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Papillary thyroid microcarcinoma (PTMC) typically has an indolent course and excellent prognosis. Nonetheless, a subset of PTMC carries a risk of lymph node metastasis (LNM) and local recurrence. PTC from the Middle Eastern population is unique with respect to demographic and clinico-pathological characteristics as compared to other ethnicities of the world. The risk factors of LNM in PTMC patients of Middle Eastern ethnicity have not been fully explored. The present study aims to investigate the influencing factors of LNM in Middle Eastern PTMC patients and its predictive impact on patient’s outcome. A total of 226 confirmed PTMC cases were selected in this retrospective study. The correlation between clinico-pathological, as well as molecular, characteristics and LNM was evaluated. Multivariate analysis was performed by logistic regression and Cox proportional hazards models. Among the 226 patients, the rate of LNM was 43.8% (99/226). Bilaterality, multifocality, gross extrathyroidal extension (ETE), and intermediate-to-high American Thyroid Association (ATA) risk tumors were significantly associated with LNM in PTMC. Multivariate logistic regression analysis showed that bilaterality and gross ETE were independent predictive factors for LNM in PTMC. The recurrence-free survival (RFS) was shorter in PTMC with LNM compared to those without LNM (p = 0.0051) and was significant on multivariate analysis. In conclusion, our study showed that bilaterality and gross ETE were independent influencing factors of LNM in Saudi patients with PTMC. LNM was also associated with shorter RFS. The identification of risk factors for LNM in patients of Middle Eastern ethnicity could help the individualization of clinical management for PTMC patients.

Thyroid cancer is the most frequent endocrine cancer with an increasing incidence rate worldwide and is the second most common malignancy among females in Saudi Arabia. Papillary thyroid cancer (PTC) is the most common subtype. Germline pathogenic variants in the proofreading domain of the POLE and POLD1 genes predispose to several types of cancers. However, the role of pathogenic variants of these two genes in PTC remains unknown. Capture sequencing, Sanger sequencing and immunohistochemistry were performed on 300 PTC cases from the Middle Eastern region. One germline pathogenic variant each of POLE (1/300, 0.33%) and POLD1 (1/300, 0.33%) genes was identified. Low expression of POLD1 was detected in 46.5% (133/286) of cases and was significantly associated with the follicular variant of PTC (P = 0.0006), distant metastasis (P = 0.0033) and stage IV tumours (P = 0.0081). However, no somatic pathogenic variant was detected in POLE gene. Furthermore, low expression of POLE was noted in 61.7% (175/284) of cases with no significant clinicopathological associations. Our study shows that pathogenic variant in the POLE and POLD1 proofreading domain is a cause of PTC and low expression of POLD1 is associated with poor prognostic markers in the Middle Eastern population. Further studies from different geographic populations are needed to determine the frequency and spectrum of proofreading domain pathogenic variants in POLE and POLD1 genes and in PTC from different ethnicities.

BACKGROUND:Data on thyroid surgery in children are scarce.OBJECTIVE:Analyze outcome data on thyroid surgery in a pediatric population.DESIGN:Medical record review.SETTING:Tertiary health care institution.PATIENTS AND METHODS:We collected demographic and clinical data on patients 18 years or younger who had thyroid surgery in the period 2000 to 2014. Descriptive data are presented.MAIN OUTCOME MEASURES:Indications for thyroidectomy, thyroid pathology, complications, length of stay, and radioactive iodine treatment and recurrences.SAMPLE SIZE:103.RESULTS:Of 103 patients who underwent 112 thyroidectomy procedures, 80 (78%) were females and the mean age at operation was 13.2 years. and 17 (16%) were associated with multiple endocrine neoplasia type 2. There was no history of radiation exposure. Eighty-one patients (78%) had fine needle aspiration (FNA) which correlated with the final histopathology in 94% of cases. Sixty-six patients (64%) had malignant cancer (61 papillary), 44 (74.6%) of 59 patients who had neck dissection had lymph node metastasis and 7 (11%) had distant metastases to the lung. Procedures included total thyroidectomy (50%), hemithyroidectomy (17%), completion (31%), and subtotal thyroidectomy (2%). Twenty-three patients (22%) developed hypocalcemia (3 permanent) and 6 (5.8%) had unilateral recurrent laryngeal nerve injury (3 permanent). Patients were followed up for a mean duration of 71.7 months (median 60 months). Of 66 patients with thyroid cancer, 43 (65%) received radioactive iodine, and 10 (15%) had recurrence.CONCLUSION:Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic. Hypocalcemia and recurrent laryngeal nerve injury are significant complications. The recurrence rate in thyroid cancer is 15%.LIMITATIONS:Retrospective.CONFLICT OF INTEREST:None.

Background: Distant metastasis is a rare occurrence in thyroid cancer, and it can be associated with poor prognosis. The genomic repertoires of various solid malignancies have previously been reported but remain underexplored in metastatic papillary thyroid cancer (PTC). Furthermore, whether distant metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. Methods: We performed whole-exome sequencing on 14 matched distant metastases, primary PTC tumors, and normal tissues. Point mutations, copy number alterations, cancer cell fractions, and mutational signatures were defined using the state-of-the-art bioinformatics methods. All likely deleterious variants were validated by orthogonal methods. Results: Genomic differences were observed between primary and distant metastatic deposits, with a median of 62% (range 21-92%) of somatic mutations detected in metastatic tissues, but absent from the corresponding primary tumor sample. Mutations in known driver genes including BRAF, NRAS, and HRAS were shared and preferentially clonal in both sites. However, likely deleterious variants affecting DNA methylation and transcriptional repression signaling genes including SIN3A, RBBP1, and CHD4 were found to be restricted in the metastatic lesions. Moreover, a mutational signature shift was observed between the mutations that are specific or enriched in the metastatic and primary lesions. Conclusions: Primary PTC and distant metastases differ in their range of somatic alterations. Genomic analysis of distant metastases provides an opportunity to identify potentially clinically informative alterations not detected in primary tumors, which might influence decisions for personalized therapy in PTC patients with distant metastasis.

