Next-generation sequencing (NGS) allows the detection of multiple genetic targets in different tumor types. This study aimed to confirm the benefits of implementing in-house NGS testing for non-small-cell lung cancer (NSCLC) samples in molecular pathology laboratories. A multi-institutional study was conducted to evaluate the analytical performance, turnaround time, and feasibility of in-house NGS testing of 50 genes from 283 NSCLC samples. The first phase was a retrospective study with inter-laboratory testing (21 samples), and the second phase was a prospective study with intralaboratory testing (262 samples). The retrospective study showed a 100% sequencing success rate for DNA and RNA, high interlaboratory concordance (95.2%), and a strong correlation (R2 Z 0.94) between observed and expected single-nucleotide variant/insertion and/or deletion variant allele fraction. The prospective study showed a sequencing success rate of 99.2% for DNA and 98% for RNA. NGS identified 285 relevant variants (81.1% single-nucleotide variants/insertion and/or deletion variants, 9.8% copy number variants, and 9.1% gene fusions). Co-mutations with potential clinical relevance were detected in 20.5% of samples positive for the main oncogenic drivers in NSCLC. Additionally, 11% of samples wild type for the main oncogenic drivers carried alterations in other relevant genes. The in-house NGS testing had a median turnaround time from sample processing to molecular report of 4 days. This study demonstrates the advantages of implementing in-house NGS testing in molecular pathology laboratories.

Poorly differentiated thyroid carcinoma (PDTC) is a rare thyroid cancer with aggressive clinical course and peculiar clinical/pathological characteristics but lacking effective therapeutic options, when surgery is not curative. We aimed at the molecular characterization of PDTC with a specific focus on the identification of potential therapeutic targets. A series of PDTC cases was selected from a multi-institutional network. Fifty-nine samples underwent wide targeted DNA and RNA next-generation sequencing (NGS) testing and immunohistochemical analysis for mismatch repair (MMR) proteins. Gene fusion analysis was enriched by 25 additional samples. Prevalence of MMR protein loss was 11.9%. The most prevalent mutations were in NRAS (25%) and TP53 (25%), mutually exclusive. TERT promoter (TERTp) mutations were detected in 19.6% of cases (10/51). NRAS-mutated cases were enriched for mutations in genes belonging to the same pathway. TP53-mutated samples lacked TERTp co-mutations, but were associated with mutations in PTEN and in genes related to MMR system and/or loss of MMR proteins. TERTp mutations were the most prevalent alterations (28%, 7/25) in a third group that lacked NRAS or TP53 mutations. Four cases harbored gene fusions, including two cases harboring the TBL1XR1::PIK3CA fusion that has never been reported in thyroid cancer, so far. In conclusion, PDTC may be genomically segregated in subgroups with specific molecular characteristics. Overall, targetable gene fusions have a prevalence of 9% (4/42). Moreover, 47% of cases are potential candidates for individualized target therapies since they harbor mutations in genes coding for potentially targetable molecules and/or have defects in the MMR system.

SUPPLEMENTARY FIGURE 1 Correlation of VIM/CDH1 ratios in different datasets. The list of 35 genes significantly correlated with EMT. Lack of correlation between AKR1B10 mRNA levels and EMT. SUPPLEMENTARY FIGURE 2 AKR1B1 levels are modulated by ZEB1, but not in a direct fashion. SUPPLEMENTARY FIGURE 3 Characteristics of the NSCLC patients stained with immunohistochemistry and impact on survival. Alterations of AKR1B1 levels influence cancer cells growth and cancer stemness. SUPPLEMENTARY FIGURE 4 Impact of SORD knockdown on cancer cells growth and invasion. The combination of the inhibition of polyol pathway gene and cisplatin suppresses in vitro growth. Oxidative consumption rate and ECAR/OCR ratios in polyol pathway-deficient cells. SUPPLEMENTARY FIGURE 5 Co-expression patterns of AKR1B1 and SORD in TCGA datasets. SUPPLEMENTARY FIGURE 6 Effects of treatment with excess glucose on the generation of polyols, on the alteration of EMT and stemness markers, and on the activation of TGF-Beta signaling and TGF-Beta-related genes.

