Understanding the dynamics of the tumor-immune ecosystem is crucial for advancing neoadjuvant strategies in cancer treatment. This study investigated alterations in the tumor-immune microenvironment related to the response to preoperative combination therapy with paclitaxel, carboplatin, and cetuximab in patients with advanced head and neck squamous cell carcinoma. Thirty patients underwent combination therapy. Biopsy or surgical specimens were obtained before and after treatment. Single-cell-based, 14-marker multiplex immunohistochemistry and image cytometry were employed to assess changes in immune cell densities and profiles. Three distinct immune profiles were identified: hypo-, lymphoid-, and myeloid-inflamed. Significant decreases in tumor volume and increases in CD45+ cells and programmed cell death ligand 1 (PD-L1) scores were observed in the hypo- and lymphoid-inflamed groups, whereas the myeloid-inflamed group showed minimal changes. After treatment, increased calreticulin expression in tumor cells, together with increased lymphoid cell lineages, was observed in non-recurrent cases. The myeloid-inflamed group exhibited higher expression of hypoxia inducible factor 1α and zinc finger E-box-binding homeobox 2, suggesting the presence of a hypoxic and metastasis-promoting environment. Spatial analysis revealed that responders had a high infiltration of T cells within tumor cell nests, whereas non-responders had fewer T cells, with β-catenin expression in cancer cells. Upregulated lymphocyte activation gene 3 in the myeloid-inflammation group, and PD-L1 dynamics suggest potential targets for further therapy. These findings highlight the need for targeted neoadjuvant strategies based on immune profiling.

T cell phenotypes and kinetics are emerging as crucial factors associated with immunotherapeutic responses in a wide range of solid cancer types. However, challenges remain in understanding the spatial and temporal profiles of T cells with differential phenotypes due to difficulties in single-cell analysis with preserved tissue structures. Here, we provide an optimized 12-marker multiplex immunohistochemical (IHC) panel and single-cell-based quantitative assessment to identify the spatial distributions of T cell phenotypes in formalin-fixed paraffin-embedded sections. This panel revealed differential T cell populations with spatial localizations in human tonsil tissue, where the percentages of CD8+ T cell-expressing programmed death receptor-1 (PD-1), T cell immunoglobulin and mucin domain 3 (TIM3), and other T cell phenotypic markers vary by tonsillar tissue components such as follicles, parenchyma, and epithelium. A specimen from salivary gland adenocarcinoma during hyper-progression, followed by anti-PD-1 treatment, exhibited the exclusion of CD8+ T cells from the intratumoral regions. Although the vast majority of peritumoral CD8+ T cells exhibited proliferative effector T cell phenotypes with PD-1−TIM3−Ki67+CD45RA+, intratumoral CD8+ T cells showed exhausted phenotypes with PD-1+TIM3− and increased Eomes expression, which might be related to poor therapeutic response in this case. To verify these findings in the context of temporal changes, we analyzed six longitudinal samples from a single patient with maxillary sinus cancer, observing increased T cell exhaustion along with metastasis and progression. Together, highly multiplexed IHC can be applied to analyze the spatiotemporal phenotypes of T cells, potentially contributing to the understanding of the mechanisms of resistance to immunotherapy.

BackgroundIn view of improving biomarkers predicting the efficacy of immunotherapy for head and neck squamous cell carcinoma (R/M HNSCC), this multicenter retrospective study aimed to identify clinical, tumor microenvironmental, and genomic factors that are related to therapeutic response to the anti- Programmed cell death protein 1 (PD-1) antibody, nivolumab, in patients with R/M HNSCC. MethodsThe study compared 53 responders and 47 non-responders, analyzing formalin-fixed paraffin-embedded samples using 14-marker multiplex immunohistochemistry and targeted gene sequencing.ResultsOf 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-1 ligand (PD-L1) expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. The frequency of natural killer cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Age-stratified analysis showed nivolumab response was linked to high CPS and lymphoid-inflamed profiles in patients aged ≥ 65. In contrast, lower NLR in peripheral blood counts was associated with response in patients aged < 65. Notably, TP53 mutation-positive group had lower CPS and T cell densities, suggesting an immune-excluded microenvironment. Patients with altered tumor suppressor gene pathways, including TP53, CDKN2A, and SMAD4 mutations, had lower CPS, higher smoking index, and were associated with poor responses. ConclusionNivolumab treatment efficacy in HNSCC is influenced by a combination of clinical factors, age, prior treatment, immune environmental characteristics, and gene mutation profiles.

