Background Programmed death‐ligand 1 (PD‐L1) expression is emerging as an important predictive biomarker in anti–PD‐L1 cancer immunotherapy. Its role has been clearly defined in various human cancers and is linked to a poor prognosis and resistance to anticancer therapies. The role of PD‐L1 in thyroid cancers has not been well defined in fine‐needle aspiration cytology (FNAC). The authors examined the performance of PD‐L1 immunostaining in liquid‐based cytology (LBC) to determine whether it could be a biomarker of malignancy or aggressive disease. Methods From January 2018 to March 2019, 236 thyroid lesions, which had been diagnosed by FNAC as indeterminate lesions, suspicious for malignancy (SFM), and malignant, were enrolled. PD‐L1 immunostaining was performed on both LBC and corresponding histology samples. Results The FNAC cohort included 50 benign negative controls, 42 samples of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), 33 samples of follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 53 samples that were suspicious for malignancy (SFM), and 58 malignant samples. AUS/FLUS samples included 3 goiters, 32 follicular adenomas (FAs), 1 noninvasive follicular thyroid neoplasm with papillary‐like nuclear features (NIFTP), 5 invasive follicular variants of papillary thyroid carcinoma (I‐FVPTCs), and 1 follicular carcinoma; whereas FN/SFN samples included 24 FAs and 9 malignancies (4 I‐FVPTCs, 1 NIFTP, 3 papillary thyroid carcinomas [PTCs], and 1 oncocytic follicular carcinoma). The 53 SFM samples were diagnosed on histopathology as 2 FAs, 5 NIFTPs, 15 I‐FVPTCs, and 31 PTCs; whereas the 58 malignant specimens included 5 NIFTPs, 5 I‐FVPTCs, and 48 PTCs. Increased plasma membrane and cytoplasmic PD‐L1 expression was found in 79 cases (38.5%), including 61 PTCs (conventional and variants). Negative PD‐L1 expression was found in NIFTPs and FAs. A BRAF V600E mutation was identified in 15% of PD‐L1–positive malignancies. Conclusions The current data suggest that PD‐L1 expression in the thyroid gland might represent a marker of malignancy that correlates with PTC, but not with NIFTP. Thyroid neoplasms with PD‐L1 expression also ae enriched with BRAF V600E mutations, suggesting that they are associated with more aggressive behavior.

Childhood ependymomas are heterogenous chemoresistant neoplasms arising from aberrant stem-like cells. Epigenome deregulation plays a pivotal role in ependymoma pathogenesis, suggesting that epigenetic modifiers hold therapeutic promise against this disease. Bromodomain and extraterminal domain (BET) proteins are epigenome readers of acetylated signals in histones and coactivators for oncogenic and stemness-related transcriptional networks, including MYC/MYCN (Proto-Oncogene, BHLH Transcritpion Factor)-regulated genes. We explored BET inhibition as an anticancer strategy in a panel of pediatric patient-derived ependymoma stem cell models by OTX015-mediated suppression of BET/acetylated histone binding. We found that ependymoma tissues and lines express BET proteins and their targets MYC and MYCN. In vitro, OTX015 reduced cell proliferation by inducing G0/G1-phase accumulation and apoptosis at clinically tolerable doses. Mechanistically, inhibitory p21 and p27 increased in a p53-independent manner, whereas the proliferative driver, phospho-signal transducer and activator of transcription 3 (STAT3), decreased. Upregulation of apoptosis-related proteins and survivin downregulation were correlated with cell line drug sensitivity. Minor alterations of MYC/MYCN expression were reported. In vivo, OTX015 significantly improved survival in 2/3 orthotopic ependymoma models. BET proteins represent promising targets for pharmaceutical intervention with OTX015 against ependymoma. The identification of predictive determinants of sensitivity may help identify ependymoma molecular subsets more likely to benefit from BET inhibitor therapies.

