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Self-medication of pain with alcohol is prevalent, and expectancies for alcohol analgesia (EAA) likely influence pain relief and alcohol consumption. Hazardous alcohol use has been associated with greater delay discounting rates; however, little is known about the relationship between delay discounting and EAA. Therefore, the present study examined sex differences in associations between delay discounting and EAA. Healthy drinkers without chronic pain (N = 53) completed measures of EAA, alcohol use, and alcohol outcome expectancies. A five-trial adjusting-delay discounting task (DDT) for monetary outcomes was also administered. Regression analyses revealed that sex moderated the relationship between delay discounting and EAA. Steeper delay discounting rates were associated with weaker EAA among men when adjusting for average alcohol consumption. Among women, nonsignificant associations between delay discounting rates and EAA were observed. These findings provide initial evidence of sex differences in associations between delay discounting and EAA. The directionality of these associations was unexpected and may have implications for patterns of self-medication with alcohol. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Figure S1 shows a box and whisker plot visualizing RG Ratios stratified by pathological category.

Thyroid carcinomas with mutations involving PI3K/AKT pathway

Figure S1: Nonspecific VE1 immunohistochemical staining in normal tissue Table S1: VE1 immunohistochemistry results in colorectal carcinoma using the Bond method according to BRAF mutation status by sequence analysis Table S2: VE1 immunohistochemistry results in colorectal carcinoma using the Ventana method according to BRAF mutation status by sequence analysis Table S3: Additional molecular analyses of colorectal carcinoma with BRAF wild-type by sequencing and moderate to strong cytoplasmic staining in {greater than or equal to}20% by VE1 immunohistochemistry using the Bond method

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PDF file - 161K, Figure 1: Schematic diagrams of the imaging systems used in this study. Supplementary Figure 2: Multimodal imaging at the floor-of-mouth in a 73-year old female patient.

PDF file - 30K, Table 1: Criteria used by the surgeon to classify measurement sites Supplementary Table 2: Scoring criteria for IHC stained sites Supplementary Table 3: Distribution of measurement sites by anatomical location Supplementary Table 4: Distribution of measurement sites by clinical impression and pathology diagnosis

Patients with oral potentially malignant disorders (OPMD) must undergo regular clinical surveillance to ensure that any progression to malignancy is detected promptly. Autofluorescence imaging (AFI) is an optical modality that can assist clinicians in detecting early cancers and high-grade dysplasia. Patients with OPMD undergoing surveillance for the development of oral cancer were examined using AFI at successive clinic visits. Autofluorescence images acquired at 133 clinical visits from sites in 15 patients who met inclusion criteria were analyzed quantitatively using an algorithm to calculate the red-to-green pixel intensity (RG ratio). A quantitative AFI threshold for high risk of progression was defined based on the RG ratio and was compared with expert clinical impression and with histopathology when available. Patients were divided into two groups based on their endpoint: surveillance (n = 6) or surgery (n = 9). In the surveillance group, 0 of 6 (0%) of patients were clinically identified as high risk for progression prior to the study endpoint, whereas 1 of 6 (17%) of patients were deemed at high risk for progression based on AFI during the same time period. In the surgery group, 9 of 9 (100%) of patients were clinically identified as high risk prior to the study endpoint, whereas 8 of 9 (89%) of patients were at high risk for progression based on AFI during the same time period. AFI results tracked over time were comparable with expert clinical impression in these patient groups. AFI has the potential to aid clinicians in noninvasively monitoring oral precancer and evaluating OPMDs that require increased surveillance.

Explanation about how RG Ratio thresholds were found from previously published data.

Next-generation sequencing (NGS) for molecular diagnostics allows simultaneous testing of activating oncogenes and tumor suppressor mutations in multiple signal pathways. Extended mutational profiling of advanced thyroid cancers may enhance considerations for targeted therapies. We analyzed clinically derived molecular profiling of 216 patients with advanced thyroid carcinoma using NGS (Ion Torrent Personal Genome Machine) from April 2012 to February 2014. We examined substitutions and small indels in 46 or 50 cancer-related genes using Ampliseq Cancer Hotspot panel in respect to tumor diagnosis and clinical correlations.Mutations were common in advanced thyroid carcinomas 154 (71%) predominately in targetable MAPK pathway (146/216, 68%), and several PI3K/AKT pathway (8, 4%; six as comutations). BRAF V600E mutation associated with papillary (94/139, 68%), poorly differentiated (4/39, 10%), and anaplastic (3/12, 25%) carcinomas. NRAS mutations occurred in follicular (5/12, 42%) and poorly differentiated thyroid carcinoma (12/39, 31%). Tumor suppressor mutations (16, 7%) occurred predominantly in TP53 in Hurthle cell (2/5, 40%, the only mutation), in anaplastic (3/12, 25%) and poorly differentiated thyroid carcinoma (4/39, 10%) some as comutations and in papillary thyroid carcinoma (5/139, 4%) always a comutation. Kaplan–Meier analysis of patients with poorly differentiated thyroid carcinoma containing activating mutations who received targeted therapeutics showed improved survival compared to similarly treated patients without mutations in targetable pathways (P = 0.02). In conclusion, MAPK pathway is the predominant target for therapy in advance thyroid carcinomas; adding NGS enables the identification of comutations associated with resistance (PI3K/AKT). Within poorly differentiated thyroid carcinoma, the molecular profile may hold prognostic value in the era of targeted therapy. Mol Cancer Ther; 17(7); 1575–84. ©2018 AACR.

