoing liquid biopsy (LB) with the FoundationOne Liquid CDX panel. The Molecular Tumor Board discussed cases, calculating Tumor Fraction (TF) based on aneuploidy, categorizing it as high or low with a 10% cutoff. Results: The analysis encompassed 239 LBs from 214 patients with relapsed or metastatic head and neck cancers. Of these, 59 LBs were from 57 patients showing stability or positive response (SD/R), while 180 were from 167 patients at diagnosis or disease progression (BL/PD). Notably, 156 (73%) were male, with a median age of 61 years. Within the cohort, 65% of LBs were from patients with squamous cell carcinomas (SCC), 15% adenoid cystic carcinomas (ACC), and 20% other histologies mainly adenocarcinomas. The anatomical distribution included 59 LBs from salivary gland (SG) cancers, 49 oropharynx (OP), 48 oral cavity (OC), and others including larynx, hypopharynx, sinus and nasopharynx. Among LBs performed at BL/PD, 72% were informative, while only 36% were so at SD/R. The proportion of informative biopsies varied across anatomical sites. For SCC, TP53 mutations were most prevalent (71%), followed by TERT and PIK3CA variants, and FGF19 and FGF3 amplifications. ACCs showed NOTCH1 mutations (6/19, 32%) as the most common, followed by TP53 and various other variants. In the entire cohort, clonal hematopoiesis-related variants were identified, with 40% showing at least one of DNMT3A, CHEK2, or ASXL1. Patients with low TF at BL/PD exhibited significantly improved overall survival (21.6 vs. 7.6 months, HR 0.35, 95% CI 0.23 - 0.53, p &amp;lt; 0.0001), consistent across subgroups except for OP.Conclusion: This study demonstrates the feasibility of liquid biopsy in HNSCC and rare head and neck tumors, providing valuable therapeutic and prognostic insights. The findings underscore the potential of liquid biopsy as a valuable tool in clinical decision-making for head and neck cancer patients. Citation Format: Filippo Gustavo Dall'Olio, Damien Vasseur, Lea Loriguet, Francois Regis Ferrand, Antoine Moya-Plana, Yungan Tao, Mariana Iacob, Karim Benihoud, Odile CASIRAGHI, Catherine Brenner, Pierre Busson, Philippe Gorphe, Santiago Ponce, Pierre Blanchard, Etienne Rouleau, Yohann Loriot, Ludovic Lacroix, Antoine Italiano, Ingrid Breuskin, Caroline Even. Feasibility and clinical utility of blood based NGS in head and neck carcinomas: A single center experience from precision medicine program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2414.

Abstract 27: A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity

Abstract Background: Genetically engineered T cells with T cell receptor (TCR-T) cell therapy have shown great potential for cancer treatment by targeting tumor-associated antigens. Despite its success in treating various tumor types, such as synovial sarcoma, head and neck tumors, and cervical cancer, its application in liver cancer is limited. Alpha-fetoprotein (AFP), a highly expressed tumor-associated antigen in liver cancer, is an ideal target for TCR-T cell therapy. Although early human trials have confirmed the safety of AFP-targeting TCR-T products, their efficacy is still modest, highlighting the need for further research and development. Methods: We developed a robust strategy combining ex vivo stimulation and single-cell paired TCR sequencing to identify AFP-recognizing TCRs in liver cancer patients. To optimize TCR expression and minimize mispairing, we employed codon optimization and replaced the constant regions with murine counterparts. Furthermore, structural analysis guided the affinity optimization of the CDR3 region to enhance the antitumor function of the selected TCR. The anti-tumor efficacy of TCR-T cells was rigorously evaluated through in vitro cytotoxicity assays using the Real-Time Cell Analyzer (RTCA) system and in vivo antitumor assessment in immunodeficient mouse xenograft models. Antigen specificity, HLA restriction, and other potential toxicity were carefully evaluated in both in vitro and in vivo models. Results: We successfully cloned a highly effective TCR that recognizes the AFP158-166 epitope presented by HLA-A*0203. After sequence optimization, we further changed a single amino acid on the TCR vβ chain based on AI prediction. The expression and functionality of this TCR was significantly improved. When introduced into human T cells, the single amino acid mutated TCR demonstrated robust and specific anti-tumor activity in various models, including cultured tumor cells, patient-derived organoids, and mouse xenografts models. Notably, this TCR also redirected CD4 T cells to recognize tumor cells, induced the secretion of multiple cytokines, and maintained long-lasting anti-tumor effects. Our rigorous screening assays confirmed that the TCR showed no cross-reactivity with the human proteome or major HLA subtypes. Additionally, extensive preclinical safety assessments in tumor-bearing animals treated with ST08A01-AM14 TCR-T cells showed no significant safety concerns. Conclusion: In this study, we have developed an HLA-A*0203 restrict AFP158-166 specific TCR-T therapy, which exhibits exceptional anti-tumor effects while ensuring a favorable safety profile. The promising results support the further investigation against AFP-positive solid tumors in clinic. Citation Format: Chen-Song Huang, shudan Ou, Jun Li, Junjun Chu, Yanyan Han. A single amino acid mutated AFP specific T cell receptor uniting synergistic functionalities of CD4 and CD8 T Cells with no cross-activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 27.