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Grade 3 meningiomas are rare malignant tumors that can originate de novo or from the progression of lower grade meningiomas. The molecular bases of anaplasia and progression are poorly known. We aimed to report an institutional series of grade 3 anaplastic meningiomas and to investigate the evolution of molecular profile in progressive cases. Clinical data and pathologic samples were retrospectively collected. VEGF, EGFR, EGFRvIII, PD-L1; and Sox2 expression; MGMT methylation status; and TERT promoter mutation were assessed in paired meningioma samples collected from the same patient before and after progression using immunohistochemistry and PCR. Young age, de novo cases, origin from grade 2 in progressive cases, good clinical status, and unilateral side, were associated with more favorable outcomes. In ten progressive meningiomas, by comparing molecular profile before and after progression, we identified two subgroups of patients, one defined by Sox2 increase, suggesting a stem-like, mesenchymal phenotype, and another defined by EGFRvIII gain, suggesting a committed progenitor, epithelial phenotype. Interestingly, cases with Sox2 increase had a significantly shortened survival compared to those with EGFRvIII gain. PD-L1 increase at progression was also associated with worse prognosis, portending immune escape. We thus identified the key drivers of meningioma progression, which can be exploited for personalized treatments.

714 Background: Intermediate IMDC group is the largest and most heterogeneous group of mRCC. Current first-line (1L) therapy options for these patients (pts) are based on either an anti-angiogenic agent (VEGFR-TKI) combined with immunotherapy (IO), or a combo of IO (ipilimumab+nivolumab [I/N]). No biomarkers (BM) for selecting the most effective regimen have been identified so far. Methods: Immunohistochemical expression of PBRM1, PD-L1, CD31, and CD4/CD8 ratio was evaluated on histological samples of intermediate-risk mRCC pts treated with VEGFR-TKI monotherapy, and then in pts receiving a VEGFR-TKI-based therapy or the immune doublet I/N. PBRM1 positivity score was based on the percentage of positive cells and on the intensity of nuclear expression; PD-L1 positivity was defined as CPS≥10; CD31 high-density had moderate to strong nuclear staining; and the CD4/CD8 ratio cut-off for positivity was >0.2. Cox model was used to assess the correlation between BM and outcomes; PFS and OS were estimated by Kaplan-Meier method. Results: After screening of tumor tissues from 150 pts, a total of 111 were included in the final analysis (Table). In pts treated with VEGFR-TKI monotherapy, a significant correlation with PFS was observed with loss of PBRM1 expression (HR 0.58, p=0.035), PD-L1 negativity (HR 0.44, p=0.048), and high CD4/CD8 ratio (HR 0.62, p=0.073). CD31 density did not significantly correlate with PFS. A profile potentially predictive of angiogenesis (AP+) was defined based on the PBRM1 loss, PD-L1 negative, and high CD4/CD8. In pts treated with VEGFR-TKI monotherapy, tumors with the AP+ (43% of all cases) had a significantly longer median PFS (mPFS 23.8 vs. 11.8 months, p=0.003) and mOS (41.5 vs. 26.9 months, p=0.024) compared to the others. The AP+ retained its significant correlation with PFS (mPFS 23.8 vs. 11.1 months, p<0.001) and OS (41.5 vs. 24.9, p=0.006) in pts receiving VEGFR-TKI-based therapies. The rate of AP+ tumors was 55.6% and 32.7% in pts with one or two IMDC risk factors, respectively (p=0.022). In the small cohort of pts treated with I/N, no differences were observed in PFS (p=0.64) and OS (p=0.75) between AP+ and AP-negative. Conclusions: The AP+ signature (loss of PBRM1, PD-L1 negative, and CD4/CD8 high ratio) was associated with improved clinical outcomes in mRCC pts at IMDC intermediate prognosis treated with VEGFR-TKI-based therapy; this correlation was significant regardless from the addition of IO to VEGFR-TKI monotherapy. Prospective validation of this signature is required for guiding the selection of the most appropriate 1L therapy. [Table: see text]