Standard surgery followed by radioactive iodine (131I, RAI) therapy are not curative for 5–20% of papillary thyroid carcinoma (PTC) patients with RAI refractory disease. Early predictors indicating therapeutic response to RAI therapy in PTC are yet to be elucidated. Whole-exome sequencing was performed (at median depth 198x) on 66 RAI-refractory and 92 RAI-avid PTCs with patient-matched germline. RAI-refractory tumors were significantly associated with distinct aggressive clinicopathological features, including positive surgical margins (p = 0.016) and the presence of lymph node metastases at primary diagnosis (p = 0.012); higher nonsilent tumor mutation burden (p = 0.011); TERT promoter (TERTp) mutation (p < 0.0001); and the enrichment of the APOBEC-related single-base substitution (SBS) COSMIC mutational signatures 2 (p = 0.030) and 13 (p < 0.001). Notably, SBS13 (odds ratio [OR] 30.4, 95% confidence intervals [CI] 1.43–647.22) and TERTp mutation (OR 41.3, 95% CI 4.35–391.60) were revealed to be independent predictors of RAI refractoriness in PTC (p = 0.029 and 0.001, respectively). Although SBS13 and TERTp mutations alone highly predicted RAI refractoriness, when combined, they significantly increased the likelihood of predicting RAI refractoriness in PTC. This study highlights the APOBEC SBS13 mutational signature as a novel independent predictor of RAI refractoriness in a distinct subgroup of PTC.

BackgroundPapillary Thyroid Cancer (PTC) is the most common endocrine malignancy, with recurrence rate as high as 30%. A great deal of controversy surrounds the significance of microscopic extrathyroidal extension (m-ETE) as a prognostic factor. The most recent edition (8th) of American Joint Committee on Cancer (AJCC) staging system has removed m-ETE from the definition of pT3, which suggests that m-ETE may lack prognostic impact in PTC patients. Moreover, data about m-ETE prevalence and clinical impact on Middle Eastern PTC remains unknown. We therefore investigate the prevalence of m-ETE and its clinico-pathological correlation and prognostic impact in Middle Eastern PTC. We also compared the AJCC 7th and 8th staging systems and their prognostic performance.MethodsPTCs from 1430 consecutive adult (> 18 years) patients from single tertiary care hospital were included in this study. A retrospective analysis of PTC patients’ survival and recurrence were compared between AJCC 8th and AJCC 7th staging systems using Proportion of Variation Explained (PVE) and Harrell’s C-index.ResultsMedian follow up of the study cohort was 9.3 years. 31.2% (446/1430) of patients had m-ETE. In the overall cohort, m-ETE was associated with multiple adverse features such as older age (p < 0.0001), male sex (p = 0.0245), tall cell variant (p < 0.0001), bilateral tumors (p < 0.0001), multifocality (p < 0.0001), lymphovascular invasion (p < 0.0001), lymph node metastasis (p < 0.0001), distant metastasis (p = 0.0166), tumor recurrence (p < 0.0001), radioactive iodine refractoriness (p < 0.0001), BRAF mutation (p < 0.0001) and reduced recurrence-free survival (RFS; HR = 1.75; 95% CI = 1.30 – 2.35; p < 0.0001) irrespective of tumor size. Of the 611 patients with T3 disease based on AJCC 7th edition, 359 (58.8%) were down-staged in AJCC 8th edition classification. Overall, the prognostic performance of AJCC 8th edition was inferior to AJCC 7th on the basis of lower PVE (3.04% vs. 3.73%) and lower C-index (0.40 vs. 0.48).ConclusionsIn Middle Eastern PTC, m-ETE is significantly associated with compromised survival and acts as an independent predictor of RFS. Given these findings, m-ETE should be included in the thyroid cancer treatment guidelines.

BackgroundProphylactic central lymph node dissection (PCLND) for adult patients with papillary thyroid carcinoma (PTC) is still a matter of debate. Data on incidence, risk and benefits of PCLND in Middle Eastern patients is lacking. Therefore, we aimed to identify the incidence and predictive clinico-pathological and molecular marker of PCLND in adult patients with clinically node negative (cN0) Middle Eastern PTC.MethodsThis retrospective study included 942 adult Middle Eastern patients with cN0 PTC who underwent total thyroidectomy (TT) or TT+PCLND. Clinico-pathological associations of central lymph node metastasis (CLNM) were assessed. Multivariate analysis was performed using logistic regression and Cox proportional hazards model.Results213 patients underwent PCLND and 38.0% (81/213) had positive CLNM. Multivariate analysis demonstrated age ≤55 years (Odds Ratio (OR) = 7.38; 95% Confidence Interval (CI) = 1.59 – 34.31; p = 0.0108), tumor bilaterality (OR = 3.01; 95% CI = 1.01 – 9.21; p = 0.0483), lymphovascular invasion (OR = 2.92; 95% CI = 1.18 – 7.23; p = 0.0206) and BRAF mutation (OR = 3.24; 95% CI = 1.41 – 7.49; p = 0.0058) were independent predictors of CLNM in adult PTC. Furthermore, patients who underwent PCLND showed significant association with improved recurrence-free survival (RFS; p = 0.0379). Multivariate analysis demonstrated that PCLND was an independent predictor of improved recurrence-free survival.ConclusionscN0 Middle Eastern PTC patients treated with PCLND showed a significantly better prognosis. PCLND was effective in improving RFS in Middle Eastern PTC patients and should be encouraged for patients with potential risk factors for CLNM.

