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Pediatric thyroid carcinomas (TCs) are rare and mainly approached based on data extrapolated from adults. We retrospectively reviewed 222 pediatric TCs (patient age less than or equal to 21 y). Lymph node (LN) disease volume at presentation was considered high if the largest positive LN measured ≥1 cm and/or5 LNs were positive. High-grade follicular cell-derived thyroid carcinoma (HGFCTC) were defined by the presence of marked mitotic count and/or tumor necrosis and considered as high-risk histology along with papillary thyroid carcinomas (PTC) diffuse sclerosing variant (DSV). Disease-free survival (DFS) was analyzed. LN involvement at presentation was significantly associated with male sex, larger tumor size, lymphatic invasion, positive surgical margins, and distant metastases at presentation. Five- and 10-year DFS was 84% and 77%, respectively. Only 1 patient with HGFCTC died of disease. Within PTC variants, PTC-DSV was associated with adverse histopathologic parameters and higher regional disease spread, unlike PTC tall cell variant which did not portend worse behavior. The presence of necrosis conferred worse DFS ( P =0.006), while increased mitotic activity did not. While the entire HGFCTC group did not correlate with outcome ( P =0.071), HGFCTC with necrosis imparted worse DFS ( P =0.006). When restricted to PTC-DSV and HGFCTC with necrosis, high-risk histologic classification emerged as an independent prognostic parameter of DFS ( P =0.020). The excellent prognosis of pediatric TCs differs from that of adult TCs showing similar histologic features. While neither increased mitotic activity nor PTC tall cell variant histology predict adverse outcome, PTC-DSV and tumors with necrosis constitute high-risk histologic variants with an increased risk of protracted disease.

Intra-operative stimulated Raman histology (SRH) is a novel technology that uses laser spectroscopy and color-matching algorithms to create images similar to the formalin-fixed paraffin-embedded (FFPE) section. We aim to assess the accuracy of SRH in a novel range of sinonasal and skull base tumors.Select patients undergoing sinonasal and skull base surgery using the Invenio Imaging™ Nio™ Laser Imaging SRH system between June 2020 and September 2021 were assessed. The SRH images were reviewed for pathologic features similar to frozen section (FS) and FFPE. Time taken for results and diagnostic concordance was assessed.Sixty-seven SRH images from 7 tumor types in 12 patients were assessed. Pathologies included squamous cell carcinoma, rhabdomyosarcoma, inverted papilloma, adenoid cystic carcinoma, SMARCB1-deficient sinonasal carcinoma, mucosal melanoma, metastatic colonic adenocarcinoma, and meningioma. Tumor was identified in 100% of lesional specimens, with characteristic diagnostic features readily appreciable on SRH. Median time for diagnosis was significantly faster for SRH (4.3 min) versus FS (44.5 min; p = .0001). Where SRH sample site matched precisely to FS (n = 32/67, 47.8%), the same diagnosis was confirmed in 93.8%. Sensitivity, specificity, precision, and overall accuracy of SRH were 93.3%, 94.1%, 93.8%, and 93.3%, respectively. Near-perfect concordance was seen between SRH and FS (Cohen's kappa [κ] = 0.89).Stimulated Raman histology can rapidly produce images similar to FFPE HE in sinonasal and skull base tumors. This technology has the potential to act as an adjunct or alternative to standard FS.4 Laryngoscope, 132:2142-2147, 2022.

This study is the largest analysis of DNA viruses in solid tumors with associated genomics. To achieve this, a novel method for discovery of DNA viruses from matched tumor/normal next-generation sequencing samples was developed and validated. This method performed comparably to reference methods for the detection of high-risk (HR) human papilloma virus (HPV) (area under the receiver operating characteristic curve = 0.953). After virus identification in 48,148 consecutives samples from 42,846 unique patients, novel virus tumor associations were established by segregating tumor types to determine whether each DNA virus was enriched in each of the tumor types compared with the remaining cohort. All firmly established solid tumor-virus associations (eg, HR HPV in cervical cancer) were confirmed, and the novel associations discovered included: human herpes virus 6 in neuroblastoma, human herpes virus 7 in esophagogastric cancer, and HPV42 in digital papillary adenocarcinoma. These associations were confirmed in an independent validation cohort. HR HPV– and Epstein-Barr virus–associated tumors showed newly discovered genomic associations, including a lower tumor mutation burden. The study demonstrated the ability to study the role of DNA viruses in human cancer from clinical genomics data and established the largest cohort that can be utilized as a validation set for future discovery efforts.

Positively enriched gene signatures by GSEA using MsigDB-HALLMARKS and CANONICAL PATHWAYS genesets in CAL62-splice vs CAL62 cells.

Targeted inhibition of BRAF V600E achieves tumor control in a subset of advanced thyroid tumors. Nearly all tumors develop resistance, and some have been observed to subsequently undergo dedifferentiation. The molecular alterations associated with thyroid cancer dedifferentiation in the setting of BRAF inhibition are unknown. We analyzed targeted next-generation sequencing data from 639 advanced, recurrent and/or metastatic thyroid carcinomas, including 15 tumors that were treated with BRAF inhibitor drugs and had tissue sampled during or posttreatment, 8 of which had matched pretherapy samples. Pre- and posttherapy tissues from one additional patient were profiled with whole-exome sequencing and RNA expression profiling. Mutations in genes comprising the SWI/SNF chromatin remodeling complex and the PI3K–AKT–mTOR, MAPK, and JAK–STAT pathways all increased in prevalence across more dedifferentiated thyroid cancer histologies. Of 7 thyroid cancers that dedifferentiated after BRAF inhibition, 6 had mutations in these pathways. These mutations were mostly absent from matched pretreatment samples and were rarely detected in tumors that did not dedifferentiate. Additional analyses in one of the vemurafenib-treated tumors before and after anaplastic transformation revealed the emergence of an oncogenic PIK3CA mutation, activation of ERK signaling, dedifferentiation, and development of an immunosuppressive tumor microenvironment. These findings validate earlier preclinical data implicating these genetic pathways in resistance to BRAF inhibitors, and suggest that genetic alterations mediating acquired drug resistance may also promote thyroid tumor dedifferentiation.Implications:The possibility that thyroid cancer dedifferentiation may be attributed to selective pressure applied by BRAF inhibitor–targeted therapy should be investigated further.