Intratumoral immune profiles are related to prognosis and therapeutic efficacy, and could result in personalized treatments based on biomarkers. To develop a multiplex, quantitative, and rapid tissue evaluation method based on the clinically established standard immunohistochemistry (IHC), a 6marker rapid multiplex IHC was developed based on our previously reported 14-marker multiplex IHC by reducing the number of labels and accelerating the staining procedure. First, fewer labels were required to identify the same immunological features linked to prognosis in 14-marker multiplex IHC analyses. The six selected markers showed a significant correlation with the 14 markers in the immune classification. Next, a rapid staining protocol was developed by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 h and 49 min, as opposed to the several days required for conventional multiplex IHC. Validation of benign tonsil and head and neck cancer tissues revealed a significant correlation between rapid and conventional 6-makrer multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cell ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable quantitative assessment of the tumor-immune microenvironment on a clinically feasible time scale, which promotes the development of tissue biomarker-guided therapeutic strategies.

Understanding longitudinal changes of tumor-immune microenvironment during chemo/targeted therapies contributes to the development of optimized combinations of immunotherapy with chemotherapy and targeted therapy for patients with head and neck squamous cell carcinoma (HNSCC). We previously reported a chromogenic sequential immunohistochemical (IHC) platform enabling quantitative and spatial assessment of 29+ biomarkers in a single tissue section (Tsujikawa T et al. Cell Reports 2017, Banik G et al. Methods Enzymology 2020). Using this platform, densities, phenotypes, and distributions of tumor and immune cells were evaluated, comparing baseline and post-treatment specimens from the same individual treated by paclitaxel, carboplatin, and cetuximab (PCE) for advanced HNSCC (N = 30). Immune cell density analyses based on CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, macrophages, dendritic cells, mast cells, granulocytes revealed the presence of differential immune cell compositions, where immune profiles were divided into hypo-, lymphoid-, and myeloid-inflamed groups according to the same criteria as in our previous report (Tsujikawa et al. Cell Reports 2017). Lymphoid and hypo-inflamed groups exhibited significant tumor volume reduction, increased CD45+ immune cell densities and elevated combined positive scores of PD-L1 at the post-treatment status, suggesting the potential involvement of immunogenic mechanisms related to therapeutic response. On the other hand, the myeloid group exhibited no significant tumor volume reduction, together with higher expression of HIF1α and ZEB2 on tumor cells which are potentially associated with hypoxia and epithelial-mesenchymal transition. In conclusion, longitudinal tissue-based monitoring revealed the presence of differential tumor-immune complexity profiles related to therapeutic efficacy and resistance. Hypo-inflamed profiles might require upfront chemo/targeted therapy before immunotherapy, and myeloid-inflamed profiles might require myeloid cell-targeted therapies, mandating the establishment of rapid clinical assessment of tumor-immune microenvironment. Citation Format: Alisa Kimura, Takahiro Tsujikawa, Junichi Mitsuda, Aya Miyagawa-Hayashino, Hiroki Morimoto, Sumiyo Saburi, Kanako Yoshimura, Gaku Ohmura, Sigeyuki Mukudai, Hikaru Nagao, Yoichiro Sugiyama, Hiroshi Ogi, Saya Shibata, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Tumor-immune microenvironmental profiling during chemo- and targeted therapy for head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5172.

Neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is an emerging biomarker candidate of immunotherapy in a wide range of cancers. However, little is known about the potential relationships between the tumor immune microenvironment and systemic inflammatory markers including NLR. Here we have explored systemic and tumor-immune microenvironmental characteristics related to treatment outcomes of immune checkpoint inhibition, based on 29 consecutive patients with recurrent/metastatic head and neck squamous cell carcinoma who received pembrolizumab between 2020 and 2021. NLR greater than 4.5 at pretreatment status significantly correlated with short overall survival (OS). Although NLR did not show a significant association with tumor volumes, high NLR exhibited significant correlations with malnutrition status characterized by CONUT (controlling nutritional status), and GNRI (geriatric nutrition risk index). Among the patients whose NLR was greater than 4.5 at pretreatment status, those whose NLR decreased to less than 4.5 at day 21 had a better OS than those whose NLR did not decrease, indicating that longitudinal changes in NLR correlate with prognosis. To investigate association with tumor-immune microenvironment, 14-marker multiplex immunohistochemistry was performed to quantitatively evaluate intratumoral CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, macrophages, dendritic cells, mast cells, and granulocytes. Notably, NLR at pretreatment status significantly correlated with intratumoral immune cell densities, where high NLR correlated with low lymphoid cells and high tumor associated macrophages in tissue. NLR in peripheral blood significantly correlated with myeloid to lymphoid cell ratios in tissue, suggesting the presence of association between circulating and intratumoral immune complexity profiles. This study highlights that the association between intratumoral myeloid predominance and systemic nutritional and inflammatory status might be a possible factor for resistance to immunotherapy. Understanding immune dynamics in tissue and blood during immunotherapy potentially contributes to the establishment of predictive biomarkers and monitoring for immunotherapy. Citation Format: Hiroki Morimoto, Takahiro Tsujikawa, Aya Miyagawa-Hayashino, Alisa Kimura, Sumiyo Saburi, Junichi Mitsuda, Kanako Yoshimura, Gaku Ohmura, Shigeyuki Mukudai, Hikaru Nagao, Yoichiro Sugiyama, Shibata Saya, Hiroshi Ogi, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Neutrophil-to-lymphocyte ratio associates with intratumoral myeloid predominance and clinical outcomes of pembrolizumab in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4453.

Abstract Background: Biomarkers predicting therapeutic response to immunotherapy have been widely explored via monitoring the liquid and tissue-derived components. Increasing treatment options for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) mandates prediction of the therapeutic response of anti-PD-1 antibody alone as well as optimization of the treatment sequence. In view of improving biomarkers predicting the efficacy of immunotherapy for R/M HNSCC, we hypothesized that biomarkers can be personalized depending on clinicopathological backgrounds and treatment sequence. Methods: In this study, we retrospectively included formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood cell counts at treatment, clinicopathological information, and outcome data for patients with R/M HNSCC receiving nivolumab across 22 institutions in Japan (N = 100). FFPE samples were subjected to 14-marker multiplex immunohistochemistry (IHC) and image cytometry analysis (Tsujikawa T et al. Cell Reports, 2017) to quantitatively evaluate CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, CD66b+ granulocytes, mast cells, programmed death ligand 1 (PD-L1) and PD-1 expression in a single slide. Intratumoral and circulating immune cell frequencies were comparatively analyzed between responders (CR, n = 14; PR, n = 39) and non-responders (SD, n = 2; PD, n = 45). Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-L1 expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. Next, focusing on the history of prior therapy, stratified analysis revealed that the frequency of NK cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Furthermore, stratified analysis by patient age revealed that nivolumab response was significantly associated with high CPS and lymphoid-inflamed profiles based on cell densities of nine immune cell lineages in the group aged 65 years or older, but not in the group under 65 years of age. On the contrary, the neutrophil/lymphocyte ratios (NLR) in peripheral blood counts at nivolumab treatment were significantly lower in responders (mean 4.96) than those in non-responders (mean 10.46) in the group under 65 years of age, but not in those over 65 years of age (7.41 versus 8.47). Conclusions: Using peripheral blood data and tumor tissue profiling stratified by patient age and prior treatment might provide better predictive biomarkers in nivolumab response to HNSCC. Further preclinical and clinical studies elucidating immune mechanisms in different patient backgrounds will be warranted. Citation Format: Takahiro Tsujikawa, Kazuchika Ohno, Sumiyo Saburi, Junichi Mitsuda, Kanako Yoshimura, Alisa Kimura, Hiroki Morimoto, Gaku Ohmura, Akihito Arai, Hiroshi Ogi, Saya Shibata, Yosuke Ariizumi, Akihisa Tasaki, Ryosuke Takahashi, Yumiko Tateishi, Hiroaki Kawabe, Sadakatsu Ikeda, Kei-ichi Morita, Tatsuhiko Tsunoda, Takumi Akashi, Morito Kurata, Issei Imoto, Yasushi Shimizu, Akihito Watanabe, Yukinori Asada, Ryuichi Hayashi, Yuki Saito, Hiroyuki Ozawa, Kiyoaki Tsukahara, Nobuhiko Oridate, Arata Horii, Takashi Maruo, Nobuhiro Hanai, Hidenori Inohara, Hiroshi Iwai, Takashi Fujii, Ken-ichi Nibu, Shigemichi Iwae, Tsutomu Ueda, Ryuji Yasumatsu, Hirohito Umeno, Muneyuki Masuda, Kyoko Itoh, Shigeru Hirano, Takahiro Asakage. Tumor immune characterization identifies age-stratified biomarkers for nivolumab in patients with head and neck squamous cell carcinoma: A nationwide collaborative study in Japan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5210.