Abstract 1154: Spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma revealed immune cell type infiltration into the TME as a predictor of immunotherapy response

Abstract Background: Immunotherapy has shown promising results in advanced head and neck cancer patients, however, only a subset of patients are responsive to immune checkpoint blockade therapy. The molecular and cellular characterization of the tumor microenvironment (TME) may reveal clues as to why some head and neck tumors are responsive or resistant to the therapy. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were collected from 34 patients at two major Queensland Hospitals with advanced head and neck squamous cell carcinoma (HNSCC), prior to immunotherapy. To investigate the cellular and molecular characteristics of the HNSCC TME, we used the Nanostring GeoMx Digital Spatial Profiler (DSP) spanning modules in an immune-oncology panel, which included immune cell typing and profiling, cell death, PI3K/AKT signaling, drug target, immune activation, and pan tumor protein biomarkers. A deeper structural analysis was performed using the Nanostring whole transcriptome atlas (WTA) panel of tertiary lymphoid structures (TLSs) and germinal centers (GCs). Results: Out of 34 patients, 3 had a complete response (CR), 7 had a partial response (PR), 5 had stable disease (SD), and 19 had progressive disease (PD), based on the Response Evaluation Criteria in Solid Tumors (RECIST). When we compared patient responders (patients with CR, PR, and SD) to patient non-responders (patients with PD), we found that the former had a higher infiltration of immune cell type markers representatives of CD8+ T cells, dendritic cells, and macrophages, into their tumor microenvironment than the latter. Then, to investigate differentially expressed genes between patients with complete response and patients with partial response to immunotherapy, we performed DE analyses and we found that patients with PR had higher Ki-67 expression than those with CR. Spatial transcriptomic mapping of TLS’s (peripheral, intra-tumoral) and normal germinal centers revealed interferon pathway enrichment in the TLS’s. In a subset, these appear to be linked with HPV viral infection. Conclusion: In this study, we found that immune cell type infiltration into the tumor microenvironment of head and neck cancers could be predictive of response to immunotherapy. Furthermore, it was revealed that patients with a high expression of Ki-67 might be less likely to respond completely to immunotherapy. Spatially resolved whole transcriptome analysis of HNSCC tissues revealed the presence of tertiary lymphoid structures with a higher enrichment of interferon pathway genes. Citation Format: Habib Sadeghirad, Chin Wee Tan, Ning Liu, James Monkman, Caroline Cooper, Ken O’Byrne, Melissa Davis, Brett Hughes, Arutha Kulasinghe. Spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma revealed immune cell type infiltration into the TME as a predictor of immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1154.