Supplemental Figure 1. Increased clonal heterogeneity in anaplastic metastases (ATC Met 1, 5 clones Supplemental Figure 2. Copy-number alterations for the pre-vemurafenib papillary thyroid carcinoma sample Supplemental Figure 3. Differentially expressed gene analyses comparing pre-treatment PTC sample and ATC metastases in MSK-THY1. Supplemental Figure 4. Prevalence of genetic alterations linked to BRAF inhibitor resistance and/or ATC pathogenesis in primary TCGA PTCs and metastatic/recurrent PTCs, PDTCs, and ATCs sequenced by MSK-IMPACT. Supplemental Figure 5. Histology and immunohistochemical studies for MSK-THY6. Supplemental Figure 6. Mutational landscape of tumors that underwent dedifferentiation subsequent to BRAF inhibitor targeted therapy with vemurafenib or dabrafenib.

Supplemental Table 1. Genomic alterations observed in MSK-THY1 Supplemental Table 2. Cellular prevalence of single nucleotide variants in MSK-THY Supplemental Table 3. Normalized gene expression data from patient MSK-THY1 Supplemental Table 4. Mutational data for 639 thyroid tumors sequenced by MSK-IMPACT Supplemental Table 5. Mutational data for BRAFi treated thyroid cancers with post-treatment sequencing

Figure S12

EIF1AX mutations in fatal non-anaplastic thyroid cancer.

List of 410 genes in IMPACT sequencing platform

Supplementary legend

All IMPACT mutation calls. Tumors previously published by Landa et al (1) are highlighted in blue.

Figure S1. Coverage and quality of whole exome sequencing. Figure S2. Coverage and quality of RNA sequencing. Figure S3. Detection of somatic mutations by RNA sequencing. Figure S4. Focal copy number alterations. Figure S5. Fusion genes. Figure S6. SDC histology and immunohistochemistry. Figure S7. Genetic alterations in cohort 2. Figure S8. Consensus clustering of SDC and basal-like breast cancer. Table S2. PCR primers used in this study. Table S5. Clinical information cohort 2. Table S6. Missense mutations in HRAS and PIK3CA. Table S7. Potentially targetable alterations.

Purpose: The molecular epidemiology of most EGFR and KRAS mutations in lung cancer remains unclear.Experimental Design: We genotyped 3,026 lung adenocarcinomas for the major EGFR (exon 19 deletions and L858R) and KRAS (G12, G13) mutations and examined correlations with demographic, clinical, and smoking history data.Results:EGFR mutations were found in 43% of never smokers and in 11% of smokers. KRAS mutations occurred in 34% of smokers and in 6% of never smokers. In patients with smoking histories up to 10 pack-years, EGFR predominated over KRAS. Among former smokers with lung cancer, multivariate analysis showed that, independent of pack-years, increasing smoking-free years raise the likelihood of EGFR mutation. Never smokers were more likely than smokers to have KRAS G > A transition mutation (mostly G12D; 58% vs. 20%, P = 0.0001). KRAS G12C, the most common G > T transversion mutation in smokers, was more frequent in women (P = 0.007) and these women were younger than men with the same mutation (median 65 vs. 69, P = 0.0008) and had smoked less.Conclusions: The distinct types of KRAS mutations in smokers versus never smokers suggest that most KRAS-mutant lung cancers in never smokers are not due to second-hand smoke exposure. The higher frequency of KRAS G12C in women, their younger age, and lesser smoking history together support a heightened susceptibility to tobacco carcinogens. Clin Cancer Res; 18(22); 6169–77. ©2012 AACR.

Supplementary figure and table legends

Table S3

Purpose:Salivary gland carcinomas (SGC) are rare, aggressive cancers with high rates of recurrence and distant metastasis. These factors, and a lack of active systemic therapies, contribute to poor clinical outcome. Response rates with immune checkpoint blockade have been low, although clinical data remain sparse. To improve the efficacy of therapies, a more comprehensive understanding of relevant molecular alterations and immunologic processes is needed.Experimental Design:To characterize the immune microenvironment and neoantigen landscape of SGCs, we performed RNA sequencing (RNA-seq) in 76 tumors representing the three most lethal histologies: adenoid cystic carcinoma (ACC), myoepithelial carcinoma (MECA), and salivary duct carcinoma (SDC). We analyzed transcriptomic profiles, tumor-infiltrating immune cell populations, and measures of T-cell activation/dysfunction. In 37 cases also undergoing exome sequencing, we analyzed somatic mutations and neoantigens.Results:SDCs exhibited high levels of immune infiltration, with corresponding higher levels of T-cell dysfunction, and higher mutational load. In contrast, ACCs were characterized by an immune-excluded microenvironment, the presence of M2-polarized macrophages and myeloid-derived suppressor cells, and very low mutational load. MECAs were more heterogeneous, with both immune-low and immune-high phenotypes represented. Across all SGCs, levels of immune infiltration were associated with mutation- and fusion-derived neoantigens, and with aggressive clinical behavior.Conclusions:These findings provide new insights into the immune microenvironment and neoantigen landscape of SGCs, showing that mechanisms of immune escape appear to differ by histology. These data nominate potential immunologic vulnerabilities and may help guide the next steps of investigation in precision immunotherapy for these difficult-to-treat cancers.