Poorly differentiated thyroid carcinoma (PDTC) represents a rare subtype of thyroid cancer with aggressive clinical course and peculiar clinical and pathological characteristics. Recent genetic studies highlighted the main molecular pathways involved in its pathogenesis but data are affected by heterogeneity of case selection and relatively limited sample size. Using a multimodal molecular approach, aim of this project was to implement the PDTC molecular characterization and to identify novel prognostic biomarkers and potential therapeutic targets. A total of 84 cases of PDTC classified according to the Turin proposal were selected from a multi-Institutional series. Fifty-nine samples underwent DNA, RNA and protein analyses. Mismatch repair (MMR) defects were tested using immunohistochemistry for MLH1, MSH2, MSH6 and PMS2. DNA and RNA analyses were performed by means of next generation sequencing (NGS) using Oncomine Comprehensive assay V3. Due to the high failure rate in RNA analysis, gene fusion analysis was enriched by 25 additional samples. MMR protein loss was observed in 7/59 samples (11.9%). In NGS DNA analyses, 51/59 cases were adequate. The most prevalent mutations were in NRAS (13/51, 25%) and TP53 (13/51, 25%), all mutually exclusive each other. TERT promoter (TERTp) mutations were detected in 11/51 of cases (21.6%). Other genes with a relevant prevalence were PTEN (15.7%), NF1 (13.7%), ATM (13.7%), NOTCH3 (11.8%) and BAP1 (11.8%). Mean number of alterations was higher in TP53-mutated cases (5.8 mutations/case) than in RAS-mutated cases (2.8 mutations/case). TP53-mutated samples lacked TERTp co-mutations but were associated with mutations in PTEN and in genes related to MMR system and/or loss of MMR proteins. PIK3CA was the most prevalent co-mutated gene (three samples) in RAS-mutated cases. A third group (25 cases) lacked RAS or TP53 mutations, had a low mean number of alterations (2.7 mutations/case) and was enriched for TERTp mutations (up to 32%). Copy number variations were not detected. Among the 43 cases adequate for NGS RNA analysis, gene fusions in PAX8-PPARG (one case) and TBL1XR1-PIK3CA (two cases) were detected. This latter fusion has never been reported in thyroid cancer, and was validated by fluorescence in situ hybridization (FISH). In conclusion, PDTC are genomically clustered into RAS-mutated tumors (with low mutational burden and co-mutations affecting genes involved in the same pathway), TP53-mutated cancers (with high mutational burden, absence of TERTp mutations and strong association with MMR defects) and a third “double negative” group enriched for TERTp mutations. Overall, targetable gene fusions have a prevalence of 7%. Moreover, 38% of overall cases harbor mutations in genes coding for tyrosine kinases potentially targetable and/or have defects in the MMR that claim a potential role for immunotherapy. Citation Format: Vanessa Zambelli, Marta Fornaro, Giulia Orlando, Ida Rapa, Francesca Napoli, Susanna Cappia, Lorenzo Daniele, Simonetta Piana, Mauro Papotti, Marco Volante. Identification of therapeutic targets in poorly differentiated carcinoma through a multimodal molecular analysis. [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 5285.