Abstract 1377: The role of TIM3 and PD1 blockades in head and neck cancer

Abstract Background: Our group has recently developed and validated a novel prognostic score for human papillomavirus (HPV+) oropharyngeal cancer called UWO3, which stratifies patients into low risk (immune rich), intermediate risk (immune mixed), and high risk (immune desert) groups. The high-risk immune desert group had a paucity of immune cells, however, that population of lymphocytes expressed high levels of the checkpoint molecule TIM3. To follow up on this, we assessed the therapeutic effect of anti-TIM3 mAb alone and in combination with anti-PD-1 in a syngenic murine HPV+ head and neck cancer model using the mERR. T-cell immunoglobulin mucin 3 (TIM3) and Programmed death 1 (PD-1) are negative immune checkpoint receptors that are expressed on tumor-infiltrating lymphocytes (TIL), play a crucial role in tumor-induced immune suppression. Given the critical role that the tumour microenvironment (TME) has on the prognosis of head and neck squamous cell carcinoma (HNSCC), immune checkpoint inhibitor immunotherapy approaches, specifically anti-PD1 and/or anti-TIM3 antibody therapy, may be very effective treatment strategies and may offer improved survival outcomes. Methods:Immunocompetent C57BL/6 mice were injected in one flank with the syngeneic mouse HPV+ cell line, mEERL (106 cells), for tumour generation. Mice were then treated with either IgG2a isotype control, anti-PD1 (200μg), anti-TIM3 (200 μg) antibodies or a combination of anti-PD-1 and anti-TIM3 were administered via intraperitoneal injection 3 times per week following tumour establishement for 6 weeks (n = 7 mice per arm, total of 28 mice). Tumour volumes were measured every 2 days. Three representative tumors from each treatment group were collected for bulk RNA sequencing, scRNA-seq, and flow cytometry to characterise immune cell populations and transcriptional changes and monitor their impact on the TME of HNSCC. Results:Anti-PD1 and anti-TIM3 both restricted tumour growth by size and weight relative to the IgG2a control (p&amp;lt;0.01, linear mixed model [lmm]). The combination of anti-PD1 and anti-TIM3 was more effective than either monotherapy alone (p&amp;lt;0.05, lmm). RNA sequencing analysis revealed differential abundance in CCL6, CCL9, CFD, and MAP3K5 transcripts relative to control for anti-PD1, anti-TIM3, and combination checkpoint inhibition. Conclusion:Targeting both PD-1 and TIM3 enhanced the anti-tumor immune response in HPV+ head and neck tumours, providing further support for dual targeting of these molecules for more effective cancer immunotherapy. Our results suggest that immune checkpoint inhibitor therapy (PD1 and TIM3 blockades) could be a promising treatment strategy for HPV+ HNSCC, and the expression of these immune checkpoint molecules could serve as a predictive biomarker of patient outcome in HPV+ HNSCC. Citation Format: Mushfiq Shaikh, Amir Karimi, Peter Zeng, Harrison Pan, John Barrett, Anthony Nichols. The role of TIM3 and PD1 blockades in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1377.

Abstract 3061: A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness

Abstract Both GLS1 and c-Myc are upregulated in head and neck squamous cell carcinoma (HNSCC) primary tumors and further increased in metastatic tumors. However, the regulation between these two molecules remains largely unknown. GLS1 is a critical enzyme that regulates glutamate, which plays an important role in cancer metabolism that supports cancer growth and survival. Our bioinformatic analysis of the TCGA HNSCC cohort revealed a strong correlation between c-Myc and GLS1 expression. We describe the importance of c-Myc in regulating GLS1 and vice versa. c-Myc protein directly binds to the promoter of the GLS1 gene and upregulates its expression at the transcriptional level. Interestingly, blocking GLS1 signaling in HNSCC cells by lentiviral shRNA knockdown or CB-839 treatment downregulates USP1, one of the best characterized human DUBs, which in turn reduces c-Myc protein stability via the ubiquitin-proteasome pathway. The GLS1-c-Myc axis thus represents a novel positive feedback loop that is critical for driving the aggressiveness of HNSCC. As the treatment of HNSCC remains a major challenge, these novel findings provide the molecular basis for combining GLS1-specific inhibitors with c-Myc-targeted therapy for the treatment of HNSCC patients. Citation Format: Jianqiang Yang, Fanghui Chen, Fan Yang, Yong Teng. A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3061.