Perspectives on this Article from Objective Detection and Delineation of Oral Neoplasia Using Autofluorescence Imaging

Purpose: c-Src inhibition in cancer cells leads to an abrogation of invasion but a variable effect on apoptosis. The pathways downstream of c-Src promoting survival are not well characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal, we sought to characterize the mechanisms of resistance to c-Src inhibition.Experimental Design: c-Src was inhibited in a panel of oral cancer cell lines and subsequent survival and signaling measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and an in vitro kinase assay. Cytotoxicity was measured and the Chou–Talalay combination index calculated. An orthotopic model of oral cancer was used to assess the effects of c-Met and c-Src inhibitors.Results: Inhibition of c-Src resulted in c-Met inhibition in sensitive cells lines, but not in resistant cell lines. Isolated c-Met was a c-Src substrate in both sensitive and resistant cells, but there was no interaction of c-Src and c-Met in intact resistant cells. To examine the biological consequences of this mechanism, we demonstrated synergistic cytotoxicity, enhanced apoptosis, and decreased tumor size with the combination of c-Src and c-Met inhibitors.Conclusions: Sustained c-Met activation can mediate resistance to c-Src inhibition. These data suggest that the differences between c-Met and c-Src signaling in sensitive and resistant cells are due to distinct factors promoting or inhibiting interactions, respectively, rather than to intrinsic structural changes in c-Src or c-Met. The synergistic cytotoxic effects of c-Src and c-Met inhibition may be important for the treatment of head and neck cancers. Clin Cancer Res; 17(3); 514–24. ©2010 AACR.

Purpose: Salivary duct carcinoma overexpresses epidermal growth factor receptor (EGFR) and HER-2, although the underlying mechanisms remain undefined. Because of the potential utilization of these markers as treatment targets, we evaluated protein and gene status by several techniques to determine complementary value.Experimental Design: A tissue microarray of 66 salivary duct carcinomas was used for immunohistochemical analysis of HER-2 and EGFR expression (semiquantitatively evaluated into a three-tiered system), and fluorescence in situ hybridization for gene copy number, and chromosomes 7 and 17 ploidy status. Sequencing of exons 18, 19, and 21 of the EGFR gene for mutations was carried out.Result: For EGFR, 46 (69.7%) of the 66 tumors showed some form of EGFR expression (17 at 3+, 17 at 2+, 12 at 1+) but none gene amplification. Five (9.4%) of 53 tumors showed mutations in exon 18 (n = 3) and exon 19 (n = 2). Polysomy of chromosome 7 (average >2.5 copies/cell) was detected in 15 (25.0%) of 60 tumors (6 at 3+, 5 at 2+, 2 at 1+, 2 at 0+ expression) and correlated with poor 3-year survival (P = 0.015). For HER-2, 17 (25.8%) of 66 tumors expressed HER-2 (10 at 3+, 3 at 2+, 4 at 1+). Eight tumors showed HER-2 gene amplification (6 at 3+, 1 at 1+, 1 at 0+ protein expression). Chromosome 17 polysomy was found in 8 (15.7%) of 51 tumors; two had HER-2 expression (3+, 1+).Conclusion: Our study shows that salivary duct carcinomas (a) harbor EGFR gene mutations in a subset of tumors that may guide therapy, (b) pursue an aggressive clinical course in cases with chromosome 7 polysomy and high EGFR expression, and (c) with HER-2 gene amplification and protein high expression, may be selected for targeted therapy. Clin Cancer Res; 16(8); 2266–74. ©2010 AACR.

Supplementary Data from Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively).Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

Supplemental data

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC.Experimental Design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC.Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2–M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas.Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643–50. ©2016 AACR.

Supplementary Figures S1-S8.

Purpose: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes.Experimental Design: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34).Results: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years.Conclusions: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. Clin Cancer Res; 17(7); 2035–43. ©2011 AACR.

Supplementary Figures S1-S3; Supplementary Tables S1-S4.

Supplemental methods Supplementary Figure 1. Total and phospho-PTK2/FAK is overexpressed in selected radioresistant HNSCC cells compared to radiosensitive cell lines via immunoblot. Supplementary Figure 2. A.) Inhibition of PTK2/FAK expression via stable expression of shRNA to PTK2/FAK in HN31 HNSCC cells. B.) Stable inhibition of PTK2/FAK leads to radiosensitization in vitro. C & D.) The combination of PF562271 and radiation leads to C.) increased γ-H2AX expression and D.) higher levels of G2/M arrest and sub-G1 staining in Fadu HNSCC cells. Supplementary Figure 3. A & B.) PTK2/FAK is highly expressed in a large public database of HNSCC cell lines (www.oncomine.com) (A.) and tumor specimens (TCGA) (B.) compared to other tumor types. Supplementary Table 1. HNSCC cell lines included in RPPA. Supplementary Table 2. Tumor characteristics of the institutional cohorts. Supplementary Table 3. Univariate and multivariate analysis of PTK2/FAK copy number and disease free survival (DFS) in the two institutional cohorts and the Head and Neck Cancer TCGA cohort. NA - not included in the model. HR - Hazard ratio. 95% CI- 95% Confidence Interval. Supplementary Table 4. Tumor characteristics of the HNSCC TCGA cohort.