生命・機能予後の改善をめざし,口腔癌を含む局所進行頭頸部癌に対する導入化学療法・免疫療法を検証する臨床試験が国内外で複数進行している。1切片から14マーカーを可視化・定量化可能な多重免疫染色により,頭頸部癌においてリンパ球系優位,低免疫細胞,骨髄系優位の3種類の免疫特性の存在が示され,これらの免疫特性が免疫療法のみでなく,導入化学療法の効果と関連することが示唆された。免疫特性をふくむ組織バイオマーカーに基づいて適切な症例選択が可能になれば,口腔癌における将来的な導入化学・免疫療法や術式を含む治療方針の最適化が期待される。

症例は26歳男性，前頸部腫脹を自覚し初診。MRIでflow voidを認め動静脈奇形（AVM）が疑われた。病変は甲状腺左葉から頭側に進展，喉頭内に進入し上喉頭動脈と連続していた。手術2日前に血管造影・動脈塞栓術を施行，病変は上，下甲状腺動脈，上喉頭動脈により栄養されていたが，手術時の出血リスクを減少させるため下甲状腺動脈のみを塞栓した。その他の流入血管を結紮後，甲状腺左葉を含むAVM摘出術を施行した。甲状軟骨板の一部を一旦切除し，喉頭内に進展する病変を追跡，摘出した。下喉頭神経は神経刺激装置で手術終了時まで反応を確認し温存した。術直後は一時的な声帯固定を認めたが，手術4か月後に声帯の可動性は回復した。

Metastatic intrathyroid thymic carcinoma (ITTC) is a rare cancer with no effective drugs for controlling. This case report has shown genomic and immune microenvironment profiles in metastatic ITTC and emphasized an immunosuppression via a PD-1/PD-L1 pathway, possibly strengthening the rationale for immune checkpoint blockade as a novel treatment.

Background Functional interactions between immune cells and neoplastic cells in the tumor immune microenvironment have been actively pursued for both biomarker discovery for patient stratification, as well as therapeutic anti-cancer targets to improve clinical outcomes. Although accumulating evidence indicates that intratumoral infiltration of immune cells has prognostic significance, limited information is available on the spatial infiltration patterns of immune cells within intratumoral regions. This study aimed to understand the intratumoral heterogeneity and spatial distribution of immune cell infiltrates associated with cell phenotypes and prognosis in head and neck squamous cell carcinoma (HNSCC). Methods A total of 88 specimens of oropharyngeal squamous cell carcinoma, categorized into discovery (n = 38) and validation cohorts (n = 51), were analyzed for immune contexture by multiplexed immunohistochemistry (IHC) and image cytometry-based quantification. Tissue segmentation was performed according to a mathematical morphological approach using neoplastic cell IHC images to dissect intratumoral regions into tumor cell nests versus intratumoral stroma. Results Tissue segmentation revealed heterogeneity in intratumoral T cells, varying from tumor cell nest-polarized to intratumoral stroma-polarized distributions. Leukocyte composition analysis revealed higher ratios of TH1/TH2 in tumor cell nests with higher percentages of helper T cells, B cells, and CD66b+ granulocytes within intratumoral stroma. A discovery and validation approach revealed a high density of programmed death receptor-1 (PD-1)+ helper T cells in tumor cell nests as a negative prognostic factor for short overall survival. CD163+ tumor-associated macrophages (TAM) provided the strongest correlation with PD-1+ helper T cells, and cases with a high density of PD-1+ helper T cells and CD163+ TAM had a significantly shorter overall survival than other cases. Conclusion This study reveals the significance of analyzing intratumoral cell nests and reports that an immune microenvironment with a high density of PD-1+ helper T cells in tumoral cell nests is a poor prognostic factor for HNSCC.