Purpose: Salivary duct carcinoma (SDC) is an aggressive salivary malignancy, which is resistant to chemotherapy and has high mortality rates. We investigated the molecular landscape of SDC, focusing on genetic alterations and gene expression profiles.Experimental Design: We performed whole-exome sequencing, RNA sequencing, and immunohistochemical analyses in 16 SDC tumors and examined selected alterations via targeted sequencing of 410 genes in a second cohort of 15 SDCs.Results: SDCs harbored a higher mutational burden than many other salivary carcinomas (1.7 mutations/Mb). The most frequent genetic alterations were mutations in TP53 (55%), HRAS (23%), PIK3CA (23%), and amplification of ERBB2 (35%). Most (74%) tumors had alterations in either MAPK (BRAF/HRAS/NF1) genes or ERBB2. Potentially targetable alterations based on supportive clinical evidence were present in 61% of tumors. Androgen receptor (AR) was overexpressed in 75%; several potential resistance mechanisms to androgen deprivation therapy (ADT) were identified, including the AR-V7 splice variant (present in 50%, often at low ratios compared with full-length AR) and FOXA1 mutations (10%). Consensus clustering and pathway analyses in transcriptome data revealed striking similarities between SDC and molecular apocrine breast cancer.Conclusions: This study illuminates the landscape of genetic alterations and gene expression programs in SDC, identifying numerous molecular targets and potential determinants of response to AR antagonism. This has relevance for emerging clinical studies of ADT and other targeted therapies in SDC. The similarities between SDC and apocrine breast cancer indicate that clinical data in breast cancer may generate useful hypotheses for SDC. Clin Cancer Res; 22(18); 4623–33. ©2016 AACR.

Table S3. All mutations in cohort 1.

Table S4. All mutations and copy number alterations in cohort 2.

Purpose: Patients with anaplastic thyroid cancer (ATC) have a very high death rate. In contrast, deaths from non-anaplastic thyroid (NAT) cancer are much less common. The genetic alterations in fatal NAT cancers have not been reported.Experimental Design: We performed next-generation sequencing of 410 cancer genes from 57 fatal NAT primary cancers. Results were compared with The Cancer Genome Atlas study (TCGA study) of papillary thyroid cancers (PTCs) and to the genomic changes reported in ATC.Results: There was a very high prevalence of TERT promoter mutations, comparable with that of ATC, and these co-occurred with BRAF and RAS mutations. A high incidence of chromosome 1q gain was seen highlighting its importance in tumor aggressiveness. Two novel fusion genes DLG5–RET and OSBPL1A–BRAF were identified. There was a high frequency of mutations in MED12 and these were mutually exclusive to TERT promoter mutations and also to BRAF and RAS mutations. In addition, a high frequency of mutations in RBM10 was identified and these co-occurred with RAS mutations and PIK3CA mutations. Compared with the PTCs in TCGA, there were higher frequencies of mutations in TP53, POLE, PI3K/AKT/mTOR pathway effectors, SWI/SNF subunits, and histone methyltransferases.Conclusions: These data support a model, whereby fatal NAT cancers arise from well-differentiated tumors through the accumulation of key additional genetic abnormalities. The high rate of TERT promoter mutations, MED12 mutations, RBM10 mutations, and chromosome 1q gain highlight their likely association with tumor virulence. Clin Cancer Res; 23(19); 5970–80. ©2017 AACR.

Table S1. List of genes and additional intronic regions included in the MSK-IMPACT next-generation sequencing panel.

PDF file - 225K, Supplementary Figure 1. (A) Gender differences in frequency of EGFR mutations by stage. Men with EGFR mutations presented at the late stage more often than women (118/170, 69% vs. 235/423, 56%; p=0.002); women predominated at stage I (31% vs. 19%, p=0.004). (B) Stage distribution of EGFR exon 19 del and EGFR L858R. EGFR L858R were significantly more frequent at stage I than exon 19 del (83/246, 34% vs. 82/347, 24%; p=0.009); Fisher exact test, P value T transversions had smoked less (average 34 pack-years vs. 40 pack-years, p=0.001) and were younger than men with the same nucleotide change (median age 64 vs. 67, p=0.006). Fisher exact test, P value

Acinic cell carcinoma (AciCC) is traditionally considered as a low-grade salivary gland carcinoma. However, a subset demonstrates high-grade features with a higher mortality rate and distant metastasis. In this large retrospective study of 117 cases, we aimed to establish a histologic grading scheme for AciCC. Adverse independent prognostic factors identified on the multivariate analysis included older age, tumor necrosis, nuclear anaplasia, lymphovascular invasion, absence of tumor-associated lymphoid stroma, and high American Joint Committee on Cancer (AJCC) pT and pN stages. A 3-tiered grading scheme using 4 pathologic parameters (mitotic index, necrosis, tumor border, and fibrosis at the frankly invasive front) was subsequently applied. Compared with low/intermediate-grade, high-grade AciCC defined as a mitotic index ≥5/10 HPFs and/or necrosis was an independently adverse prognostic factor. The 5-year overall survival was 50% in high-grade AciCCs, and 100% in low-grade or intermediate-grade AciCCs. Compared with low-grade or intermediate-grade AciCC, high-grade tumors were associated with older age, larger tumor size, focal rather than diffuse zymogen granules, solid architecture, infiltrative tumor border, fibrosis at the frankly invasive front, lymphovascular invasion, perineural invasion, positive margin, high pT, and pN stages. NR4A3 was a highly sensitive and specific immunohistochemical stain for diagnosing AciCC with a sensitivity and specificity of 96% and 93%, respectively. In conclusion, although we proposed a 2-tiered grading system for AciCC with high-grade tumors defined by a mitotic count ≥5/10 HPFs and/or necrosis, more studies are needed to assess the prognostic value of intermediate grade. NR4A3 immunohistochemical stain is a useful diagnostic marker for AciCC.

Targeted inhibition of BRAF V600E achieves tumor control in a subset of advanced thyroid tumors. Nearly all tumors develop resistance, and some have been observed to subsequently undergo dedifferentiation. The molecular alterations associated with thyroid cancer dedifferentiation in the setting of BRAF inhibition are unknown. We analyzed targeted next-generation sequencing data from 639 advanced, recurrent and/or metastatic thyroid carcinomas, including 15 tumors that were treated with BRAF inhibitor drugs and had tissue sampled during or posttreatment, 8 of which had matched pretherapy samples. Pre- and posttherapy tissues from one additional patient were profiled with whole-exome sequencing and RNA expression profiling. Mutations in genes comprising the SWI/SNF chromatin remodeling complex and the PI3K–AKT–mTOR, MAPK, and JAK–STAT pathways all increased in prevalence across more dedifferentiated thyroid cancer histologies. Of 7 thyroid cancers that dedifferentiated after BRAF inhibition, 6 had mutations in these pathways. These mutations were mostly absent from matched pretreatment samples and were rarely detected in tumors that did not dedifferentiate. Additional analyses in one of the vemurafenib-treated tumors before and after anaplastic transformation revealed the emergence of an oncogenic PIK3CA mutation, activation of ERK signaling, dedifferentiation, and development of an immunosuppressive tumor microenvironment. These findings validate earlier preclinical data implicating these genetic pathways in resistance to BRAF inhibitors, and suggest that genetic alterations mediating acquired drug resistance may also promote thyroid tumor dedifferentiation. Implications: The possibility that thyroid cancer dedifferentiation may be attributed to selective pressure applied by BRAF inhibitor–targeted therapy should be investigated further.