<p>List of differentially expressed genes in all analyzed datasets</p>

e21064 Background: Testing for EGFR mutations is an essential recommendation in both the ESMO and ASCO guidelines for metastatic non-squamous NSCLC, and is considered mandatory in European countries. However, in practice, considerable challenges are often faced when performing routine biomarker testing, including access to testing, inadequate tissue samples, and long turnaround times (TATs). Methods: In order to evaluate the real-world EGFR testing practices of thoracic pathology laboratories across Europe, an online survey was set up and validated by The Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories across Europe. The retrospective survey focused on the laboratory organisation and daily practice of pathologists and molecular biologists between 2018 and 2021, and covered the clinical circumstances in which EGFR testing was performed, sample types used, molecular biology techniques performed, average TATs for determining EGFR status, annual EGFR testing rates, laboratory accreditation, and external quality assessment. Results: TATs varied greatly depending on different parameters (notably relating to testing practices and methods used) both between and within countries. TATs were slightly (but non-significantly) faster for liquid biopsy samples (Mean: 7 days, IQR 4.5-12.5) when compared to tissue samples (mean 7 days, IQR 5-15) but were reported to take as long as 20 or 30 days, respectively. Discrepancies in TATs may be due in part to reflex testing practices, as 20% (11/54) of laboratories performed EGFR testing only at the request of the clinician. Many laboratories across Europe still favour targeted sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques, and 45.1% indicated they used targeted sequencing followed by NGS, depending on the case. Reported testing volumes were consistent over time with no significant decrease in the number of EGFR tests performed observed in 2020, despite the increased pressure and difficulties faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42% of molecular biology laboratories for targeted sequencing, and by 42.3% for NGS. 92% (49/53) of laboratories indicated they regularly participate in an EQA scheme. Conclusions: The results of the survey highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there remains large variability in testing capabilities, reflex testing practice and laboratory accreditation, which stresses the urgent need to harmonize budgets and reimbursement in Europe, in addition to harmonization of the technologies and decision-making algorithms.

PDF file, 30KB, ERCC1 gene expression under mitotane treatment in ACC cancer cells.

Supplementary Figures S1-S4.

The standard front-line treatment for pleural mesothelioma (PM) is pemetrexed-based chemotherapy, whose major target is thymidylate synthase (TS). In several cancer models, miR-215 and miR-375 have been shown to target TS, while information on these miRNAs in PM are still limited although suggest their role in epithelial to mesenchymal transition. Seventy-one consecutive PM tissues (4 biphasic, 7 sarcomatoid, and 60 epithelioid types) and 16 commercial and patient-derived PM cell lines were screened for TS, miR-215, and miR-375 expression. REN and 570B cells were selected for miR-215 and miR-375 transient transfections to test TS modulation. ZEB1 protein expression in tumor samples was also tested. Moreover, genetic profile was investigated by means of BAP1 and p53 immunohistochemistry. Expression of both miR-215 and miR-375 was significantly higher in epithelioid histotype. Furthermore, inverse correlation between TS protein and both miR-215 and miR-375 expression was found. Efficiently transfected REN and 570B cell lines overexpressing miR-215 and miR-375 showed decreased TS protein levels. Epithelioid PM with a mesenchymal component highlighted by reticulin stain showed significantly higher TS and ZEB1 protein and lower miRNA expression. A better survival was recorded for BAP1 lost/TS low cases. Our data indicate that miR-215 and miR-375 are involved in TS regulation as well as in epithelial-to-mesenchymal transition in PM.

BACKGROUND:The molecular characterization of thyroid nodules in cytological samples has so far been focused on discriminating between benign and malignant forms in a purely diagnostic setting. The evidence on the impact of molecular biomarkers to determine the risk of aggressiveness in cytologically "neoplastic" lesions is limited to genomic alterations (such as BRAF and TERT mutations). The aim of our study was to assess the preoperative role of microRNAs (miRNAs) in predicting the nodal status of patients with papillary thyroid cancer.METHODS:A pilot series of histological samples of papillary thyroid carcinoma with (6 cases) or without (6 cases) lymph node metastases, matched for other major clinical and pathological features, was analyzed for global miRNA expression in a screening phase. A set of miRNAs was then validated in a series of 63 consecutive cytological samples of papillary carcinomas: 48 pN-negative and 15 pN-positive at histology.RESULTS:Unsupervised cluster analysis segregated surgical pN-negative and pN-positive samples, except for 1 case. The 45 differentially expressed miRNAs in pN-positive versus pN-negative cases were predicted to regulate a wide range of cellular pathways, enriched for Wnt, gonadotropin-releasing hormone receptor, and cerulein/cholecystokinin receptor signaling. In agreement with their profiles in surgical samples, 4 miRNAs of the 10 selected for validation (miR-154-3p, miR-299-5p, miR-376a-3p, and miR-302E) had a significant differential expression in cytological samples of papillary carcinoma with lymph node metastases and predicted the positive nodal status with a relatively good performance.CONCLUSIONS:MiRNA profiling is a potential promising strategy to define papillary carcinoma aggressiveness in the preoperative setting.

