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Prognosis after chemoradiotherapy (CRT) for anal squamous cell carcinoma (ASCC) shows marked differences among patients according to TNM subgroups, however individualized risk assessment tools to better stratify patients for treatment (de-) escalation or intensified follow-up are lacking in ASCC.Patients' data from eight sites of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG), comprising a total of 605 patients with ASCC, treated with standard definitive CRT with 5-FU/Mitomycin C or Capecitabine/Mitomycin C between 2004-2018, were used to evaluate prognostic factors based on Cox regression models for disease-free survival (DFS). Evaluated variables included age, gender, Karnofsky performance score (KPS), HIV-status, T-category, lymph node status and laboratory parameters. Multivariate cox models were separately constructed for the whole cohort and the subset of patients with early-stage (cT1-2 N0M0) tumors.After a median follow-up of 46 months, 3-year DFS for patients with early-stage ASCC was 84.9%, and 67.1% for patients with locally-advanced disease (HR 2.4, p 0.001). T-category (HR vs. T1: T2 2.02; T3 2.11; T4 3.03), N-category (HR versus N0: 1.8 for N1-3), age (HR 1.02 per year), and KPS (HR 0.8 per step) were significant predictors for DFS in multivariate analysis in the entire cohort. The model performed with a C-index of 0.68. In cT1-2N0 patients, T-category (HR 2.14), HIV status (HR 2.57), age (1.026 per year), KPS (HR 0.7 per step) and elevated platelets (HR 1.3 per 100/nl) were associated with worse DFS (C-index of 0.7).Classical clinicopathologic parameters like T-category, N-category, age and KPS remain to be significant prognostic factors for DFS in patients treated with contemporary CRT for ASCC. HIV and platelets were significantly associated with worse DFS in patients with early stage ASCC.

PURPOSE/OBJECTIVE(S) Tumor-infiltrating lymphocytes (TILs) correlate with an improved outcome of head-and-neck squamous cell cancer (HNSCC) patients undergoing radiotherapy and are influenced by the tumor microenvironment and tumor-associated hypoxia. The present analysis is based on a prospective imaging trial and analyzes the value of TILs and tumor-associated hypoxia to stratify HNSCC patients according to their response to radiotherapy. MATERIALS/METHODS A total of 49 patients with locoregionally advanced HNSCCs were prospectively enrolled in this trial and underwent longitudinal hypoxia PET imaging using fluoro-18 misonidazole ([18F]FMISO) in weeks 0, 2 and 5 during chemoradiation. Tumor hypoxia was assumed for a tumor-to-background SUV (contralateral sternocleidomastoid muscle) exceeding 1.4, and an early hypoxia response was defined as a decrease in the normalized maximum intratumoral SUV (FMISO-SUVindex) between weeks 0 and 2. Pre-treatment tumor biopsies were analyzed for TILs and the hypoxia tissue marker carbonic anhydrase IX (CAIX). Trial patients were stratified into 4 subgroups based on their TIL numbers (> 100 vs. < 100 TILs/HPF) and expression of CAIX (above vs. below median H-score), and locoregional control (LRC) and progression-free survival (PFS) rates for all subgroups were assessed using Cox and subsequent concordance analyses (Harrell's C). RESULTS High TIL levels correlated with improved LRC (HR = 0.279, P = 0.011) and PFS (HR = 0.276, P = 0.006). Similarly, a decrease in the FMISO-SUVindex within the first 2 weeks of treatment corresponded with better LRC (HR = 0.321, P = 0.015) and PFS (HR = 0.402, P = 0.043). Harrell's C was 0.68, when TILs and early hypoxia PET response were combined. The hypoxia PET-based hypoxia-immune classifier separated 3 distinct prognostic patient subgroups, a favorable (TILhigh/ early PET response), and intermediate (TILhigh/no early PET response or TILlow/early PET response) and a poor (TILlow/no early PET response) prognostic group with 2-year LRC of 71%, 33% and 0%, respectively. Low pre-treatment tissue levels of CAIX were also prognostic for improved LRC (HR = 0.352, P = 0.050) but not PFS (HR = 0,468, P = 0,087). Harrell's C was 0.66 for CAIX and TILs separately and 0.71 for the combination. The immunohistochemistry-based immune-hypoxia classifier similarly stratified between a favorable (CAIXlow/TILhigh), an intermediate (CAIXlow/TILlow or CAIXhigh/TILhigh) and a poor (CAIXhigh/TILlow) subgroup with 2-year LRC rates of 73%, 62% and 11%, respectively. CONCLUSION We developed a hypoxia PET-based hypoxia-immune classifier that was able to separate HNSCC patients into 3 distinct prognostic subgroups based on their tumor biology. These subgroups could also be stratified in a clinically feasible, immunohistochemistry-based classifier with comparable model accuracy. Therefore, this hypoxia-immune classifier could help to stratify prognoses of HNSCC patients undergoing radiotherapy pending validation in an external cohort.