BackgroundNeoadjuvant checkpoint inhibition (CPI) has recently demonstrated impressive outcomes in patients with stage 3 cutaneous melanoma. However, the safety, efficacy, and outcome of neoadjuvant CPI in patients with mucosal melanoma (MM) are not well studied as MM is a rare melanoma subtype. CPI such as combination nivolumab and ipilimumab achieves response rates of 37-43% in unresectable or metastatic MM but there is limited data regarding the efficacy of these agents in the preoperative setting. We hypothesize that neoadjuvant CPI is a safe and feasible approach for patients with resectable MM.MethodUnder an institutionally approved protocol, we identified adult MM patients with resectable disease who received neoadjuvant anti-PD1 +/- anti-CTLA4 between 2015 to 2019 at our institution. Clinical information include age, gender, presence of nodal involvement or satellitosis, functional status, pre-treatment LDH, tumor mutation status, and treatment data was collected. Outcomes include event free survival (EFS), overall survival (OS), objective response rate (ORR), pathologic response rate (PRR), and grade ≥3 toxicities.ResultsWe identified 36 patients. Median age was 62; 58% were female. Seventy-eight percent of patients received anti-PD1 + anti-CTLA4. Node positive disease or satellite lesions was present at the time of treatment initiation in 47% of patients. Primary sites of disease were anorectal (53%), urogenital (25%), head and neck (17%), and esophageal (6%). A minority of patients did not undergo surgery due to complete response (n=3, 8%) and disease progression (n=6, 17%), respectively. With a median follow up of 37.9 months, the median EFS was 9.2 months with 3-year EFS rate of 29%. Median OS had not been reached and 3-year OS rate was 55%. ORR was 47% and PRR was 35%. EFS was significantly higher for patients with objective response and for patients with pathologic response. OS was significantly higher for patients with pathologic response. Grade 3 toxicities were reported in 39% of patients.ConclusionNeoadjuvant CPI for resectable MM is a feasible approach with signs of efficacy and an acceptable safety profile. As there is currently no standard approach for resectable MM, this study supports further investigations using neoadjuvant therapy for these patients.

BackgroundDespite the use of aggressive multimodality treatment, most anaplastic thyroid carcinoma (ATC) patients die within a year of diagnosis. Although the combination of BRAF and MEK inhibitors has recently been approved for use in BRAF-mutated ATC, they remain effective in a minority of patients who are likely to develop drug resistance. There remains a critical clinical need for effective systemic agents for ATC with a reasonable toxicity profile to allow for rapid translational development.Material and MethodsTwelve human thyroid cancer cell lines with comprehensive genomic characterization were used in a high-throughput screening (HTS) of 257 compounds to select agents with maximal growth inhibition. Cell proliferation, colony formation, orthotopic thyroid models, and patient-derived xenograft (PDX) models were used to validate the selected agents.ResultsSeventeen compounds were effective, and docetaxel, LBH-589, and pralatrexate were selected for additional in vitro and in vivo analysis as they have been previously approved by the US Food and Drug Administration for other cancers. Significant tumor growth inhibition (TGI) was detected in all tested models treated with LBH-589; pralatrexate demonstrated significant TGI in the orthotopic papillary thyroid carcinoma model and 2 PDX models; and docetaxel demonstrated significant TGI only in the context of mutant TP53.ConclusionsHTS identified classes of systemic agents that demonstrate preferential effectiveness against aggressive thyroid cancers, particularly those with mutant TP53. Preclinical validation in both orthotopic and PDX models, which are accurate in vivo models mimicking tumor microenvironment, may support initiation of early-phase clinical trials in non-BRAF mutated or refractory to BRAF/MEK inhibition ATC.

Optical imaging studies of oral premalignant lesions have shown that optical markers, including loss of autofluorescence and altered morphology of epithelial cell nuclei, are predictive of high-grade pathology. While these optical markers are consistently positive in lesions with moderate/severe dysplasia or cancer, they are positive only in a subset of lesions with mild dysplasia. This study compared the gene expression profiles of lesions with mild dysplasia (stratified by optical marker status) to lesions with severe dysplasia and without dysplasia.Forty oral lesions imaged in patients undergoing oral surgery were analyzed: nine without dysplasia, nine with severe dysplasia, and 22 with mild dysplasia. Samples were submitted for high throughput gene expression analysis.The analysis revealed 116 genes differentially expressed among sites without dysplasia and sites with severe dysplasia; 50 were correlated with an optical marker quantifying altered nuclear morphology. Ten of 11 sites with mild dysplasia and positive optical markers (91%) had gene expression similar to sites with severe dysplasia. Nine of 11 sites with mild dysplasia and negative optical markers (82%) had similar gene expression as sites without dysplasia.This study suggests that optical imaging may help identify patients with mild dysplasia who require more intensive clinical follow-up. If validated, this would represent a significant advance in patient care for patients with oral premalignant lesions.