AimsSeveral papers have shown that programmed death-ligand 1 (PD-L1) expression is a relevant predictive biomarker in anti-PD-L1 cancer immunotherapy. While its role in several human cancers is correlated with poor prognosis and resistance to anticancer therapies, in thyroid cancers the role of PD-L1 remains questionable. Few articles have studied PD-L1 in thyroid fine-needle aspiration cytology (FNAC), demonstrating a possible correlation with papillary thyroid carcinoma. However, its role in oncocytic thyroid lesions remains controversial. We accordingly examine the performance of PD-L1 immunostaining in liquid based cytology (LBC) from oncocytic lesions.MethodsFrom January 2019 to March 2021, 114 thyroid lesions diagnosed by FNAC from lesions with a predominant oncocytic component, were enrolled for evaluation by PD-L1 immunostaining on both LBC and corresponding histology samples.ResultsThe FNAC cohort included 51 benign (B, negative controls), 4 atypia of undetermined significance/follicular lesions of undetermined significance (AUS/FLUS), 57 follicular lesions (follicular neoplasm/suspicious for FN, FN/SFN) and 2 suspicious for malignancy (SFM) cases. Fifty-four cases (11B, 2 AUS/FLUS, 39 FN/SFN and 2 SFM) had histological follow-up including: 1B case resulted as a hyperplastic oxyphilic nodule in Hashimoto thyroiditis (HT), 10B as goitre, 2 AUS/FLUS cases as oncocytic adenomas (OAs); 39 FN/SFN included 27 OAs, 4 FA and 8 oncocytic follicular carcinoma (OFC). The two SFM cases were diagnosed on histopathology as OAs. Increased plasma membrane and cytoplasmic PD-L1 expression were found in 47 cases of the LBC cases (41.2%). Among the histological series, 67.3% of OAs and 75% of OFC had PD-L1 expression, while negative PD-L1 was found in hyperplastic oncocytic cells in HT. A positivity in more than 30% of the neoplastic cells was found in 72.9% of the cases including six OFC.ConclusionsThese data suggest that PD-L1 expression is expressed in oncocytic thyroid lesions. While weak PD-L1 expression failed to discriminate benign from malignant lesions, OFC demonstrated more intense cytoplasmic and membranous expression.

e16304 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies for which few effective pharmacological treatments are currently available. Immunotherapy has revolutionized the treatment of several solid tumors, yet the results obtained in PDAC have been disappointing. Although PDAC is generally considered an immune “cold” cancer, different PDAC subtypes have diverse immune tissue microenvironment (TME) components, including tumor-associated macrophages (TAMs), that might differentially influence immunotherapy responsiveness and patient survival. TAM population is plastic, switching rapidly from a pro- to an anti-tumoral behaviour, based on specific cytokines in TME. A role for the chemokines/CXCR axis in inducing M1 polarization has been proposed in several tumors, and, consequently, the potential advantage of combining CXCR inhibitors with immune check-point inhibitors. Here, we evaluated the effectiveness of the CXCR1/2 inhibitor ladarixin, alone or in combination with anti-PD-1, on immune tolerance against PDAC and on tumor growth. Methods: A set of preclinical models were obtained by engrafting mouse PDAC-derived cell lines into the pancreas of recipient syngeneic immune-competent mice, as well as by orthotopically transplanting patient derived PDAC tumor into human immune system reconstituted (HIR) mice (Hu-NSG-CD34+). Tumor bearing mice were randomly assigned to receive vehicles, ladarixin, and anti-PD-1 alone or in combination with ladarixin, to evaluate the effect of treatment on tumor growth and mice survival. This work is supported by AIRC and Italian Ministry of Health grants. Results: CXCR1/2 inhibition by ladarixin reverted in vitro tumor-mediated M2 polarization and migration of Bone Marrow-Derived Macrophages. Ladarixin as a single agent significantly reduced tumor burden in cancer derived graft (CDG) models with high-immunogenic potential and significantly increased the efficacy of ICI in non-immunogenic CDG models that did not respond to anti-PD-1 treatment. In a HIR mouse model bearing the immunogenic subtype of human PDAC, ladarixin showed high efficacy as a single agent and the ability to increase the antitumor effects of anti-PD-1 with a statistically significant reduction of tumor volume compared to control and to single treatments (ctr vs lad, p = 0.0077; ctr vs comb, p = 0.0098; lad vs comb: 0.002; anti-PD-1 vs comb, p = 0.036). Moreover, ladarixin both as monotherapy and in combination setting increased survival (median survival: ctr 49.5 vs lad 74.5 days, p = 0.044; ctr 49.5 vs comb 150 days, p = 0.0108; anti-PD-1 57 vs comb 150 days, p = 0.046; lad 74.5 vs comb 150 days, p = 0.047). Conclusions: Ladarixin in combination with anti-PD-1 might represent an effective approach for the treatment of PDAC, converting a protumoral into an immunopermissive microenvironment in PDAC subtypes usually refractory to immunotherapy.