Thyroid cancer incidence has increased in recent decades. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Approximately 30% of PTC patients develop recurrence or distant metastasis and tend to have poor prognosis. Therefore, the identification of targetable biomarkers in this subset of patients is of great importance. Accumulating evidence indicates that zinc finger protein 677 (ZNF677), which belongs to the zinc finger protein family, is an important effector during the progression of multiple malignancies. However, its role in Middle Eastern PTC patients has not been fully illustrated. Here, we uncovered the molecular mechanism and the clinical impact of ZNF677 expression in a large cohort of more than 1200 Middle Eastern PTC and 15 metastatic tissues. We demonstrated that ZNF677 is frequently downregulated in primary PTC (13.6%, 168/1235) and showed that complete loss of expression of ZNF677 is significantly associated with aggressive clinico-pathological markers such as extrathyroidal extension (p = 0.0008) and distant metastases (p &lt, 0.0001). We also found a significantly higher incidence of ZNF677 loss in primary tumors with distant metastases (33.3%, p &lt, 0.0001) as well as in distant metastatic tissues (46.7%, p = 0.0002) compared to the overall cohort (13.6%). More importantly, PTC with loss of ZNF677 expression showed significantly lower metastasis-free survival (p = 0.0090). Interestingly, on multivariate logistic regression analysis, ZNF677 loss was an independent predictor of distant metastasis in PTC (Odds ratio = 2.60, 95% Confidence interval = 1.20–5.62, p = 0.0155). In addition, we found a significant association between ZNF677 loss and phospho-AKT expression (p &lt, 0.0001). Our functional molecular results suggest that ZNF677 acts as a tumor suppressor, mediating its effect by inhibiting AKT phosphorylation. Taken together, our results highlight the pivotal role played by ZNF677 during carcinogenesis and metastasis formation in Middle Eastern PTC patients.

Mitogen-activated protein kinase kinase 1 (MAP2K1) is a dual specificity protein kinase that phosphorylates both threonine and tyrosine residues in ERK. MAP2K1 mutations have been identified in several cancers. However, their role in Middle Eastern papillary thyroid cancer (PTC) and colorectal cancer (CRC) is lacking. In this study, we evaluated the prevalence of MAP2K1 mutations in a large cohort of Middle Eastern PTC and CRC using whole-exome and Sanger sequencing technology. In the discovery cohort of 100 PTC and 100 CRC cases (comprising 50 MAPK mutant and 50 MAPK wildtype cases each), we found one MAP2K1 mutation each in PTC and CRC, both of which were MAPK wildtype. We further analyzed 286 PTC and 289 CRC MAPK wildtype cases and found three MAP2K1 mutant PTC cases and two MAP2K1 mutant CRC cases. Thus, the overall prevalence of MAP2K1 mutation in MAPK wildtype cases was 1.1% (4/336) in PTC and 0.9% (3/339) in CRC. Histopathologically, three of the four MAP2K1 mutant PTC cases were follicular variant and all four tumors were unifocal with absence of extra-thyroidal extension. All the three CRC cases harboring MAP2K1 mutation were of older age (> 50 years) and had moderately differentiated stage II/III tumors located in the left colon. In conclusion, this is the first comprehensive report of MAP2K1 somatic mutations prevalence in PTC and CRC from this ethnicity. The mutually exclusive nature of MAP2K1 and MAPK mutations suggests that each of these mutation may function as an initiating mutation driving tumorigenesis through MAPK signaling pathway.

Mitogen-activated protein kinase kinase 1 (MAP2K1) is a dual specificity protein kinase that phosphorylates both threonine and tyrosine residues in ERK. MAP2K1 mutations have been identified in several cancers. However, their role in Middle Eastern papillary thyroid cancer (PTC) and colorectal cancer (CRC) is lacking. In this study, we evaluated the prevalence of MAP2K1 mutations in a large cohort of Middle Eastern PTC and CRC using whole-exome and Sanger sequencing technology. In the discovery cohort of 100 PTC and 100 CRC cases (comprising 50 MAPK mutant and 50 MAPK wildtype cases each), we found one MAP2K1 mutation each in PTC and CRC, both of which were MAPK wildtype. We further analyzed 286 PTC and 289 CRC MAPK wildtype cases and found three MAP2K1 mutant PTC cases and two MAP2K1 mutant CRC cases. Thus, the overall prevalence of MAP2K1 mutation in MAPK wildtype cases was 1.1% (4/336) in PTC and 0.9% (3/339) in CRC. Histopathologically, three of the four MAP2K1 mutant PTC cases were follicular variant and all four tumors were unifocal with absence of extra-thyroidal extension. All the three CRC cases harboring MAP2K1 mutation were of older age (> 50 years) and had moderately differentiated stage II/III tumors located in the left colon. In conclusion, this is the first comprehensive report of MAP2K1 somatic mutations prevalence in PTC and CRC from this ethnicity. The mutually exclusive nature of MAP2K1 and MAPK mutations suggests that each of these mutation may function as an initiating mutation driving tumorigenesis through MAPK signaling pathway.