Molecular characterization of adrenocortical carcinomas (ACC) by The Cancer Genome Atlas (TCGA) has highlighted a high prevalence of TERT alterations, which are associated with disease progression. Herein, 78 ACC were profiled using a combination of next generation sequencing (n = 76) and FISH (n = 9) to assess for TERT alterations. This data was combined with TCGA dataset (n = 91). A subset of borderline adrenocortical tumors (n = 5) and adrenocortical adenomas (n = 7) were also evaluated. The most common alteration involving the TERT gene involved gains/amplifications, seen in 22.2% (37/167) of cases. In contrast, “hotspot” promoter mutations (C > T promoter mutation at position -124, 7/167 cases, 4.2%) and promoter rearrangements (2/165, 1.2%) were rare. Recurrent co-alterations included 22q copy number losses seen in 24% (9/38) of cases. Although no significant differences were identified in cases with and without TERT alterations pertaining to age at presentation, tumor size, weight, laterality, mitotic index and Ki67 labeling, cases with TERT alterations showed worse outcomes. Metastatic behavior was seen in 70% (28/40) of cases with TERT alterations compared to 51.2% (65/127, p = 0.04) of cases that lacked these alterations. Two (of 5) borderline tumors showed amplifications and no TERT alterations were identified in 7 adenomas. In the borderline group, 0 (of 4) patients with available follow up had adverse outcomes. We found that TERT alterations in ACC predominantly involve gene amplifications, with a smaller subset harboring “hotspot” promoter mutations and rearrangements, and 70% of TERT-altered tumors are associated with metastases. Prospective studies are needed to validate the prognostic impact of these findings.

Secretory carcinoma (SC) is a rare form of salivary carcinoma that was first described in 2010 and is characterized by ETV6::NTRK3 fusion in most cases. In this large retrospective study, we aimed to identify adverse clinicopathologic factors and propose a prognostically relevant grading scheme for SC.A detailed clinicopathologic review was conducted on 90 SCs from the major and minor salivary glands.The median age at presentation was 50 years (range: 7-93). Sixty-nine (77%) tumours originated from major salivary glands, whereas the remaining 21 involved minor salivary glands.Six cases (7%) had cervical nodal metastasis. Only lymphovascular invasion (LVI) was associated with a risk of nodal metastasis (P 0.05). The 5-year disease-specific survival and disease-free survival (DFS) were 98% and 87%, respectively. On univariate survival analysis, adverse prognostic factors associated with decreased DFS included minor salivary gland origin, atypical mitosis, high mitotic index, high-grade transformation (HGT), necrosis, nuclear pleomorphism, infiltrative tumour border, fibrosis at the invasive front, LVI, positive margin, and advanced pT stage (P 0.05). When adjusted for pT stage and margin status, mitotic index, LVI, nuclear pleomorphism, and HGT remained as independent prognostic factors.We therefore propose a two-tiered grading system for SC. The low-grade SC is defined as those with5 mitoses /10 high-power fields and no tumour necrosis, and high-grade SC as those with ≥5 mitoses /10 high-power fields and/or necrosis. This proposed grading system can be useful to risk stratify patients with SC for appropriate clinical management.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

Introduction Pulmonary invasive mucinous adenocarcinomas (IMAs) often present with spatially separate lung lesions. Clonal relationship between such lesions, particularly those involving contralateral lobes, is not well established. Here, we used comparative genomic profiling to address this question. Methods Patients with genomic analysis performed on two IMAs located in different lung regions were identified. Molecular assays included DNA-based next-generation sequencing (NGS) for 410 to 468 genes (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets), RNA-based NGS for 62 genes (Memorial Sloan Kettering-Fusion), or non-NGS assays. Results Comparative genomic profiling was performed on two separate IMAs in 24 patients, of whom 19 had contralateral lesions. Tumors from all but one patient shared matching driver alterations, including KRAS (n = 19), NRG1 (n = 2), ERBB2 (n = 1) or BRAF (n = 1). In addition, in patients with paired tumors profiled by NGS (n = 12), shared driver alterations were accompanied by up to 4 (average 2.6) other identical mutations, further supporting the clonal relationship between the tumors. Only in a single patient separate IMAs harbored entirely nonoverlapping mutation profiles, supporting clonally unrelated, distinct primary tumors. Notably, in a subset of patients (n = 3), molecular testing confirmed a clonal relationship between the original resected IMAs and subsequent contralateral IMA presenting after an extremely long latency (8.1–11.7 y). Conclusions Comparative molecular profiling supports that nearly all separate pulmonary IMA lesions represent intrapulmonary spread arising from a single tumor and documents a subset with a remarkably protracted course of intrapulmonary progression. This study reinforces the unique biology and clinical behavior of IMAs while further highlighting the value of genomic testing for clarifying the clonal relationship between multiple lung carcinomas.