Extra-pulmonary neuroendocrine carcinomas (EPNEC) represent a group of rare and heterogenous neoplasms with adverse clinical outcome. Their molecular profile is largely unexplored. Our aim was to investigate if the major transcriptional drivers recently described in high-grade pulmonary neuroendocrine carcinomas characterize distinct molecular and clinical subgroups of EPNEC. Gene expression of ASCL1, NEUROD1, DLL3, NOTCH1, INSM1, MYCL1, POU2F3, and YAP1 was investigated in a series of 54 EPNEC (including 10 cases with mixed components analyzed separately) and in a group of 48 pulmonary large cell neuroendocrine carcinomas (P-LCNEC). Unsupervised hierarchical cluster analysis classified the whole series into four major clusters. P-LCNEC were classified into two major clusters, the first ASCL1/DLL3/INSM1-high and the second (including four EPNEC) ASCL1/DLL3-low but INSM1-high. The remaining EPNEC cases were sub-classified into two other clusters. The first showed INSM1-high and alternative ASCL1/DLL3 or NEUROD1 high expression. The second was characterized mainly by MYCL1 and YAP1 overexpression. In the ten cases with mixed histology, ASCL1, DLL3, INSM1, and NEUROD1 genes were significantly upregulated in the neuroendocrine component. Higher gene-expression levels of NOTCH1 and INSM1 were associated with lower pT stage and negative nodal status. Low INSM1 gene expression was associated with shorter overall survival in the entire case series (p = 0.0017) and with a trend towards significance in EPNEC, only (p = 0.06). In conclusion, our results show that EPNEC possess distinct neuroendocrine-lineage-specific transcriptional profiles; moreover, low INSM1 gene expression represents a novel potential unfavorable prognostic marker in high-grade NECs including those in extra-pulmonary location.

Aims Lung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak. Methods Fifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time. Results In most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing. Conclusions The workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

Pediatric primary lung carcinomas are extremely rare. Apart from known associations with congenital adenomatoid malformations, cases of primary lung adenocarcinomas after prolonged treatments of pediatric malignancy have been reported. We describe the morphological and molecular features of three cases of lung adenocarcinoma developed in adolescents aged 8 to 17 years during progression of their bone osteosarcoma or Ewing sarcomas. The morphological features overlapped those of adult lung adenocarcinoma including in situ, minimally invasive, and invasive forms. EGFR gene mutations were found in all three cases by targeted next-generation sequencing. The two patients with Ewing sarcoma had no progression of their lung cancer and no further progression of the metastatic bone tumor after additional chemo- and radio-therapy. Conversely, the osteosarcoma patient refused further treatments after thoracic surgery for metastatic osteosarcoma and locally advanced adenocarcinoma and died 2 years later of widespread distant metastases. Our results indicate that primary lung cancer might originate in pediatric patients during prolonged adjuvant therapies for primary bone neoplasm, and this possibility should be considered in the presence of suspected lung disease progression to correctly monitor the primary tumor evolution and define the appropriate therapeutic strategy at each time point. If appropriately treated, second primary lung cancer may not affect the patients’ prognosis. The pathogenetic mechanisms of these rare lung adenocarcinomas are not clear, but the presence of EGFR mutations in all three cases indicates an oncogene addiction of the lung tumor, rather than a direct cancerogenic effect of the sarcoma-related treatment.

Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 “hyper”-amplified (≥ 8 copies) tumors. We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody–drug conjugates, in a selected subgroup of HER2 “hyper”-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody–drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-“hyper”-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

The molecular mechanisms of adrenocortical carcinoma development are incompletely defined. De-regulation of cellular-to-extracellular matrix interactions and angiogenesis appear among mechanisms associated to the malignant phenotype. Our aim was to investigate, employing PCR-based array profiling, 157 molecules involved in cell-to-matrix interactions and angiogenesis in a frozen series of 6 benign and 6 malignant adrenocortical neoplasms, to identify novel pathogenetic markers. In 14 genes, a significant dysregulation was detected in adrenocortical carcinomas as compared to adenomas, most of them being downregulated. Three exceptions—hyaluronan synthase 1 (HAS-1), laminin α3 and osteopontin genes—demonstrated an increased expression in adrenocortical carcinomas of 4.46, 4.23 and 20.32-fold, respectively, and were validated by immunohistochemistry on a series of paraffin-embedded tissues, including 20 adenomas and 73 carcinomas. Osteopontin protein, absent in all adenomas, was expressed in a carcinoma subset (25/73) (p = 0.0022). Laminin α3 and HAS-1 were mostly expressed in smooth muscle and endothelial cells of the vascular network of both benign and malignant adrenocortical tumors. HAS-1 was also detected in tumor cells, with a more intense pattern in carcinomas. In this group, strong expression was significantly associated with more favorable clinicopathological features. These data demonstrate that cell-to-matrix interactions are specifically altered in adrenocortical carcinoma and identify osteopontin and HAS-1 as novel potential diagnostic and prognostic biomarkers, respectively, in adrenal cortical tumors.

Background: O6-methylguanine-methyltransferase (MGMT) is a key enzyme for the DNA repair machinery strongly associated with response to alkylating agents in different tumors. Data on its expression and related clinical impact in neuroendocrine tumors are limited to the gastro-entero-pancreatic system, with controversial results in terms of prognostic or predictive value. In lung carcinoids, although clinical efficacy of alkylating agents has been shown in small studies, very few data to date are available on MGMT status. Objective: To assess MGMT status in lung carcinoids using multiple assays and to compare data with major clinical and pathological features. Methods: A retrospective series of 95 lung carcinoids and 51 control cases of high-grade neuroendocrine lung carcinomas was analyzed for MGMT promoter methylation, MGMT gene expression, and MGMT protein expression using pyrosequencing, quantitative real-time PCR, and immunohistochemistry, respectively. Results: MGMT protein expression was inversely correlated with MGMT promoter methylation and positively with MGMT gene expression. MGMT promoter methylation progressively increased from carcinoids to high-grade carcinomas. In the carcinoid group, decreased MGMT gene expression was significantly associated with aggressive features (atypical histotype, grade G2, larger tumor size, higher T stage, and positive nodal status) but not with survival. MGMT promoter methylation was associated with lower stage and negative nodal status. Conclusions: Our study investigated MGMT status in a large series of lung carcinoids in the attempt to move forward a rational use of alkylating agents in these tumors. Interestingly, low MGMT gene expression defines a subgroup of lung carcinoids with aggressive features.

BACKGROUND:Epithelial-to-mesenchymal transition (EMT) enhances motility, stemness, chemoresistance and metastasis. Little is known about how various pathways coordinate to elicit EMT's different functional aspects in non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) has been previously correlated with EMT transcription factor ZEB1 in NSCLC and imparts resistance against anti-folate chemotherapy. In this study, we establish a functional correlation between TS, EMT, chemotherapy and metastasis and propose a network for TS mediated EMT.METHODS:Published datasets were analysed to evaluate the significance of TS in NSCLC fitness and prognosis. Promoter reporter assay was used to sort NSCLC cell lines in TSHIGH and TSLOW. Metastasis was assayed in a syngeneic mouse model.RESULTS:TS levels were prognostic and predicted chemotherapy response. Cell lines with higher TS promoter activity were more mesenchymal-like. RNA-seq identified EMT as one of the most differentially regulated pathways in connection to TS expression. EMT transcription factors HOXC6 and HMGA2 were identified as upstream regulator of TS, and AXL, SPARC and FOSL1 as downstream effectors. TS knock-down reduced the metastatic colonisation in vivo.CONCLUSION:These results establish TS as a theranostic NSCLC marker integrating survival, chemo-resistance and EMT, and identifies a regulatory network that could be targeted in EMT-driven NSCLC.