PD-L1 inhibition is a promising therapeutic target whose efficacy has been demonstrated in several cancers. Immunohistochemistry was performed to assess PD-L1 protein expression in PTC. We further conducted in vitro analysis to investigate the role of PD-L1 in regulating BRAFV600E in PTC cell lines. PD-L1 over-expression was noted in 32.4% (473/1458) of cases and significantly associated with aggressive clinico-pathological parameters. Importantly, PD-L1 was found to be an independent poorer prognostic marker. We also found PD-L1 to be significantly associated with BRAF mutation and patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival compared to patients with BRAF mutation alone. In vitro analysis showed high expression of PD-L1 in BRAF-mutated PTC cell lines compared to a BRAF wild-type cell line. Inhibition of BRAF using vemurafenib induced PD-L1 expression in BRAF-mutated cell lines without affecting cell growth. Knockdown of PD-L1 in BRAF-mutated cell lines significantly decreased the cell growth and induced apoptosis. Our data suggest that PD-L1 might represent a useful prognostic marker in Middle Eastern PTC and PD-L1 inhibition could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, BRAF mutation-positive patients that are unresponsive to standard treatment.

The Kr&uuml, ppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1&alpha, overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1&alpha, in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1&alpha, in PTC cells. We also identified KLF5-binding sites in the HIF-1&alpha, promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

Simple Summary This study was conducted to investigate the prognostic significance of programmed death-ligand 1 (PD-L1) expression in a large cohort of Middle Eastern papillary thyroid carcinoma (PTC) patients and to explore the correlation of PD-L1 and BRAFV600E mutations in PTC tumors and cell lines. We found PD-L1 over-expression in PTC patients and it was significantly associated with aggressive clinico-pathological parameters and BRAF mutation. PTC patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival. In vitro studies showed that BRAF inhibition induces PD-L1 expression in BRAF-mutated PTC cell lines via mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway activation. Silencing of PD-L1 in BRAF-mutated cell lines significantly attenuated cell growth. Our data suggest that PD-L1 could represent a useful prognostic marker for risk stratification in Middle Eastern PTC and that a programmed cell death protein 1 (PD-1)/PD-L1 inhibitor could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, BRAF mutation-positive patients that are unresponsive to standard PTC treatment. Abstract PD-L1 inhibition is a promising therapeutic target whose efficacy has been demonstrated in several cancers. Immunohistochemistry was performed to assess PD-L1 protein expression in PTC. We further conducted in vitro analysis to investigate the role of PD-L1 in regulating BRAFV600E in PTC cell lines. PD-L1 over-expression was noted in 32.4% (473/1458) of cases and significantly associated with aggressive clinico-pathological parameters. Importantly, PD-L1 was found to be an independent poorer prognostic marker. We also found PD-L1 to be significantly associated with BRAF mutation and patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival compared to patients with BRAF mutation alone. In vitro analysis showed high expression of PD-L1 in BRAF-mutated PTC cell lines compared to a BRAF wild-type cell line. Inhibition of BRAF using vemurafenib induced PD-L1 expression in BRAF-mutated cell lines without affecting cell growth. Knockdown of PD-L1 in BRAF-mutated cell lines significantly decreased the cell growth and induced apoptosis. Our data suggest that PD-L1 might represent a useful prognostic marker in Middle Eastern PTC and PD-L1 inhibition could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, BRAF mutation-positive patients that are unresponsive to standard treatment.

Predicting the pattern of recurrence in papillary thyroid cancer (PTC) is necessary to establish optimal surveillance and treatment strategies. We analyzed changes in hazard rate (HR) for tumor recurrence over time in 1201 unselected Middle Eastern PTC patients. The changes in risk were further analyzed according to clinical variables predictive of early (&le, 5 years) and late (&gt, 5 years) recurrence using Cox regression analysis to identify patient populations that remain at risk. Tumor recurrence was noted in 18.4% (221/1201) patients. The annualized hazard of PTC recurrence was highest during the first 5 years (2.8%), peaking between 1 and 2 years (3.7%), with a second smaller peak between 13 and 14 years (3.2%). Patients receiving radioactive iodine (RAI) therapy had lower recurrence hazard compared to those who did not (1.5% vs. 2.7%, p = 0.0001). Importantly, this difference was significant even in intermediate-risk PTC patients (0.7% vs. 2.3%, p = 0.0001). Interestingly, patients aged &ge, 55 years and having lymph node metastasis were at persistent risk for late recurrence. In conclusion, we confirmed the validity of the double-peaked time-varying pattern for recurrence risk in Middle Eastern PTC patients and our findings could help in formulating individualized treatment and surveillance plans.

The cyclooxygenase-2 (COX-2)&ndash, prostaglandin E2 (PGE2) pathway has been implicated in carcinogenesis, with BRAF mutation shown to promote PGE2 synthesis. This study was conducted to evaluate COX-2 expression in a large cohort of Middle Eastern papillary thyroid carcinoma (PTC), and further evaluate the prognostic significance of COX-2 expression in strata of BRAF mutation status. BRAF mutation analysis was performed using Sanger sequencing, and COX-2 expression was evaluated immunohistochemically using tissue microarray (TMA). COX-2 overexpression, noted in 43.2% (567/1314) of cases, was significantly associated with poor prognostic markers such as extra-thyroidal extension, lymph-node metastasis, and higher tumor stage. COX-2 was also an independent predictor of poor disease-free survival (DFS). Most notably, the association of COX-2 expression with DFS differed by BRAF mutation status. COX-2 overexpression was associated with poor DFS in BRAF-mutant but not BRAF wild-type PTCs, with a multivariate-adjusted hazard ratio of 2.10 (95% CI = 1.52&ndash, 2.92, p &lt, 0.0001) for COX-2 overexpressed tumors in BRAF-mutant PTC. In conclusion, the current study shows that COX-2 plays a key role in prognosis of PTC patients, especially in BRAF-mutated tumors. Our data suggest the potential therapeutic role of COX-2 inhibition in patients with BRAF-mutated PTC.