Poorly differentiated thyroid carcinoma (PDTC), defined by Turin criteria, comprises a subset of high-grade follicular-derived thyroid carcinomas with intermediate prognosis. While differentiated oncocytic thyroid carcinomas demonstrate clinicopathologic and genetic differences compared to their non-oncocytic counterparts, similar data is limited in oncocytic (Hurthle) PDTCs (OPDTCs). Here, we assessed the impact of various oncocytic cut-offs in PDTCs on clinical, histologic and survival parameters.Our bi-institutional cohort comprised 210 primary PDTCs with available slides reviewed by at least one pathologist. Histologic features, including oncocytic fraction, were recorded. Clinicopathologic data were obtained, including overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), locoregional recurrence free survival (LRRFS), and distant metastasis-free survival (DMFS). Radioactive iodine avidity data was available for 125 PDTCs based on postoperative whole-body scanning.Within our cohort, 39.0% PDTCs had any oncocytic component with 24.8% meeting the 75% World Health Organization (WHO) oncocytic definition. Any oncocytic component and 25% oncocytic cut-off correlated with decreased DSS and LRRFS, respectively, compared to non-oncocytic PDTCs (NOPDTCs) on univariate and multivariate analysis. The 100% oncocytic cut-off was significant for DSS on univariate analysis but a non-significant trend on multivariate analysis. Any oncocytic cut-off (100%, 75%, 50%, 25%, or 0%) conferred higher radioactive iodine (RAI)-refractoriness to OPDTCs compared to NOPDTCs. NF1 and PTEN alterations were enriched in OPDTCs (40% vs. 0%, and 60% vs 8%, respectively), whereas NRAS mutations were frequent in NOPDTCs (47% vs. 7%).Among PDTCs, the presence of oncocytes led to downward trend in all outcome parameters, especially for DSS and LRRFS. OPDTCs were enriched in NF1 and PTEN mutations. Consistently, all oncocytic cut-offs were associated with RAI-refractoriness. Accordingly, additional studies are needed to reassess the current 75% cut-off used to define oncocytic thyroid lesions.

Oral squamous cell carcinoma (OSCC) occasionally occurs in young patients and is likely to be distinct from OSCC in older patients. In this retrospective study, we described the clinicopathologic features and outcome of 150 OSCCs that were diagnosed in patients 40-year-old or younger. Most patients (63%) were non-smokers. The most common site of the primary tumor was oral tongue (n = 131, 87%), followed by gingiva (n = 9), buccal mucosa (n = 8) and lip (n = 2). The median patients' age at presentation was 34 (range: 16–40). Seven patients (5%) had Fanconi anemia with the gingiva being the most common location (4/7, 57%). All OSCCs were of keratinizing type. All cases tested for high-risk HPV (n = 34) were negative. On univariate analysis, high tumor budding was associated with decreased overall survival (OS) and distant metastasis free survival (DMFS), pattern of invasion correlated with OS and tumors with high stromal tumor infiltrating lymphocytes (sTIL) were associated with improved locoregional recurrence free survival (LRFS). Compared with patients 31 to 40-year-old, OSCC in the younger group was associated with significant less alcohol consumption (p = 0.011) and decreased DSS (p = 0.003) and DMFS (p = 0.023). On multivariate analysis, younger age (30 years or younger) was an independent prognostic factor for worse OS and DSS, whereas histologic grade was an independent prognostic factor for DSS. In summary, most OSCC in young patients occurred in non-smokers and did not occur in association with Fanconi anemia. Independent prognostic factors included age at presentation (30 years or younger) for OS and DSS, and histologic grade for DSS.

Mutations in the epidermal growth factor receptor (EGFR) are the most common targetable alterations in lung adenocarcinoma. To facilitate rapid testing, the Idylla EGFR assay was incorporated as a screening method before next-generation sequencing (NGS). Validation and experience using an in-house developed analysis pipeline, enhanced with a manual review algorithm is described. Results are compared with corresponding NGS results. In all, 1249 samples were studied. Validation demonstrated 98.57% (69/70) concordance with the reference methods. The limit of detection varied from 2% to 5% variant allele frequency for total EGFR quantitation cycle between 20 and 23. Of the 1179 clinical cases, 23.41% were EGFR-positive by Idylla. Concurrent NGS was successfully performed on 94.9% (799/842) requests. Concordance of Idylla with NGS was 98.62% (788/799) and 98.50% (787/799) using our in-house and Idylla analysis pipelines, respectively. Discordances involved missed mutations by both assays associated with low tumor/low input. Incorporating a manual review algorithm to supplement automated calls improved accuracy from 98.62% to 99.37% and sensitivity from 94.68% to 97.58%. Overall reporting time, from receipt of material to official clinical report, ranged from 1 to 3 days. Therefore, Idylla EGFR testing enables rapid and sensitive screening without compromising subsequent comprehensive NGS, when required. Automated calling, enhanced with a manual review algorithm, reduces false-negative calls associated with low tumor/low input samples.

The impact of the intratumoral microbiome on immune checkpoint inhibitor (ICI) efficacy in patients (pts) with non-small cell lung cancer (NSCLC) is unknown. In preclinical studies, the presence of lung intratumoral Escherichia was associated with a proinflammatory tumor microenvironment and decreased metastases within lung tissue. We sought to detect intratumoral bacteria in pts with advanced NSCLC using hybrid capture-based, next generation sequencing (NGS). We studied 849 pts treated with ICI-based therapy who underwent NGS at our center. We extracted unmapped reads from BAM files, and these were queried for bacteria (blastn alignment using the NCBI database). Putative environmental contaminants were subtracted from the analysis using “no template” controls (n=2,539) to exclude possible artifactual false positives. A custom E.Coli fluorescence in situ hybridization (FISH) probe was used to visualize Escherichia within the tumors after co-registration with H&E. In 849 pts, a median of 30 bacterial reads was detected per sample (inter-quartile range (18-85)). Among 68 pts with paired primary/metastatic samples, the bacterial spectra were similar in both sites, suggesting that tumor resident bacteria might travel with cancer cells to distant sites. Antibiotic use within 30 days of tumor sampling was associated with decreased intratumoral bacterial diversity (p=0.023 by Inverse Simpson, p=0.038 by Shannon). Intratumoral Escherichia was associated with better PFS (HR 0.78, 95% CI 0.62-0.98, p=0.036), and OS (HR 0.74, 95% CI 0.58-0.95, p=0.017) in pts treated with single-agent ICI, but not combination Chemo/ICI. In a multivariable model adjusting for prognostic features in NSCLC including PD-L1 tumor proportion score, the presence of intratumoral Escherichia was associated with better PFS (p=0.040) and OS (p=0.045) upon single-agent ICI therapy. Escherichia appeared to be intracellular based on co-registration of FISH staining and serial H&E sections. These findings warrant further investigation of the possible inter-relationships between intratumoral Escherichia, tumor immune micro-environment, and ICI therapeutic outcomes. Citation Format: Arielle Elkrief, Anita S. Bowman, Ayyuce Begum Bektas, Wenfei Kang, Katia Manova-Todorova, Jacklynn V. Egger, Hira Rizvi, Daniel Kelly, Eric Chan, Eric Rosiek, Fan Ning, Gregory J. Riely, Álvaro Quintanal Villalonga, Snjezana Dogan, Umesh Bhanot, Mithat Gonen, Matthew D. Hellmann, Adam J. Schoenfeld, Charles M. Rudin, Marc Ladanyi, Chad M. Vanderbilt. Intratumoral Escherichia is associated with response to single-agent immune checkpoint inhibition in patients with advanced non-small cell lung cancer. [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 5185.