Bone and soft tissue tumors of the head and neck are relatively uncommon tumors that often represent a diagnostic challenge because of the wide range of entities that must be considered in the differential diagnosis. Over the past few years, classification of bone and soft tissue tumors has evolved primarily because of substantial contributions from molecular genetics, with the identification of new markers that are increasingly used to complement histopathologic findings in the routine diagnostic workup. This review focuses on the recently described mesenchymal tumors that preferentially involve the head and neck region, with a focus on the most relevant novel immunohistochemical and molecular findings, including gene fusions and mutations, that can help in the diagnosis and in the assessment of clinical behavior.

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC), however the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alterations data of patients with different subtypes of thyroid cancer. The resulting model started from stress-responsive DTC cells to inflammatory ATC cells, to mitotic defective ATC cells and extended all the way to mesenchymal ATC cells. In parallel with tumor cell evolution, macrophages shifted from anti-tumor to tumor-promoting states and T cells reprogrammed from cytotoxic to exhausted states. Further, our analysis identified two important milestones: 1) diploid stage, where ATC cells were commonly diploids with non-RAS mutations and inflammatory phenotypes. 2) aneuploid stage, where ATC cells gained aneuploidy with frequent RAS mutations and mesenchymal phenotypes leading to the extreme lethal stage of ATC progression. Citation Format: Lina Lu, Jennifer Rui Wang, Ying C. Henderson, Shanshan Bai, Jie Yang, Min Hu, Yuanqing Yan, Tuan M Tran, Jianzhuo Li, Cheng-Kai Shiau, Rachel Kieser, Xiao Zhao, Jiping Wang, Roza Nurieva, Michelle D. Williams, Maria E. Cabanillas, Ramona Dadu, Naifa Lamki Busaidy, Mark Zafereo, Nicholas Navin, Stephen Y. Lai, Ruli Gao. Anaplastic transformation model in thyroid cancer revealed by single cell lineage and fate 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 3131.

Introduction Anaplastic thyroid cancer (ATC) is rare with a poor prognosis. There are no approved treatments for non-BRAF-mutated ATCs, leading to an unmet need. Phase II studies of single-agent lenvatinib or immunotherapy showed minimal benefit (lenvatinib: overall response rate (ORR) 2.9%, progression free survival (PFS) 2.6 months, overall survival (OS) 3.2 months; spartalizumab: ORR 19%, PFS 1.7 months, OS 5.9 months). Synergism between lenvatinib and pembrolizumab (L/P) has been reported. We sought to assess the efficacy and safety of L/P in ATC. Methods Thisis a retrospective, single-center study of ATC patients treated with L/P, between 1/2015-10/2021. L/P had to be started before first restaging. Those with pembrolizumab added at the time of progression on lenvatinib alone were excluded. The endpoints were ORR and PFS (primary) and OS and safety (secondary). Response evaluation was assessed by a single radiologist using RECIST v1.1. Treatment start date was the date of either lenvatinib or pembrolizumab initiation. Results 18 patients were included: 61% men; median age 64; initial stage: IVA (17%), IVB (11%), IVC (72%); common distant metastases: lung (83%), mediastinum (39%), bone (22%). Previous therapies: surgery (78%), chemoradiation (39%), cytotoxic chemotherapy (11%), palliative neck radiation (17%), BRAF-targeted therapy (6%), and immunotherapy (6%). All patients had mutation testing, and the common included TP53 (67%), PTEN (50%), TERTp (39%), NF1 (28%), RAS (22%), PIK3CA (17%), BRAF-V600E (6%). 11/12 tested tumors had a PDL-1 score >1%. Lenvatinib was started at 14-24 mg daily. With respect to lenvatinib, pembrolizumab was started from -14 to +49 days (median +6 days) at a fixed dose of 200 mg every 3 weeks. ORR was evaluable in 12 patients (outside hospital restaging scans unavailable in 6 patients): 1/12 (8%) confirmed complete response, 7/12 (58%) partial response (3 confirmed), 2/12 (17%) stable disease, and 2/12 (17%) progressive disease. The median PFS was 4.1 months (95% CI, 2.2 to NA), with treatment durations ranging from 0.5-33.5 months. The median OS of the 18 patients was 6.2 months (95% CI, 4.6 to NA). Respective 12-month PFS and OS rates were 25% and 22%. Grade III/IV toxicities were fatigue (22%); anorexia, weakness, arthralgia, dry mouth, pneumonitis (6%, each). AEs leading to death were pulmonary hemorrhage (17%), gastrointestinal bleed, pulmonary embolism (6%, each). Lenvatinib dose reduction, interruption, and discontinuation rates were 28%, 6%, and 28%, respectively. Pembrolizumab dose interruption and discontinuation rates were 6% and 11%, respectively. Conclusions The combination L/P had an ORR of 66%. Although most responses were short-lived, a quarter of patients benefited for more than 1 year. The ORR, PFS, and OS were better than lenvatinib alone. The toxicity profile was as expected. Our results represent the largest single-institution experience thus far with L/P in ATC and warrant further investigation (NCT04171622). Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Signet Ring Cell (SRC)/Histiocytoid carcinoma of the eyelid is a rare neoplasm that shares histological and immunohistochemical similarities with diffuse gastric cancer and breast lobular carcinoma. The CDH1 gene, which encodes the E-cadherin protein, is the best known gene associated with these tumors. The structural and functional integrity of E-cadherin is regulated by interconnecting molecular pathways which might participate in the development of this disease. Hence, we analyzed the protein expression in key genes in E-cadherin-related pathways associated with primary SRC/Histiocytoid carcinoma of the eyelid. SRC/Histiocytoid carcinoma diagnosed in the eyelid/orbit at MD Anderson Cancer Center from 1990 to 2016 were evaluated. Clinicopathologic findings were studied to confirm the primary site of origin. Immunohistochemical studies for the expression of E-cadherin, β-catenin, c-Myc, Cyclin D1, Src, and p53 were analyzed. Next generation sequencing for the detection of somatic mutations was performed on each tumor with matched normal tissue, examining 50 cancer-related genes. Four primary SRC/Histiocytoid carcinomas of the eyelid were diagnosed in four male patients aged 40-82 years. Immunohistochemically, two tumors with loss of E-cadherin expression had weak β-catenin and low cytoplasmic staining for Src while the other two cases with intact E-cadherin showed strong β-catenin expression and high cytoplasmic expression for Src. Cyclin D1 was focally positive in three cases. Somatic mutations in CDH1, PIK3CA, and TP53 genes were detected in two cases. Our results suggest an abnormality in the convergence of E-cadherin/β-catenin pathways which may promote tumorigenesis by inducing expression of oncogenes such as Cyclin D1 and C-Myc. Mutations in CDH1, PIK3CA, and TP53 genes could induce E-cadherin dysfunction which takes part in the development and progression of this malignancy.