Abstract 5494: Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading form of cancer worldwide and one of the most difficult and challenging malignancies, with a high level of heterogeneity and a wide range of treatment responses. Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer and accounts for over 90% of cancers that develop within the mucosal epithelium of the oral cavity. Additional carcinogenic risk factors, including tobacco, alcohol and HPV infection are also associated with the pathogenesis of oral carcinogenesis. Unfortunately, today the failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy underscore the need to identify better biomarkers of this disease. Advances, especially in spatial technology have created unprecedented opportunities to identify cell types and biomarkers of disease processes, revealing important relationships within the tumor microenvironment (TME) that allow a detailed characterization of specific cell phenotypes defined by co- or lack of expression of multiple markers that may help in predicting clinical responses and mechanisms of resistance to therapy. Recent findings indicate that abnormal expression of claudins (CLDNs) is closely related to the occurrence, progression and prognosis of many solid tumors. Specifically, CLDN6 is expressed in numerous tumors but rarely found in adult normal tissues making it a potential therapeutic target in head and neck tumor types. To investigate this further, expression of CLDN6 and the its relationship with changes within the TME of cohort OSCC tumors was determined using a proprietary comprehensive spatial workflow and gene expression analysis. Methods: A retrospective analysis was performed on biopsied Formalin-Fixed Paraffin-Embedded (FFPE) samples from OSCC patients with and without a history of smoking and treated +/- chemotherapy. Patient samples representing matched normal tissue to serve as controls were compared to samples derived from OSCC tumors. In order to perform a comprehensive expression profiling of the TME from OSCC tumors we used the spatial MultiOmyx™ immunofluorescence (IF) multiplexing assay with transcriptomic information from bulk mRNA gene expression acquired using the NanoString nCounter® PanCancer IO 360™ panel that includes the Tumor Inflammation Signature (TIS), as well as targets that characterize immune hot, desert, and cold regions in the stroma and tumor epithelium. Results: We characterized the expression of CLDN6 across the different cohorts of OSCC samples and further investigated the relationship within the tumor immune microenvironment coupled to genes related to the prognosis of OSCC. A better understanding of the TME and gene expression profiling of OSCC tumors and the effect of smoking will allow us to tailor the treatment of patients to continue to improve outcomes. Citation Format: Kirsteen Maclean, Lakshmi Chandramohan, Maryam Rohafza, Harry Nunns, Erinn Parnell, Qingyan Au, Anna Juncker-Jensen. Spatial organization between claudin and the tumor microenvironment in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5494.