Summary IDH1/2 hotspot mutations occur in glioma, cholangiocarcinoma, chondrosarcoma, sinonasal carcinoma, and T-cell lymphoma and have diagnostic, prognostic, and/or therapeutic value. Availability of immunohistochemistry (IHC) protocols for specific IDH2 mutation detection is limited. A targeted exome sequencing assay MSK-IMPACT cohort comprising >38,000 cancer cases was explored for the presence of IDH1/2 mutations in solid malignancies and select T-cell lymphomas. Seventy-four formalin-fixed paraffin-embedded IDH1/2-mutated (n = 62) and wild-type (n = 12) samples were used for testing and optimization of anti–IDH2 monoclonal antibodies (mAbs) 14H7, 3C11, and MMab1 targeting R172K, R172G, and R172M mutant proteins, respectively. IDH1/2 mutations were common in glioma (26.8% and 1.6%), intrahepatic cholangiocarcinoma (23.1% and 5.7%), chondrosarcoma (19.4% and 10.7%), sinonasal undifferentiated/large-cell neuroendocrine carcinoma (0% and 84.2%), angioimmunoblastic T-cell lymphoma (0% and 22%), and peripheral T-cell lymphoma (0 and 5.1%). In other cancers, IDH2 mutations were rare. IDH2 R172 variants included R172K (39%), R172S (29%), R172W (12%), R172G (10%), R172M (5%), and R172T (4%). 14H7, 3C11, and MMab1 detected all IDH2 R172K, R172G, and R172M, respectively, and produced a crisp, granular cytoplasmic staining pattern. 3C11 was also positive in 5 of 6 IDH1 R132G mutants showing a homogeneous, smooth cytoplasmic staining. All 3 mAbs were negative in other IDH1/2 mutant or wild-type cases. IHC using mAbs 14H7, 3C11, and MMab1 can facilitate molecular diagnosis as a reliable, fast, and inexpensive alternative for specific IDH2 variant detection. Given the distinct distribution of IDH2 R172 mutations in cancers, these mAbs could also serve as useful pathologic diagnostic markers.

Introduction: The collision of medullary (MTC) and papillary thyroid carcinoma (PTC) in the same cervical lymph node can occur, but its growth kinetics has not been reported. Case Presentation: We report a 27-year-old male patient who had collision nodal metastases from PTC and sporadic MTC in the central compartment. This was treated with total thyroidectomy and central neck dissection. The collision nodal metastasis persisted and presented with a single sonographically enlarging central compartmental lymph node postoperatively. The volume of the collision nodal metastasis increased from 226 to 507 mm3 over the first 8 months, from 507 to 572 mm3 over the next 6 months, and from 572 to 762 mm3 over the next 31 months. The calcitonin and carcinoembryonic antigen (CEA) fluctuated in the first 19 months followed by a steady increase at a doubling time of 1.97 and 8.42 years, respectively. Unstimulated thyroglobulin remained at 0.2 ng/mL or lower during the same period while thyrotropin (TSH) was not suppressed. Revision central neck dissection performed 4.5 years later resulted in undetectable serum calcitonin, CEA of 2 ng/mL, and thyroglobulin of 0.1 ng/mL from a preoperative calcitonin of 212 ng/L, CEA of 10 ng/mL, and thyroglobulin of 0.2 ng/mL. Further structural imaging 13.5 months later revealed no evidence of disease. Discussion: The growth kinetics of collision nodal metastasis from PTC and MTC can be similar to conventional PTC and MTC. Furthermore, the growth rate of such collision nodal metastases can be slow. Guided by tumor marker doubling time and regular structural imaging, surgical salvage performed after a period of active surveillance may still result in biochemical and structural remission.

Extranodal extension (ENE) is a significant prognostic factor for human papilloma virus (HPV)-negative head and neck squamous cell carcinoma and is incorporated into AJCC 8th edition pN stage. It remains controversial whether ENE or the degree of ENE is prognostically relevant in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). A detailed clinicopathologic review was conducted in a large retrospective cohort of 232 surgically treated patients with HPV-positive OPSCC and nodal metastasis. Fifty-six patients (24%) had nodal metastasis with ENE. The median vertical extent of ENE was 2.9 mm (range 0.2-20.3 mm), and the median horizontal span of ENE was 2.5 mm (range: 0.3-14.0 mm). Comparing with patients without ENE, those with ENE were associated with a higher number of positive lymph nodes, lymphovascular invasion, perineural invasion, adjuvant chemotherapy, larger primary tumor size, and shorter follow up period. Patients with ENE had shortened overall survival (OS), disease specific survival (DSS), disease free survival (DFS), distant metastasis free survival (DMFS), and regional recurrence free survival (RRFS) on univariate survival analysis. The 5-year OS, DSS, and DFS were 95%, 97%, and 90% respectively for the group without ENE, and 64%, 71%, and 65% respectively for the group with ENE. On Multivariate survival analysis, the presence of ENE was an independent adverse prognostic factor for OS, DSS, and DFS. Additionally, major ENE defined as a vertical extent of ≥4 mm or irregular soft tissue deposit independently predicted shortened OS, DSS, and RFS. In conclusion, the presence of ENE, in particular major ENE, is an independent prognostic factor in HPV-positive OPSCC. Therefore, we propose to document the presence and extent of ENE for these tumors. Consideration may be given for AJCC 9th edition to include ENE into pN stage of HPV-positive OPSCC.