Objective Fusions involving neurotrophic tyrosine receptor kinase (NTRK) are known oncogenic drivers in a broad range of tumor types. It recently gained attention as a predictor of targeted therapy since selective NTRK inhibitors are now approved in the US and Europe for patients with solid tumors harboring gene fusions. However, estimation of NTRK gene fusion/alteration frequency and its clinicopathological characteristics in papillary thyroid cancer (PTC) is limited, especially in a population with high incidence for PTC like Middle Eastern population. This study aims to characterize the NTRK gene fusion frequency and investigate the utility of pan-Trk immunohistochemistry (IHC) as predictor of NTRK fusion in a large cohort of Middle Eastern PTC. Methods FISH analysis for NTRK gene fusions and pan-Trk IHC was performed on 315 Middle Eastern PTCs. Correlation of NTRK gene fusion and protein expression with clinicopathological markers and patient outcome were determined. Results In our cohort, 6.0% (19/315) patients showed NTRK gene fusions and were significantly associated with pediatric PTC (P = 0.0143), lymph node metastasis (P = 0.0428) and BRAF WT tumors (P < 0.0001). Pan-Trk IHC was positive in 9.2% (29/315) of cases and significantly associated with NTRK fusions, with a sensitivity of 73.7% and specificity of 94.9% in this cohort. Conclusions This study confirms the presence of NTRK fusions in Middle Eastern PTC which is significantly enriched in BRAF WT as well as pediatric age group and proposes the usefulness of IHC to screen for PTC patients with NTRK fusion that might benefit from TRK inhibitors.

Background: Disparity between sexes with regards to incidence, disease aggressiveness, and prognosis has been documented in several cancers. Although various reports have documented the association between male sex and aggressive papillary thyroid carcinoma (PTC), the prognostic impact of sex on PTC has been inconsistent. The role of sex in PTC aggressiveness and outcome in Middle Eastern PTC remains unknown. Therefore, our study retrospectively analyzed the data of a large cohort of Middle Eastern PTC patients to address this issue. Methods: We compared men and women with respect to clinico-pathological characteristics, disease persistence, structural recurrence, risk stratification and prognosis. We included 1430 patient - 1085 (75.9%) women and 345 (24.1%) men. Results: The median follow up was 9.5 years. At diagnosis, 27% (93/345) of men were ≥ 55 years, compared with 17.8% (193/1085) of women (p = 0.0003). Men had significantly more advanced disease at presentation: higher stage (p = 0.0074), larger tumor size (p = 0.0069), higher rates of lymphovascular invasion (p = 0.0129), extrathyroidal extension (p = 0.0086), regional lymph node metastasis (p = 0.0279), and distant metastasis (p = 0.0101). There was a higher rate of recurrence (p < 0.0001) and TERT mutations (p = 0.0003) in male PTC patients than in female patients. Additionally, radioiodine refractoriness was higher in male PTC patients (p = 0.0014). In multivariate analysis, male sex was an independent prognostic factor for poor recurrence-free survival (RFS) (Hazard ratio = 1.59; 95% Confidence interval = 1.21 – 2.07; p = 0.0010). Conclusions: Men with PTC are more likely to present with more advanced and aggressive diseases. Importantly, male sex was an independent prognostic factor for RFS. Thus, men may benefit from more aggressive management and therapeutic interventions. Citation Format: KHAWLA S. AL-KURAYA, Sandeep Kumar Parvathareddy, Abdul K Siraj, Felisa De Vera, Padmanaban Annaiyappanaidu, Saeeda O. Ahmed, Saif S. Al-Sobhi, Fouad Al-Dayel. Male Sex is an Independent Predictor of Recurrence-Free Survival in Middle Eastern Papillary Thyroid Carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA042.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Tumor recurrence occurs in ∼20% of PTCs and some reach advanced stages. Promoter mutation in the telomerase reverse transcriptase (TERT) gene is identified to be a prognostic marker in PTC. However, the contribution of TERT promoter mutation to cancer progression in PTC patients is still not fully understood. In this study, we investigated the incidence of TERT promoter mutations and TERT protein expression and their association with clinicopathological outcomes in a large cohort of PTC samples using direct sequencing technology and immunohistochemistry. Furthermore, two PTC cell lines were utilized to investigate role of TERT mutations in mediating metastasis. Two promoter hotspot mutations C228T and C250T were identified in 18.0% (167/927) of our cohort and were significantly associated with poor 5 years disease-free survival and distant metastasis of PTC. TERT protein overexpression was noted in 20.1% of our PTC cohort and was significantly associated with poor prognostic markers such as older age, extrathyroidal extension and Stage IV tumors. A significant association was also found between TERT overexpression and epithelial-mesenchymal transition (EMT) markers. Functional analysis showed that TERT inhibition reduced cell growth, invasion, migration and angiogenesis in PTC via suppression of EMT in PTC cells. Our results suggest that TERT promoter mutation is an independent predictor of disease-free survival and might drive the metastasis, and downregulation of TERT could potentiate antitumor and antimetastatic activities in PTC.