BRAF-activating mutations are the most frequent driver mutations in papillary thyroid cancer (PTC). Targeted inhibitors such as dabrafenib have been used in advanced BRAF-mutated PTC, however, acquired resistance to the drug is common and little is known about other effectors that may play integral roles in this resistance. In addition, the induction of PTC dedifferentiation into highly aggressive KRAS-driven anaplastic thyroid cancer (ATC) has been reported. We detected a novel RAC1 (P34R) mutation acquired during dabrafenib treatment in a progressive metastatic lesion with ATC phenotype. To identify a potential functional link between this novel mutation and tumor dedifferentiation, we developed a cell line derived from the metastatic lesion and compared its behavior to isogenic cell lines and primary tumor samples. Our data demonstrated that RAC1 mutations induce changes in cell morphology, reorganization of F-actin almost exclusively at the cell cortex, and changes in cell adhesion properties. We also established that RAC1 amplification, with or without mutation, is sufficient to drive cell proliferation and resistance to BRAF inhibition. Further, we identified polyploidy of chromosome 7, which harbors RAC1, in both the metastatic lesion and its derived cell line. Copy number amplification and overexpression of other genes located on this chromosome, such as TWIST1, EGFR, and MET were also detected, which might also lead to dabrafenib resistance. Our study suggests that polyploidy leading to increased expression of specific genes, particularly those located on chromosome 7, should be considered when analyzing aggressive thyroid tumor samples and in further treatments.

Summary We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities., Graphical abstract, Highlights • Cities possess a consistent “core” set of non-human microbes • Urban microbiomes echo important features of cities and city-life • Antimicrobial resistance genes are widespread in cities • Cities contain many novel bacterial and viral species, This systematic, worldwide catalog of urban microbiomes represents a metagenomic atlas important for understanding the ecology, virulence, and antibiotic resistance of city-specific microbial communities.

Patients with oral potentially malignant disorders (OPMD) must undergo regular clinical surveillance to ensure that any progression to malignancy is detected promptly. Autofluorescence imaging (AFI) is an optical modality that can assist clinicians in detecting early cancers and high-grade dysplasia. Patients with OPMD undergoing surveillance for the development of oral cancer were examined using AFI at successive clinic visits. Autofluorescence images acquired at 133 clinical visits from sites in 15 patients who met inclusion criteria were analyzed quantitatively using an algorithm to calculate the red-to-green pixel intensity (RG ratio). A quantitative AFI threshold for high risk of progression was defined based on the RG ratio and was compared with expert clinical impression and with histopathology when available. Patients were divided into two groups based on their endpoint: surveillance (n = 6) or surgery (n = 9). In the surveillance group, 0 of 6 (0%) of patients were clinically identified as high risk for progression prior to the study endpoint, whereas 1 of 6 (17%) of patients were deemed at high risk for progression based on AFI during the same time period. In the surgery group, 9 of 9 (100%) of patients were clinically identified as high risk prior to the study endpoint, whereas 8 of 9 (89%) of patients were at high risk for progression based on AFI during the same time period. AFI results tracked over time were comparable with expert clinical impression in these patient groups. AFI has the potential to aid clinicians in noninvasively monitoring oral precancer and evaluating OPMDs that require increased surveillance.

Background Multimodal optical imaging, incorporating reflectance and fluorescence modalities, is a promising tool to detect oral premalignant lesions in real‐time. Methods Images were acquired from 171 sites in 66 patient visits for clinical evaluation of oral lesions. An automated algorithm was used to classify lesions as high‐ or low‐risk for neoplasia. Biopsies were acquired at clinically indicated sites and those classified as high‐risk by imaging, at the surgeon's discretion. Results Twenty sites were biopsied based on clinical examination or imaging. Of these, 12 were indicated clinically and by imaging; 58% were moderate dysplasia or worse. Four biopsies were indicated by imaging evaluation only; 75% were moderate dysplasia or worse. Finally, four biopsies were indicated by clinical evaluation only; 75% were moderate dysplasia or worse. Conclusion Multimodal imaging identified more cases of high‐grade dysplasia than clinical evaluation, and can improve detection of high grade precancer in patients with oral lesions.