Primary mesenchymal tumors of the thyroid gland are extremely rare, with only case reports and small case series documented in the English literature, many of which were published prior to the era of molecular pathology. In the current study, we aim to present a contemporary multi-centric cohort of thyroid mesenchymal tumors. Nineteen primary thyroid mesenchymal tumors were collected from three tertiary centers. Their clinicopathologic features, immunoprofile, molecular alterations, and outcome were described. Eight cases were classified as benign or intermediate with solitary fibrous tumor being the most common histotype (n = 3). The remaining 11 cases were malignant, including three angiosarcomas, one epithelioid hemangioendothelioma, one adamantinoma-like Ewing sarcoma, one biphasic synovial sarcoma, one malignant melanocytic peripheral nerve sheath tumor (melanotic schwannoma), one myxofibrosarcoma, and two undifferentiated pleomorphic/spindle sarcomas (one of which was radiation-induced). Six tumors showed characteristic diagnostic translocations. We herein also described the first case of thyroid malignant perivascular epithelioid cell tumor (PEComa) with RBM10-TFE3 fusion in a 35-year-old female patient. Thyroid mesenchymal tumors, benign or malignant, are rare with a broad spectrum of possible diagnoses. A comprehensive examination to include histology, immunohistochemistry, and molecular testing is essential for the correct diagnosis and to distinguish them from anaplastic thyroid carcinoma. PEComa may occur as a primary tumor of the thyroid gland, expanding the histologic spectrum of thyroid mesenchymal tumors.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid cancer with most patients dying of their disease within a few months. Only a very small percentage of long-term survivors (LTS) are alive for 2 year or longer. In this retrospective case-control study, we provided a comprehensive comparison between 46 ATC LTSs and 75 ATC control patients who suffered disease-specific mortality within 2 years, aiming to identify factors that may be associated with prolonged survival in ATC. METHODS: A comprehensive clinicopathologic and molecular comparison was performed between 46 ATC LTSs and 75 ATC control patients. Peripheral neutrophil count and neutrophil-to-lymphocyte ratio (NLR) were recorded. The composition of the tumor microenvironment was compared using immunohistochemistry. RESULTS: Compared with ATC control patients, ATC LTSs were characterized by 1) higher frequency of (primary) resection as well as clinicopathologic parameters attributed to resectability; 2) lower rate of concurrent RAS/BRAF and TERT promoter mutations; 3) lower peripheral neutrophil count and NLR; and 4) lower number of tumor-infiltrating neutrophils/myeloid-derived suppressor cells (MDSC). The survival benefits of low peripheral neutrophil counts and low NLR persisted even when controlling for distant metastasis status at presentation. CONCLUSION: In addition to traditional beneficial prognostic factors, e.g., surgical resection, factors attributed to resectability, and absence of co-existing RAS/BRAF and TERT promoter mutations, we herein show that tumor-infiltrating and circulating neutrophils/MDSC are adverse prognostic factors in ATC.

Background: Anaplastic thyroid carcinoma (ATC) is nearly always fatal. Large studies on ATC are exceedingly rare. We aimed to study the clinical, genotypic, and histologic characteristics of ATC in the largest retrospective cohort of ATC to date. Methods: Three hundred sixty patients with ATC from two tertiary centers were studied. Molecular testing was performed in 126 cases including 107 using next-generation sequencing. Results: The median patients' age was 68 years. Differentiated thyroid carcinoma (DTC) was present in 208 cases (58%), the most common being papillary carcinoma (n = 150). The 1-, 2-, 3-, and 5-year overall survival (OS) was 36%, 17%, 13%, and 11%, respectively. On univariate analysis, age, resectability, chemotherapy, radiotherapy, margin status, encapsulation, gross residual disease, gross extrathyroidal extension, percentage, and size of ATC in the primary tumor predicted OS (p

Polymorphous adenocarcinoma (PAC) shows histologic diversity with streaming and targetoid features whereas cribriform adenocarcinoma of salivary gland (CASG) demonstrates predominantly cribriform and solid patterns with glomeruloid structures and optically clear nuclei. Opinions diverge on whether CASG represents a separate entity or a variant of PAC. We aimed to assess the level of agreement among 25 expert Head and Neck pathologists in classifying these tumors. Digital slides of 48 cases were reviewed and classified as: PAC, CASG, tumors with ≥50% of papillary architecture (PAP), and tumors with indeterminate features (IND). The consensus diagnoses were correlated with a previously reported molecular alteration. The consensus diagnoses were PAC in 18/48, CASG in16/48, PAP in 3/48, and IND in 11/48. There was a fair interobserver agreement in classifying the tumors (κ= 0.370). The full consensus was achieved in 3 (6%) cases, all of which were classified as PAC. A moderate agreement was reached for PAC (κ= 0.504) and PAP (κ= 0.561), and a fair agreement was reached for CASG (κ= 0.390). IND had only slight diagnostic concordance (κ= 0.091). PAC predominantly harbored PRKD1 hotspot mutation, whereas CASG was associated with fusion involving PRKD1, PRKD2, or PRKD3. However, such molecular events were not exclusive as 7% of PAC had fusion and 13% of CASG had mutation. In conclusion, a fair to moderate interobserver agreement can be achieved in classifying PAC and CASG. However, a subset (23%) showed indeterminate features and was difficult to place along the morphologic spectrum of PAC/CASG among expert pathologists. This may explain the controversy in classifying these tumors.