Context and Objective S-phase kinase protein 2 (SKP2) is an F-box protein with proteasomal properties and has been found to be overexpressed in a variety of cancers. However, its role in papillary thyroid cancer (PTC) has not been fully elucidated. Experimental Design SKP2 expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of >1000 PTC samples. In vitro and in vivo studies were performed using proteasome inhibitor bortezomib and proapoptopic death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) either alone or in combination on PTC cell lines. Results SKP2 was overexpressed in 45.5% of PTC cases and was significantly associated with extrathyroidal extension (P = 0.0451), distant metastasis (P = 0.0435), and tall cell variant (P = 0.0271). SKP2 overexpression was also directly associated with X-linked inhibitor of apoptosis protein overexpression (P < 0.0001) and Bcl-xL overexpression (P = 0.0005) and inversely associated with death receptor 5 (P < 0.0001). The cotreatment of bortezomib and TRAIL synergistically induced apoptosis via mitochondrial apoptotic pathway in PTC cell lines. Furthermore, bortezomib and TRAIL synergistically induced reactive oxygen species (ROS) generation and caused death receptor 5 upregulation through activation of the extracellular signal-regulated kinase-C/EBP homologous protein signaling cascade. Finally, bortezomib treatment augmented the TRAIL-mediated anticancer effect on PTC xenograft tumor growth in nude mice. Conclusion These data suggest that SKP2 is a potential therapeutic target in PTC and that a combination of bortezomib and TRAIL might be a viable therapeutic option for the treatment of patients with aggressive PTC.

Papillary thyroid carcinoma (PTC) is the most common histologic subtype, accounting for nearly 90% of thyroid cancer. Chromodomain-helicase-DNA-binding protein 4 (CHD4), a core subunit of the nucleosome remodeling and deacetylation (NuRD) complex is highly expressed in several cancers. However, its role in the pathogenesis and progression of PTC has not been investigated. Immunohistochemistry was performed to assess CHD4 protein expression in PTC using a tissue microarray. We also performed in vitro analysis to investigate the role of CHD4 in regulating cancer stemness and EMT in PTC cells. CHD4 overexpression was observed in 45.3% (650/1436) of PTCs, and was associated with aggressive clinico-pathological parameters and worse outcome. Functional analysis using PTC cell lines showed that forced expression of CHD4 promoted cell proliferation, spheroid growth, migration, invasion, and progression of epithelial to mesenchymal transition (EMT) in PTC cells whereas its knockdown reversed the effect. Methylation of E-cadherin was associated with loss of expression in CHD4 expressing cells, while CHD4 depletion reactivated E-cadherin expression. Most importantly, knockdown of mesenchymal transcriptional factors, Snail1 or Zeb1, attenuated the spheroid growth in CHD4 expressing PTC cells, showing a potential link between EMT activation and stemness maintenance in PTC. These findings suggest that CHD4 might be a promising therapeutic target in the treatment of patients with an aggressive subtype of PTC. Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Sasidharan Padmaja Divya, Sandeep Kumar Parvathareddy, Roxanne Diaz, Rafia Begum, Khadija Al-Obaisi, Saravanan Thangavel, Saif S. Al-Sobhi, Fouad Al-Dayel, Khawla S. Al-Kuraya. CHD4 predicts aggressiveness in PTC patients and promotes cancer stemness and EMT in PTC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1176.

Unlike many cancers, the pattern of tumor evolution in papillary thyroid cancer (PTC) and its potential role in relapse have not been elucidated. In this study, multi-region whole-exome sequencing (WES) was performed on early-stage PTC tumors (n = 257 tumor regions) from 79 individuals, including 17 who had developed relapse, to understand the temporal and spatial framework within which subclonal mutations catalyze tumor evolution and its potential clinical relevance. Paired primary-relapse tumor tissues were also available for a subset of individuals. The resulting catalog of variants was analyzed to explore evolutionary histories, define clonal and subclonal events, and assess the relationship between intra-tumor heterogeneity and relapse-free survival. The multi-region WES approach was key in correctly classifying subclonal mutations, 40% of which would have otherwise been erroneously considered clonal. We observed both linear and branching evolution patterns in our PTC cohort. A higher burden of subclonal mutations was significantly associated with increased risk of relapse. We conclude that relapse in PTC, while generally rare, does not follow a predictable evolutionary path and that subclonal mutation burden may serve as a prognostic factor. Larger studies utilizing multi-region sequencing in relapsed PTC case subjects with matching primary tissues are needed to confirm these observations.

Background Thyroid cancer is the second most common cancer affecting Saudi women after breast cancer, with papillary thyroid carcinoma (PTC) accounting for 80–90% of thyroid cancers. DNA methyltransferases affect DNA methylation, and it is thought that they play an important role in the malignant transformation of various cancers. Methods We sought to evaluate the frequency of DNA methyltransferase 3A (DNMT3A) alterations in a large cohort of >1000 PTC cases using exome sequencing, capture sequencing, immunohistochemistry and methylation-specific polymerase chain reaction. We also performed in vitro analysis to investigate the role of DNMT3A methylation in PTC cell lines. Results DNMT3A pathogenic mutations were noted in 1.2% (12/1013) of PTC cases. Reduced/loss of DNMT3A expression was seen in 59.8% (579/968) of PTC cases and was significantly associated with the DNMT3A mutation (p = 0.0120). DNMT3A alterations (mutation and/or loss of expression) were associated with aggressive clinical parameters and a poor outcome. The promoter region of the DNMT3A gene was methylated in 57.1% of PTC cases tested and was significantly associated with reduced DNMT3A protein expression (p = 0.0253). Treatment of the methylated PTC cell line with 5-aza-2′-deoxycytidine resulted in demethylation of the DNMT3A gene, leading to restoration of its expression. Demethylation significantly potentiated the TRAIL-mediated apoptosis in PTC cells. Interestingly, silencing of DNMT3A using siRNA suppressed TRAIL-mediated apoptosis. Conclusion These findings suggest that DNMT3A alterations play an important role in PTC pathogenesis and demethylation agents can be used to restore the function of DNMT3A in a subset of patients with PTC.