AIM:To validate the prognostic role of a panel of genes previously uncovered by our group to be specific targets of miRNAs differentially expressed in lung carcinoids with aggressive pathological features.METHODS:Four genes, namely, cyclic AMP response element binding protein-1 (CREBP1), activin A receptor type 2B (ACVR2B), LIM homeobox 2 (LHX2), and Krüppel-like factor 12 (KLF12), were identified in a previous study by our group using in silico analysis to be regulated by 3 miRNAs (miR-409-3p, miR-409-5p, and miR-431-5p) that were shown to be downregulated in aggressive lung carcinoids. These genes were analyzed using real-time PCR in a cohort of 102 lung carcinoids. Fifty high-grade lung carcinomas served as control group. Their expression was correlated with the expression of miR-409-3p, miR-409-5p, and miR-431-5p and with clinical pathological parameters and disease-free survival.RESULTS:The expression of all but CREBP1 gene was significantly different between lung carcinoids and high-grade neuroendocrine carcinomas. ACVR2B and LHX2 were significantly inversely correlated with miR-409-3p and miR-409-5p. High levels of ACVR2B and LHX2 were significantly associated with atypical histotype, high tumor grade, and higher proliferation Ki-67 index (all p < 0.05). Low levels of KLF12 were significantly associated with the presence of necrosis and positive nodal status (all p < 0.05). Finally, low KLF12 expression was associated with shorter disease-free survival in lung carcinoids as a whole and in atypical carcinoids, only (all p < 0.001).CONCLUSIONS:ACVR2B, LHX2, and KFL12 are novel potential biomarkers associated with aggressive features in lung carcinoids.

BACKGROUND:Poorly differentiated thyroid cancer (PDTC) is a rare, follicular cell-derived neoplasm with an unfavorable prognosis. The oncocytic variant of PDTC may be associated with even more adverse outcome than classical PDTC cases, but its specific molecular features are largely unknown. Our aim was to explore the immune-related gene expression profile of oncocytic and classical PDTC, in correlation with clinical and pathological characteristics (including programmed death ligand 1 [PD-L1] expression) and outcome, and in comparison with a control group of well-differentiated follicular carcinomas (WDFCs), including conventional follicular carcinomas (FTCs) and Hürthle cell carcinomas (HCCs).METHODS:A retrospective series of 48 PDTCs and 24 WDFCs was analyzed by means of NanoString technology employing the nCounter PanCancer Immune Profiling panel. Gene expression data were validated using quantitative real-time polymerase chain reaction.RESULTS:Oncocytic PDTCs showed a specific immune-related gene expression profile, with higher expression of LAIR2, CD274, DEFB1, IRAK1, CAMP, LCN2, LY96, and APOE, and lower expression of NOD1, as compared to conventional PDTCs. This molecular signature was associated with increased intratumoral lymphocytic infiltration, PD-L1 expression, and adverse outcome. Three of these genes, CD274, DEFB1, and IRAK1, as well as PD-L1 expression, were also the hallmarks of HCCs as compared to FTCs. By contrast, the panel of genes differentially regulated in PDTCs as compared to WDFCs was unrelated to the oncocytic phenotype.CONCLUSIONS:Our results revealed a distinctive immune-related gene expression profile of oncocytic PDTC and confirmed a more aggressive outcome in this cancer subtype. These findings may provide guidance when exploring novel immunotherapeutic options for oncocytic PDTC patients.