Background: When achieved, complete surgical resection improves outcomes in anaplastic thyroid carcinoma (ATC). However, most ATC patients present with advanced inoperable disease, often with impending airway obstruction, increased hemorrhage risk, and significant dysphagia. Novel treatment strategies are critically needed to improve disease control and decrease locoregional morbidity. The objective of this study was to determine the feasibility and effectiveness of a neoadjuvant regimen by using dabrafenib with trametinib followed by surgical resection in patients with initially unresectable BRAFV600E-mutated ATC. Methods: Case series of six consecutive patients with BRAFV600E-mutated ATC diagnosed between January 2017 and February 2018. Pathologic confirmation of ATC was obtained before treatment. BRAFV600E status was ascertained via immunohistochemistry or sequencing of circulating tumor DNA. All patients received dabrafenib and trametinib (DT) followed by surgical resection and adjuvant chemoradiation. Three patients also received pembrolizumab. Results: Complete surgical resection was achieved in all patients. Histopathologic analyses of resected specimens showed high pathologic response rates with significantly decreased ATC viability and residual papillary thyroid carcinoma components. Overall survival at six months and one year was 100% and 83%, respectively. Locoregional control rate was 100%. Two patients died of distant metastases without evidence of locoregional disease at 8 and 14 months from diagnosis. The remaining four patients had no evidence of disease at the last follow-up. Conclusions: We report the first series in the literature of BRAFV600E-mutated ATC patients with locoregionally advanced disease treated with DT followed by surgical resection. We demonstrated feasibility of complete resection, decreased need for tracheostomy, high pathologic response rates, and durable locoregional control with symptom amelioration.

Background Alterations in the epidermal growth factor receptor and PI3K pathways in head and neck squamous cell carcinomas (HNSCCs) are frequent events that promote tumor progression. Ectopic expression of the epidermal growth factor receptor–targeting microRNA (miR), miR-27a* (miR-27a-5p), inhibits tumor growth. We sought to identify mechanisms mediating repression of miR-27a* in HNSCC, which have not been previously identified. Methods We quantified miR-27a* in 47 oral cavity squamous cell carcinoma patient samples along with analysis of miR-27a* in 73 oropharyngeal and 66 human papillomavirus–positive (HPV+) samples from The Cancer Genome Atlas. In vivo and in vitro TP53 models engineered to express mutant TP53, along with promoter analysis using chromatin immunoprecipitation and luciferase assays, were used to identify the role of TP53 and TP63 in miR-27a* transcription. An HNSCC cell line engineered to conditionally express miR-27a* was used in vitro to determine effects of miR-27a* on target genes and tumor cells. Results miR-27a* expression was repressed in 47 oral cavity tumor samples vs matched normal tissue (mean log2 difference = −0.023, 95% confidence interval = −0.044 to −0.002; two-sided paired t test, P = .03), and low miR-27a* levels were associated with poor survival in HPV+ and oropharyngeal HNSCC samples. Binding of ΔNp63α to the promoter led to an upregulation of miR-27a*. In vitro and in vivo findings showed that mutant TP53 represses the miR-27a* promoter, downregulating miR-27a* levels. ΔNp63α and nucleoporin 62, a protein involved in ΔNP63α transport, were validated as novel targets of miR-27a*. Conclusion Our results characterize a negative feedback loop between TP63 and miR-27a*. Genetic alterations in TP53, a frequent event in HNSCC, disrupt this regulatory loop by repressing miR-27a* expression, promoting tumor survival.

The presence of tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment has been demonstrated to be of prognostic value in various cancers. In this systematic review and meta-analysis, we investigated the prognostic value of TIL in laryngeal squamous cell carcinoma (LSCC). We performed a systematic search in PubMed for publications that investigated the prognostic value of TIL in LSCC. A meta-analysis was performed including all studies assessing the association between TIL counts in hematoxylin-eosin (HE)-stained sections, for CD8+ and/or CD3+/CD4+ TIL and overall survival (OS) or disease-free survival (DFS). The pooled meta-analysis showed a favorable prognostic role for stromal TIL in HE sections for OS (HR 0.57, 95% CI 0.36–0.91, p = 0.02), and for DFS (HR 0.56, 95% CI 0.34–0.94, p = 0.03). High CD8+ TIL were associated with a prolonged OS (HR 0.62, 95% CI 0.4–0.97, p = 0.04) and DFS (HR 0.73, 95% CI 0.34–0.94, p = 0.002). High CD3+/CD4+ TIL demonstrated improved OS (HR 0.32, 95% CI 0.16–0.9, p = 0.03) and DFS (HR 0.23, 95% CI 0.10–0.53, p = 0.0005). This meta-analysis confirmed the favorable prognostic significance of TIL in LSCC. High stromal TIL evaluated in HE sections and intra-tumoral and stromal CD3+, CD4+ and/or CD8+ TIL might predict a better clinical outcome.