Papillary thyroid carcinoma (PTC) has a wide geographic variation in incidence; it is most common in Saudi Arabia, where it is only second to breast cancer as the most common cancer among females. Genomic profiling of PTC from Saudi Arabia has not been attempted previously. We performed whole-exome sequencing of 101 PTC samples and the corresponding genomic DNA to identify genes with recurrent somatic mutations, then sequenced these genes by using a next-generation gene-panel approach in an additional 785 samples. In addition to BRAF, N-RAS, and H-RAS, which have previously been shown to be recurrently mutated in PTC, our analysis highlights additional genes, including thyroglobulin (TG), which harbored somatic mutations in 3% of the entire cohort. Surprisingly, although TG mutations were not exclusive to mutations in the RAS-MAP kinase pathway, their presence was associated with a significantly worse clinical outcome, which suggests a pathogenic role beyond driving initial oncogenesis. Analysis of metastatic PTC tissue revealed significant enrichment for TG mutations (p < 0.001), including events of apparent clonal expansion. Our results suggest a previously unknown role of TG somatic mutations in the pathogenesis of PTC and its malignant evolution.

Background: PTEN gene at chromosomes 10q23.3 is a tumour suppressor gene that is inactivated in many types of human cancers. The known mechanisms of PTEN inactivation are rendered to mutation, epigenetic silencing by aberrant methylation or gene deletion. Although PTEN role has been documented in many cancers, PTEN alteration in papillary thyroid carcinoma (PTC) has not been fully elucidated. The aim of this study is to comprehensively investigate PTEN alterations in a large cohort of Middle Eastern papillary thyroid cancer by immunohistochemistry and fluorescent in situ hybridisation (FISH). Methods: PTEN protein expression was analysed by immunohistochemistry in a tissue microarray (TMA) format in a large cohort of more than 1000 patients with papillary thyroid cancer. Copy number changes in PTEN were analysed by FISH and data were correlated with clinicopathological parameters along with survival analysis. Results: PTEN inactivation reflected by complete absence of staining was seen in 24.5% of PTC samples, whereas PTEN deletion was seen only in 4.8% of the tested samples by FISH. No association was seen between PTEN loss of protein expression and PTEN gene deletion. However, interestingly, PTEN loss of expression was significantly associated with the follicular variant subset of papillary thyroid cancer. Conclusion: Our study confirmed that PTEN might have a role in pathogenesis in a subset of PTC. PTEN loss of protein expression is a more common event in follicular variant of papillary thyroid cancer. Lack of association between PTEN loss of protein expression and PTEN gene deletion might indicate that gene deletion may not be the sole cause for PTEN loss of expression and these results might raise the possibility of other mechanism such as promoter methylation-mediated gene silencing to be responsible for PTEN inactivation.

Our ability to identify germline variants in hereditary cancer cases remains challenged by the incomplete cataloging of relevant genes and lack of consensus on who should be tested. We designed a panel [hereditary oncogenesis predisposition evaluation (HOPE)] that encompasses most of the genes known to be associated with cancer development and tested its yield on more than 1300 samples of cancer patients. Pathogenic or likely pathogenic variants in high and intermediate risk genes were identified in 16, 23.9, 9.7 and 2.7%, respectively, of peripheral blood or normal tissue samples taken from patients with breast, ovarian, colorectal and thyroid cancer. To confirm specificity of these findings, we tested an ethnically matched cohort of 816 individuals and only identified pathogenic or likely pathogenic variants in 1.59% (0.98% in high risk and 0.61% in intermediate risk). Remarkably, pathogenic or likely pathogenic alleles in DNA repair/genomic instability genes (other than BRCA2, ATM and PALB2) accounted for at least 16.8, 11.1, 50 and 45.5% of mutation-positive breast, ovarian, thyroid and colorectal cancer patients, respectively. Family history was noticeably lacking in a substantial fraction of mutation-positive cases (63.7, 81.5, 42.4 and 87.5% in breast, ovarian, colorectal and thyroid, respectively). Our results show high contribution of germline mutations to cancer predisposition that extends beyond “classical” hereditary cancer genes. Family history was lacking in 63.5% mutation-positive cases, shows that hereditary cancer need not appear familial and suggests that relaxed selection of cancer patients for hereditary cancer panels should be considered.

Distant metastasis is a rare occurrence in thyroid cancer, associated with dismal prognosis. The genomic repertoires of various solid malignancies have previously been reported but remain under-explored in metastatic Papillary Thyroid Cancer (PTC). Furthermore, whether distant metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole exome sequencing on 14 matched distant metastases, primary PTC tumors and normal tissues. Point mutations, copy number alterations, cancer cell fractions and mutation signatures were defined using state-of-the-art bioinformatics methods. All likely deleterious variants were validated by orthogonal methods. Genomic differences were observed between primary and distant metastatic deposits with a median of 62% (range 21% - 92%) of somatic mutations detected in metastatic tissues, but absent from the corresponding primary tumor sample. Mutations in known driver genes including BRAF, NRAS and HRAS were shared and preferentially clonal in both sites. On the other hand, likely deleterious variants affecting DNA methylation and transcriptional repression signaling genes including SIN3A, RBBP1 and CHD4 were found to be restricted in the metastatic lesions. Moreover, mutational signature shift was observed between the mutations that are specific or enriched in the metastatic and primary lesions. Primary PTC and distant metastases differ in their range of somatic alterations. Genomic analysis of distant metastases provides an opportunity to identify potentially clinically informative alterations not detected in primary tumors, which might influence decisions for personalized therapy in PTC patients with distant metastasis. Citation Format: Tariq Masoodi, Abdul K. Siraj, Sarah Siraj, Saud Azam, Zeeshan Qadri, Wafaa N. Albalawy, Sandeep Kumar Parvathareddy, Saif S. Al-Sobhi, Fouad Al-Dayel, Fowzan S. Alkuraya, Khawla S. Al-Kuraya. Exome sequencing of primary papillary thyroid cancer and their distant metastases reveals the role of DNA methylation and transcriptional repression genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3427.