Abstract 1734: Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC

Abstract Background: Approximately 75% of Human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors carry genetic and expression alterations in the family of protein methyltransferases and demethylases, underscoring the importance of these enzymes in the pathogenesis of HNSCC. SMYD3 a lysine methyltransferase that is overexpressed in HPV-negative HNSCC. We recently reported that nuclear SMYD3 plays dual role in immune-related gene regulation through H3K4me3 or H4K20me3, leading to gene activation or silencing, respectively in HPV-negative HNSCC. While SMYD3 has been reported to write and bind to H3K4me3 activating target gene expression, its function in gene silencing is understudied. This study aims to determine the mechanism(s) through which SMYD3 functions both as a transcriptional activator and a repressor within the same cell context, specifically in HPV-negative HNSCC. Methods: immunoprecipitation (IP) of SMYD3 was performed from nuclear extracts isolated from HN-6 cells to identify candidate interacting partners of SMYD3. The nuclear immunoprecipitates were sent for MS analysis to identify candidate nuclear interacting partners of SMYD3. CUT&amp;RUN assays for selected candidate interacting proteins and SMYD3 will be performed in control HN-6 cells versus SMYD3 KO cells to assess genomic co-occupancy with SMYD3. Further, CUT&amp;RUN assays for repressive marks H4K20me3, H3K27me3 and H3K9me3 have been performed in HN-6 and SMYD3 KO cells to assess co-occupancy with SMYD3 as well as its effect of SMYD3 on the genome-wide mapping of these marks. Finally, CUT&amp;RUN assays for SMYD3 followed by re-CUT&amp;RUN of repressive histone marks are ongoing to identify co-occupied loci. Results: Preliminary analysis of our nuclear SMYD3 IP results has identified a chromatin modifier with known repressive function. Co-immunoprecipitation experiments to validate this interaction are planned. CUT&amp;RUN assays for repressive histone marks in HN-6 versus SMYD3 KO cells have revealed impact on H4K20me3 and H3K27me3. Conclusions: Our preliminary results suggest that SMYD3 may exert its silencing function by regulating the genome-wide deposition of H4K20me3.Our nuclear SMYD3 IP analysis has identified candidate interacting partners that may assist SMYD3 in promoting the deposition of these repressive marks. CUT&amp;RUN assays for SMYD3 followed by re-CUT&amp;RUN for H4K20me3 and H3K27me3 bound chromatin may provide further insight about whether SMYD3 co-occupies specific genomic regions together with these repressive marks. This project is expected to provide mechanistic insights into the bifaceted functions of SMYD3 within the same cell context using HPV-negative HNSCC cell lines as an experimental model system.. Citation Format: Jawad Akhtar, Sohyoung Kim, Vassiliki Saloura. Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1734.

Spatial relationships in the urothelial and head and neck tumor microenvironment predict response to combination immune checkpoint inhibitors

Immune checkpoint inhibitors (ICI) can achieve remarkable responses in urothelial cancer (UC), which may depend on tumor microenvironment (TME) characteristics. However, the relationship between the TME, usually characterized by immune cell density, and response to ICI is unclear. Here, we quantify the TME immune cell densities and spatial relationships (SRs) of 24 baseline UC samples, obtained before pre-operative combination ICI treatment, using multiplex immunofluorescence. We describe SRs by approximating the first nearest-neighbor distance distribution with a Weibull distribution and evaluate the association between TME metrics and ipilimumab+nivolumab response. Immune cell density does not discriminate between response groups. However, the Weibull SR metrics of CD8+ T cells or macrophages to their closest cancer cell positively associate with response. CD8+ T cells close to B cells are characteristic of non-response. We validate our SR response associations in a combination ICI cohort of head and neck tumors. Our data confirm that SRs, in contrast to density metrics, are strong biomarkers of response to pre-operative combination ICIs.

Enhancing Diagnostic Precision: Evaluation of Preprocessing Filters in Simple Diffusion Kurtosis Imaging for Head and Neck Tumors.

Background: Our initial clinical study using simple diffusion kurtosis imaging (SDI), which simultaneously produces a diffusion kurtosis image (DKI) and an apparent diffusion coefficient map, confirmed the usefulness of SDI for tumor diagnosis. However, the obtained DKI had noticeable variability in the mean kurtosis (MK) values, which is inherent to SDI. We aimed to improve this variability in SDI by preprocessing with three different filters (Gaussian [G], median [M], and nonlocal mean) of the diffusion-weighted images used for SDI. Methods: The usefulness of filter parameters for diagnosis was examined in basic and clinical studies involving 13 patients with head and neck tumors. Results: The filter parameters, which did not change the median MK value, but reduced the variability and significantly homogenized the MK values in tumor and normal tissues in both basic and clinical studies, were identified. In the receiver operating characteristic curve analysis for distinguishing tumors from normal tissues using MK values, the area under curve values significantly improved from 0.627 without filters to 0.641 with G (σ = 0.5) and 0.638 with M (radius = 0.5). Conclusions: Thus, image pretreatment with G and M for SDI was shown to be useful for improving tumor diagnosis in clinical practice.