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid cancer that requires a rapid diagnosis and treatment to achieve disease control. Gene mutation profiling of circulating cell-free DNA (cfDNA) in peripheral blood may help to facilitate early diagnosis and treatment selection. The relatively rapid turnaround time compared with conventional tumor mutation testing is a major advantage. The objectives of this study were to examine the concordance of ATC-related mutations detected in cfDNA with those detected in the corresponding tumor tissue, and to determine the prognostic significance of cfDNA mutations in ATC patients. Methods: The ATC patients who were diagnosed and treated at The University of Texas MD Anderson Cancer Center between January 2015 and February 2018 and who had cfDNA testing were included in this study. cfDNA was collected by blood draw and was analyzed by next-generation sequencing (NGS) using the Guardant360-73 gene platform. Results: A total of 87 patients were included in the study. The most frequently mutated genes detected in cfDNA were TP53, BRAF, and PIK3CA. In 28 treatment naive ATC patients, the concordance rate of detected mutations in TP53, BRAF(V600E), and PIK3CA between cfDNA and matched tissue NGS was 82.1%, 92.9%, and 92.9%, respectively. Patients with a PIK3CA mutation detected on cfDNA had worse overall survival (OS) (p = 0.03). This association was observed across various treatment modalities, including surgery, cytotoxic chemotherapy, radiation, and BRAF inhibitor (BRAFi) therapy. With regard to treatment, BRAFi therapy significantly improved ATC OS (p = 0.003). Conclusions: cfDNA is a valuable tool to evaluate a tumor's molecular profile in ATC patients. We identified high concordance rates between the gene mutations identified via cfDNA analysis and those identified from the NGS of the corresponding tumor tissue sequencing. Identified mutations in cfDNA can potentially provide timely information to guide treatment selection and evaluate the prognosis in patients with ATC.

Gain-of-function point mutations in the receptor tyrosine kinase RET, a driver oncogene in medullary thyroid carcinoma (MTC), prevent apoptosis through inhibition of ATF4, a critical transcriptional regulator of endoplasmic reticulum stress. However, the critical regulatory mechanisms driving RET-dependent oncogenesis remain elusive, and there is a clinical need to identify a transcriptional RET inhibitor. Here, we found that RET depletion decreased IGFBP2 and VEGFR2 mRNA and protein expression in MTC cells. IGFBP2 knockdown decreased cell survival and migration of MTC cells. In patients, IGFBP2 expression increased in metastatic MTC, and high IGFBP2 associated with poor overall survival. VEGFR2 protein levels were positively associated with RET expression in primary tumors, and VEGF-mediated increased cell viability was RET dependent. The small-molecule ONC201 treatment of MTC cells caused apoptotic cell death, decreased transcription of RET, VEGFR2, IGFBP2, increased mRNA levels of ATF4, and ATF4 target genes including DDIT3, BBC3, DUSP8, MKNK2, KLF9, LZTFL1, and SESN2. Moreover, IGFBP2 depletion increased ONC201-induced cell death. ONC201 inhibited tumor growth at a well-tolerated dose of 120 mg/kg/week administered by oral gavage and decreased MTC xenograft cell proliferation and angiogenesis. The protein levels of RET, IGFBP2, and VEGFR2 were decreased in ONC201-treated xenografts. Our study uncovered a novel ONC201 mechanism of action through regulation of RET and its targets, VEGFR2 and IGFBP2; this mechanism could be translated into the clinic and represent a promising strategy for the treatment of all patients with MTC, including those with TKI-refractory disease and other cancer with RET abnormalities.

Background Targeted kinase inhibitors have been increasingly utilized in the treatment of advanced medullary thyroid cancer (MTC) over the last decade. Recently, highly potent next generation selective RET inhibitors have been clinically validated, and selpercatinib was recently Food and Drug Administration (FDA)‐approved for advanced MTC. The advent of highly selective, potent RET inhibitors is broadening the treatment options for patients with RET‐mutated cancers. Methods We report the first published case of neoadjuvant selpercatinib followed by surgery for a patient with initially unresectable, widely metastatic, RET‐mutated MTC who was treated on a single patient protocol. Results After greater than 50% RECIST response, the patient underwent complete surgical resection followed by selpercatinib resumption. He remains locoregionally disease‐free 21 months after starting therapy with stable metastatic disease (after initial partial response); and calcitonin/CEA continue to decline. Conclusion This novel treatment strategy for locoregionally advanced RET‐mutated MTC warrants further study in clinical trials.

Purpose: In vivo optical imaging technologies like high-resolution microendoscopy (HRME) can image nuclei of the oral epithelium. In principle, automated algorithms can then calculate nuclear features to distinguish neoplastic from benign tissue. However, images frequently contain regions without visible nuclei, due to biological and technical factors, decreasing the data available to and accuracy of image analysis algorithms. Approach: We developed the nuclear density-confidence interval (ND-CI) algorithm to determine if an HRME image contains sufficient nuclei for classification, or if a better image is required. The algorithm uses a convolutional neural network to exclude image regions without visible nuclei. Then the remaining regions are used to estimate a confidence interval (CI) for the number of abnormal nuclei per mm 2 , a feature used by a previously developed algorithm (called the ND algorithm), to classify images as benign or neoplastic. The range of the CI determines whether the ND-CI algorithm can classify an image with confidence, and if so, the predicted category. The ND and ND-CI algorithm were compared by calculating their positive predictive value (PPV) and negative predictive value (NPV) on 82 oral biopsies with histopathologically confirmed diagnoses. Results: After excluding the images that could not be classified with confidence, the ND-CI algorithm had higher PPV (65% versus 59%) and NPV (78% versus 75%) than the ND algorithm. Conclusions: The ND-CI algorithm could improve the real-time classification of HRME images of the oral epithelium by informing the user if an improved image is required for diagnosis.