Purpose As both tumor hypoxia and an immunosuppressing tumor microenvironment hamper the anti-tumor activity of radiotherapy in head-and-neck squamous cell carcinoma (HNSCC), we aimed to develop an immunohistochemistry-based hypoxia-immune classifier. Methods 39 patients receiving definitive chemoradiation for HNSCC within a prospective trial were included in this analysis. Baseline tumor samples were analyzed for the hypoxia marker carbonic anhydrase IX (CAIX) and tumor-infiltrating lymphocytes (TILs) and were correlated with [18F]-misonidazole ([18F]FMISO) PET measurements. The impact of the biomarkers on the locoregional control (LRC) was examined using Cox analyses and concordance index statistics. Results Low CAIX (HR = 0.352, 95%CI 0.124–1.001, p = 0.050) and high TIL levels (HR = 0.308, 95%CI 0.114–0.828, p = 0.020) were independent parameters for improved LRC and did not correlate with each other (Spearman’s ρ = 0.034, p = 0.846). Harrell’s C was 0.66 for CAIX and TIL levels alone and 0.71 for the combination. 2-year LRC was 73%, 62% and 11% for the prognostically good (CAIXlow/TILhigh), intermediate (CAIXlow/TILlow or CAIXhigh/TILhigh) and poor groups (CAIXhigh/TILlow), respectively (p = 0.001). Focusing on T lymphocytes, the hypoxia-immune classifier could still stratify between favorable (CAIXlow/CD3 + TILhigh), intermediate (CAIXlow/CD3 + TILlow or CAIXhigh/CD3 + TILhigh) and poor subgroups (CAIXhigh/CD3 + TILlow) with a 2-year LRC of 80%, 59% and 14%, respectively (p = 0.001). There was a positive correlation between baseline CAIX levels and [18F]FMISO SUV in week 2 of chemoradiation (ρ = 0.324, p = 0.050), indicating an association between higher baseline CAIX expression and tumor hypoxia persistence. Conclusion We developed a clinically feasible hypoxia-immune prognostic classifier for HNSCC patients based on pre-treatment immunohistochemistry. However, external validation is required to determine the prognostic value and the potential usage for personalized radiation oncology.

PURPOSE/OBJECTIVE(S) Elderly head-and-neck squamous cell carcinoma (HNSCC) patients form a heterogeneous cohort, and survival estimation is often challenging due to underlying comorbidities. Prognostic classifiers and nomograms may therefore be useful for overall survival (OS) estimation in order to provide shared decision-making in the clinical routine. MATERIALS/METHODS A total of 284 elderly HNSCC patients aged ≥65 years who received a curative (chemo)radiation between 2010 and 2020 at a tertiary cancer center were used for the development of a survival classifier. On the basis of a multivariate Cox regression analysis, significant parameters were identified for which points were given according to the beta regression values. The derived classifier was then validated in a second, external cohort consisting of 217 elderly HNSCC patients undergoing (chemo)radiation. Based on the cumulative data of 501 patients, a nomogram for the 2-year and 4-year OS was created. We then examined in a third independent cohort whether the classifier could also stratify the prognosis of surgically treated elderly HNSCC patients without adjuvant (chemo)radiation (n = 169). RESULTS In the multivariate backward stepwise Cox regression with likelihood ratio tests using P < 0.1 as inclusion criterion, the Karnofsky Performance Status (KPS, HR = 2.654, P < 0.001), the age-adjusted Charlson Comorbidity Index (CCI, HR = 2.598, P < 0.001) and the pre-radiotherapy CRP serum concentration (HR = 1.634, P = 0.064) were significant prognostic parameters for OS. Following the beta regression values, a KPS ≤70% and a CCI ≥6 points were given 1 point, while a CRP concentration ≥5 mg/L was given 0.5 points. The median OS was 107 (0 points), 34 (0.5 points), 28 (1 point), 11 (1.5 points), 9 (2 points) and 6 months (2.5 points), respectively. In order to obtain considerably distinct prognostic subgroups, 3 prognostic groups were created: A favorable (0 points), an intermediate (0.5-2 points) and a poor (2.5 points) subgroup. While the median OS for the favorable group amounted to 107 months, it was 28 and 6 months for the intermediate and poor