Recent advances made in treatment for head and neck squamous cell carcinoma (HNSCC) highlight the need for new prediction tools to guide therapeutic strategies. In this study, we aimed to develop a HNSCC-targeting multiplex immunohistochemical (IHC) panel that can evaluate prognostic factors and the intratumor heterogeneity of HNSCC. To identify IHC-based tissue biomarkers that constitute new multiplex IHC panel, a systematic review and meta-analysis were performed to analyze reported IHC biomarkers in laryngeal and pharyngeal SCC in the period of 2008–2018. The Cancer Genome Atlas (TCGA) and Reactome pathway databases were used to validate the prognostic and functional significance of the identified biomarkers. A 14-marker chromogenic multiplex IHC panel including identified biomarkers was used to analyze untreated HNSCC tissue. Forty-five high-quality studies and thirty-one candidate tissue biomarkers were identified (N = 7062). Prognostic validation in TCGA laryngeal and pharyngeal SCC cohort (N = 205) showed that β-catenin, DKK1, PINCH1, ADAM10, and TIMP1 were significantly associated with poor prognosis, which were related to functional categories such as immune system, cellular response, cell cycle, and developmental systems. Selected biomarkers were assembled to build a 14-marker panel, evaluating heterogeneity and polarized expression of tumor biomarkers in the tissue structures, which was particularly related to activation of Wnt/β-catenin pathway. Integrated IHC analysis based on a systemic review and meta-analysis provides an in situ proteomics tool to assess the aggressiveness and intratumor heterogeneity of HNSCC.

Spatial profiles of the tumor-immune microenvironment are associated with disease progression and clinicopathological factors in various cancers. Follicular thyroid carcinoma (FTC) is the second most common thyroid cancer, where the presence of capsular invasion or angioinvasion determines the pathological diagnosis; however, little is known about the immune microenvironment profiles associated with the acquisition of invasive potential of FTC. In this study, we focused on FTC with minimal capsular invasion, and the spatially resolved immune microenvironment of FTC was studied in the discovery (n = 13) and validation cohorts (n = 40). CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, tumor-associated macrophages, CD66+ granulocytes, mature dendritic cells, and mast cells were quantitatively evaluated in single tissue sections, via a 12-marker multiplex immunohistochemistry and image cytometry. Cell densities and compositions of immune cells were spatially stratified by six tissue regions including tumor center, subcapsular region, capsular invasion, adjacent stroma of capsular invasion, peritumoral stroma, and adjacent normal. Lymphoid cell lineages in the tumor center and subcapsular regions were significantly lower than those in adjacent normal and peritumoral stroma, potentially related to the lymphoid lineage exclusion from the intratumoral regions of FTC. Interestingly, immune cell composition profiles in the capsular invasive front were distinct from those of intratumoral region. The ratios of T cells to CD66b+ granulocytes with capsular invasion were significantly higher than those without capsular invasion, suggesting the presence of a unique immune microenvironment at the invasive front between tumor foci and stroma. In addition, tumor cells at the capsular invasive front showed significantly higher expression of tumor programmed cell death ligand 1 (PD-L1) than those at the tumor center. This study revealed spatial immune profiles associated with capsular invasion of FTC, providing new insights into the mechanisms underlying its development and initial invasion.