High-grade endometrial stromal sarcomas (HGESSs) are more aggressive and have higher rates of resistance to endocrine therapy than low-grade endometrial stromal sarcomas (LGESSs). The pathogenesis of hormonal resistance in these lesions has yet to be defined. Here we sought to histologically and genetically characterize 3 LGESSs and their recurrences that underwent histologic high-grade transformation following endocrine therapy. For this, DNA from primary tumors and select subsequent recurrences were subject to massively parallel sequencing targeting 468 cancer-related genes. Somatic mutation analyses were performed using validated bioinformatics methods. In addition, RNA from each case was evaluated for the presence of gene fusions using targeted RNA-sequencing. All patients initially presented with LGESS, developed HGESS recurrences, and received at least 2 lines of hormonal suppressive therapy. Gene fusions classically described as associated with LGESS were identified in all 3 cases, including JAZF1-PHF1, EPC1-PHF1 and JAZF1-SUZ12 fusions for Cases 1, 2 and 3, respectively. Targeted sequencing analysis revealed that none of the primary LGESS, however the HGESS recurrences of Cases 1 and 3, and the LGESS and HGESS recurrences of Case 2 post endocrine treatment harbored ESR1 p.Y537S hotspot mutations. These ESR1 ligand-binding domain mutations have been found as a mechanism of acquired endocrine resistance in breast cancer. Also, a reduction in estrogen receptor (ER) expression was observed in recurrences. Our findings suggest that the ESR1 p.Y537S hotspot mutation in LGESS with histologic high-grade transformation may be associated with endocrine resistance in these lesions. Furthermore, our data suggest that genetic analyses may be performed in recurrent LGESS following hormonal therapy, development of high-grade morphology, and/or altered/diminished ER expression.

Salivary duct carcinoma (SDC) is an aggressive salivary gland malignancy with poor survival. Approximately 30% SDC harbor HER2 amplification and response to trastuzumab has been reported. However, a systematic approach for HER2 status assessment in this tumor type has not been established. A total of 67 tumor samples were evaluated for HER2 protein overexpression or ERBB2 gene amplification using at least 2 methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and/or targeted exome next-generation sequencing (NGS). NGS assessed ERBB2 copy number fold change (FC) and total copy number (TCN). HER2 status was first determined by IHC/FISH according to the 2018 ASCO/CAP breast cancer guidelines. FISH results, the "gold standard", were compared with the NGS results. All (15/15) IHC positive, 35% (6/17) equivocal, and no (0/19) IHC negative SDC were HER2 amplified by FISH. HER2 FISH signal/cell showed a good correlation with FC (Spearman correlation: 0.708, R

Secretory carcinoma of the thyroid gland is histologically and genetically similar to its mammary and salivary gland counterparts. Unlike differentiated thyroid carcinomas of follicular cell origin, thyroid SC is not a thyroglobulin-producing tumor and would not be amenable to radioactive iodine therapy. Instead, these carcinomas may respond to targeted therapy with TRK inhibitors, which further emphasizes the importance of their recognition among morphologically similar thyroid entities. Based on eleven cases reported to date, most primary thyroid SC tend to present as locally advanced malignancies and are characterized by frequent recurrences and long-term survival. High-grade histologic features, increased mitotic count and necrosis have been described but their impact on clinical course and outcome remains unclear. We hereby report the case of a primary SC with high-grade features arising in the thyroid of a 49-year-old man, who was treated with Larotrectinib for his second recurrence. The patient achieved a durable response that lasted for 18 months but then he continued to progress and died of disease 181 months after the diagnosis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12105-021-01386-6.

Molecular characterization of adrenocortical carcinomas (ACC) by The Cancer Genome Atlas (TCGA) has highlighted a high prevalence of TERT alterations, which are associated with disease progression. Herein, 78 ACC were profiled using a combination of next generation sequencing (n = 76) and FISH (n = 9) to assess for TERT alterations. This data was combined with TCGA dataset (n = 91). A subset of borderline adrenocortical tumors (n = 5) and adrenocortical adenomas (n = 7) were also evaluated. The most common alteration involving the TERT gene involved gains/amplifications, seen in 22.2% (37/167) of cases. In contrast, "hotspot" promoter mutations (C T promoter mutation at position -124, 7/167 cases, 4.2%) and promoter rearrangements (2/165, 1.2%) were rare. Recurrent co-alterations included 22q copy number losses seen in 24% (9/38) of cases. Although no significant differences were identified in cases with and without TERT alterations pertaining to age at presentation, tumor size, weight, laterality, mitotic index and Ki67 labeling, cases with TERT alterations showed worse outcomes. Metastatic behavior was seen in 70% (28/40) of cases with TERT alterations compared to 51.2% (65/127, p = 0.04) of cases that lacked these alterations. Two (of 5) borderline tumors showed amplifications and no TERT alterations were identified in 7 adenomas. In the borderline group, 0 (of 4) patients with available follow up had adverse outcomes. We found that TERT alterations in ACC predominantly involve gene amplifications, with a smaller subset harboring "hotspot" promoter mutations and rearrangements, and 70% of TERT-altered tumors are associated with metastases. Prospective studies are needed to validate the prognostic impact of these findings.

PURPOSE Telomerase reverse transcriptase ( TERT) promoter mutations are prognostic in many cancers and have been observed in human papillomavirus (HPV)–negative head and neck squamous cell carcinomas (HNSCCs). However, the role of TERT promoter mutations in HPV-negative HNSCCs remains poorly understood in these cancers, which have increased risk for locoregional failure (LRF). PATIENTS AND METHODS We retrospectively identified patients who were diagnosed with HNSCC between July 1, 2004, and October 12, 2017, at Memorial Sloan Kettering Cancer Center and whose tumors underwent next-generation sequencing using the MSK-IMPACT panel. Patients with HPV-positive oropharyngeal squamous cell carcinoma (SCC) were excluded. Cumulative incidence of LRF, patterns of failure, and overall survival were measured. RESULTS We identified 117 patients with SCC of the oral cavity (OSCC), larynx, hypopharynx, or HPV-negative oropharynx whose tumors underwent next-generation sequencing. Sequencing was performed on 95 tumors that were obtained after recurrence and 22 that were obtained before recurrence. TERT promoter mutations were enriched in OSCC compared with laryngopharyngeal cancers (81.1% v 7.0%; P < .001), which was the largest genetic difference between these anatomic disease subsites. TERT promoter mutations were associated with LRF in OSCCs (Gray's test, P < .001) and in the overall cohort (Gray's test, P < .001). On multivariate analysis, TERT promoter mutations were associated with an increased risk for LRF (subdistribution hazard ratio, 2.82; 95% CI, 1.47 to 5.42; P = .0019), independent of oral cavity primary site and TP53 mutation status. CONCLUSION TERT promoter status is associated with the cumulative incidence of LRF and patterns of failure. TERT promoter mutations may define a subset of OSCCs with unique pathogenesis that is associated with an increased risk of LRF. Validation in prospective cohorts is warranted.