Recognizing rare but clinically significant toxicity of immunotherapy is critical. Here we describe the first detailed case of severe osteonecrosis of the jaw due to anti-PD-1. A 75-year-old man with metastatic melanoma, with no prior radiation or treatment with bone-targeting agents, experienced jaw pain 1 week after his first dose of nivolumab. Imaging studies were negative, and treatment was resumed after pain was controlled. 4 months later, the patient experienced acute exacerbation of pain and malocclusion of the jaw. Imaging showed bilateral fractures of the angle of mandible with extensive disruption of the normal trabecular architecture, requiring total mandibulectomy. The patient’s metastatic melanoma responded to treatment and remains controlled >20 months after treatment cessation without further therapy.

IMPORTANCE: Anaplastic thyroid carcinoma (ATC) historically has a 4-month median overall survival (OS) from time of diagnosis, with disease-specific mortality approaching 100%. The association between recent major advancements in treatment and OS has yet to be evaluated. OBJECTIVE: To evaluate rates of OS in patients with ATC over the last 2 decades. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study in a single tertiary care institution. Patients with histopathological confirmation of ATC from January 2000 to October 2019 were included and divided into 3 groups according to date of presentation: 2000-2013, 2014-2016, and 2017-2019. MAIN OUTCOMES AND MEASURES: Overall survival compared among different treatment eras and differing therapies, including targeted therapy, immunotherapy, and surgery. RESULTS: Of 479 patients (246 men [51%]; median age, 65.0 [range, 21.1-92.6] years) with ATC evaluated, 52 (11%) were stage IVA, 172 (36%) stage IVB, and 255 (53%) stage IVC at presentation. The median OS of the entire cohort was 0.79 years (9.5 months), ranging from 0.01 to 16.63. The OS at 1 and 2 years was 35% (95% CI, 29%-42%) and 18% (95% CI, 13%-23%) in the 2000-2013 group (n = 227), 47% (95% CI, 36%-56%) and 25% (95% CI, 17%-34%) in the 2014-2016 group (n = 100), and 59% (95% CI, 49%-67%) and 42% (95% CI, 30%-53%) in the 2017-2019 group (n = 152), respectively (P

Acinic cell carcinoma (AcCC) is a morphologically distinctive salivary gland malignancy often associated with chromosome rearrangements leading to overexpression of the NR4A3 transcription factor. However, little is known about how NR4A3 contributes to AcCC biology. Detailed RNA-sequencing of 21 archived AcCC samples revealed fusion reads arising from recurrent t(4, 9), t(9, 12), t(8, 9) or t(2, 4) chromosomal translocations, which positioned highly active enhancers adjacent to the promoter of the NR4A3 gene or the closely related NR4A2 gene, resulting in their aberrant overexpression. Transcriptome analyses revealed several distinct subgroups of AcCC tumors, including a subgroup that overexpressed both NR4A3 and MSANTD3. A poor survival subset of the tumors with high-grade transformation expressed NR4A3 and POMC as well as MYB, an oncogene that is the major driver in a different type of salivary gland tumor, adenoid cystic carcinoma. The combination of NR4A3 and MYB showed cooperativity in regulating a distinct set of genes. In addition, the ligand binding domain of NR4A3 directly bound the Myb DNA binding domain. Transformation assays indicated that, while overexpressed NR4A3 was sufficient to generate transformed colonies, the combination of NR4A3 plus Myb was more potent, leading to anchorage-independent growth and increased cellular invasiveness. The results confirm that NR4A3 and NR4A2 are the main driver genes of AcCC and suggest that concurrent overexpression of NR4A3 and MYB defines a subset of AcCC patients with high-grade transformation that display exceptionally poor outcome.

Standardized pathologic reporting for cancers improves patient care and prognostic determination. However, access in many countries is limited. To address this issue, the International Collaboration on Cancer Reporting (ICCR), a not-for-profit organization, has the mission to develop and disseminate standardized data sets for global use. Within endocrine organs, the parathyroid gland has rarely been included in formal pathologic data sets. Utilizing an expert international panel of eleven members, an evidence-based data set was developed for parathyroid carcinoma and atypical parathyroid neoplasms. This data set consists of sixteen core (required) elements viewed as essential for documentation of these conditions. Characterizing parathyroid carcinomas and atypical neoplasms begins with correlative clinical information, the operative procedure, specimens submitted, and site of the disease. The pathologic features essential to document include parathyroid weight, size, classification, and, when a carcinoma, the tumor grade. Histologic grade of parathyroid carcinoma incorporates other core elements including necrosis, mitotic count, perineural invasion, and lymphovascular invasion. Documenting the extent of disease locally into adjacent organs, regionally, and distally is critical for staging. Pathologic staging is now included as part of the American Joint Committee on Cancer 8th edition and is included in this data set. Ancillary studies should be recorded when performed as noncore elements. Standardized pathologic data sets for endocrine organs including the parathyroid gland are now available through the ICCR website. These essential resources enhance international standardization for documenting these rare tumors for both patient care and future guidelines.