cohorts, respectively (P < 0.001, log-rank test). In the external cohort, the median OS was found to range at 130, 29 and 9 months for the favorable, intermediate and poor group, respectively (P = 0.005). Using the aggregated data of both cohorts, a nomogram based on the KPS, CCI and CRP was created for the 2-year and 4-year OS that exhibited a concordance index of 0.65 (Harrell's C). For the surgically treated cohort of elderly HNSCC patients, only the KPS ≤70% (HR = 5.950, P = 0.002) but not the CCI ≥6 points (HR = 1.937, P = 0.232) or the baseline CRP serum value (HR = 1.743, P = 0.328) were significant prognosticators. CONCLUSION We developed and externally validated a clinically feasible survival score for elderly HNSCC patients undergoing (chemo)radiation. Both the score and nomogram may be useful aiding shared decision-making of radiation oncologists and medical oncologists treating elderly HNSCC patients.

Purpose/objective(s) Transgelin-2 (TAGLN2) is an oncogene and actin-binding protein known to induce actin polymerization, thereby playing a critical role in cell proliferation, migration/invasion, and therapeutic resistance in GBM and other malignancies. In our clinical correlative studies, TAGLN2 was found to be one of the top candidate genes associated with worse survival response and its expression at significantly higher level in GBM patients with wild-type IDH1/2 compared to mutant IDH1/2 patients. The standard treatment has remained relatively ineffective partly due to their invasive phenotype leading to tumor recurrence. Currently, there is no bonafide pharmacologic inhibitor that specifically blocks the Transgelin-2 functions. We hypothesize that targeting Transgelin-2 by small molecule inhibitors (SMIs) may improve patient outcome by circumventing therapeutic resistance mechanisms. This study aims to identify novel SMIs of Transgelin-2 and demonstrate the potential of these inhibitors as a novel strategy for therapeutic intervention in human glioblastoma. Materials/methods Structure-based computational screening of SMIs libraries using docking algorithms- AutoDock Vina and Glide XP was employed to identify SMIs of Transgelin-2, which can bind to Transgelin-2. The compounds were further screened for their ability to be able to permeate BBB, using SwissADME webtool. A series of in-vitro binding and functional assays were employed to measure the potential of these inhibitors to bind and inhibit Transgelin-2-dependent actin-polymerization and subsequent cell proliferation/survival, invasion, neurosphere and colony formation in PDX GBM cell lines. Further, the potential of these inhibitors to sensitize these cells towards standard TMZ and/or RT was evaluated. Results In-silico virtual screenings identified potential Transgelin-2 binding small molecule compounds that bind to Transgelin-2. These candidate molecules were predicted to permeate BBB using in-silico approach. In vitro binding study confirmed that these compounds bind to Transgelin-2 and inhibit the Transgelin-2-dependent actin-polymerization. We found that these compounds significantly compromised the proliferation, survival, migration/invasion and clonal expansion of GBM 08-387 and 3359 PDX cell lines. These compounds however showed no toxicity to normal human cells. These inhibitors also reduced the level of Transgelin-2 protein in these two cell lines in a dose- and time- dependent fashion. Our in-vitro data also suggest that these inhibitors significantly sensitized these GBM PDX cell lines towards temozolamide and/or radiation treatment in a dose- and time-dependent fashion. Conclusion Our study identified novel SMIs of Transgelin-2 and validated their potential to bind and significantly inhibit Transgelin-2-actin mediated cell proliferation, invasion and therapeutic resistance without any normal cell toxicity, thereby serving as potential candidates for preclinical trials. Author disclosure A. Kumar: None. P. Rajasekera: None. S. Biehn: None. S. Beyer: None. J. McElroy: None. A. Becker: None. B. Johnson: None. T. Cui: None. E. Sebastian: None. A. Grosu: None. S. Lindert: None. E.H. Bell: None. S.J. Haque: None. A. Chakravarti: None.