Fatty acid synthase (FASN) is an enzyme that plays a critical role in de novo synthesis of fatty acids. FASN is overexpressed in variety of human cancers, but its role has not been elucidated in papillary thyroid carcinoma (PTC).Our objective was to investigate the role of FASN and its relationship with phosphatidylinositol 3-kinase/AKT activation in a large series of PTC in a tissue microarray format followed by studies using PTC cell lines and Nude mice.Analysis of apoptosis and cell cycle were evaluated by flow cytometry and DNA fragmentation assays. FASN and phospho-AKT protein expression was determined by immunohistochemistry and Western blotting.Our data show that expression of FASN is associated with activated AKT (phospho-AKT) in a subset of PTC. Treatment of PTC cell lines (NPA-187, ONCO-DG-1, and B-CPAP) with C-75, an inhibitor of FASN, suppresses growth and induces apoptosis in all cell lines. Treatment of PTC cells with C-75 or expression of FASN small interfering RNA causes down-regulation of FASN and inactivation of AKT activity. Furthermore, treatment of PTC cell lines with C-75 results in apoptosis via the mitochondrial pathway involving the proapoptotic factor Bad, activation of Bax, activation of caspases, and down-regulation of antiapoptotic proteins. Finally, treatment of NPA-187 xenografts with C-75 results in growth inhibition of tumors in Nude mice via down-regulation of FASN expression and inactivation of AKT.Our results suggest that FASN and activated AKT pathway may be a potential target for therapeutic intervention for the treatment of PTC.

Papillary thyroid cancer (PTC) is the second most common cancer in females in Saudi Arabia. However, the pathogenesis of PTC is still not fully elucidated.To identify potential genes that play important role in progression of PTC, we studied the role of X-linked inhibitor of apoptosis protein (XIAP) as a potential prognostic marker and therapeutic target in a large cohort of PTC samples and cell lines.A DNA microarray chip was used to screen for gene copy number. XIAP expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of 1022 clinical samples. In vitro and in vivo studies were performed using Embelin and/or LY294002 on PTC cell lines.XIAP was found to be amplified in 14 of 29 and overexpressed in 48.8% of PTC cases. XIAP overexpression was significantly associated with old age, extrathyroidal extension, tumor size, nodal involvement, tall-cell variant, advanced stage disease, and significantly poor disease-free survival (P = .0341). XIAP was also significantly associated with phosphorylated AKT (P.0001), Bcl-Xl (P.0001), and Ki67 (P = .0006) proteins. Embelin treatment caused growth inhibition and apoptosis in PTC cell lines and induced tumor regression in PTC xenograft in nude mice. Finally, the combination of suboptimal doses of Embelin and LY294002 induced a synergistic apoptotic response in PTC cells.XIAP dysregulation in PTC confers an aggressive phenotype with poor outcome. In vitro and in vivo studies using an XIAP inhibitor suggest that this subgroup of PTC with overexpression of XIAP can be therapeutically targeted, either alone or in combination, to induce efficient apoptosis in these cancers.

Surgical resection of persistent/recurrent (P/R) papillary thyroid cancer (PTC), when localized to the neck, is generally recommended; however, its impact on the course of the disease is not clear. We introduced a new technique in the form of preoperative neck ultrasonographic mapping (US-M) to improve the outcome of the surgical resection of P/R PTC. A total of 19 patients had undergone regional (central, lateral, or both) neck dissection before introducing the current technique (group 1, or G1), and 26 patients (group 2, or G2) had limited lymph node resection guided by US-M with findings accurately plotted on a standard diagram. All of the operations were performed by a single surgeon. The surgical outcomes of the two groups were compared. The resected lesions were positive for PTC in 17 patients (89.5%) in G1 and in 25 patients (96.2%) in G2. In G2, the intraoperative findings exactly matched the US-M in 23 patients (88.5%). Postoperatively, neck US became negative in 50% in G1 and in 83.3% in G2 (p = 0.02). Thyroglobulin (Tg) became undetectable in 37.5% in G1 and 52.3% in G2 (p = 0.37). Whole-body iodine scans (WBS) became negative in one of six patients (16.7%) in G1, and in three of four patients (75%) in G2, (p = 0.06). After a mean follow-up of 23.8+/-7.1 months in G1 and 9.8+/-4.7 months in G2, 6 patients (31.6%) in G1 and 15 patients (62.5%) in G2 were in remission (p = 0.04), whereas the disease persisted in 13 cases (68.4%) in G1 and 9 (37.5%) in G2 (p = 0.04). In conclusion, US-M improved the surgical outcome, as evidenced by the postoperative US, Tg, and WBS findings and the higher remission rate for the G2 patients than for the G1 patients.

Airway invasion is a life-threatening complication of thyroid cancer. An important issue that deserves better attention is the differentiation between the clinical features of tracheal wall invasion versus those of an obstructive endotracheal lesion. We present information on the clinical course, diagnostic modalities utilized, management instituted, along with the prognosis, and follow-up data on a group of patients presenting with obstructive endotracheal lesions of thyroid cancer. Two thousand four hundred and eighty-nine thyroid cancer patients were seen at our institution from December 1975 to May 2000. Thirteen patients presented with symptoms of respiratory distress related to obstructive endotracheal lesions. At presentation, 11 patients underwent endoscopic examination. Imaging studies consisting of I123 whole body scan (WBS), computed tomography/magnetic resonance imaging (CT/MRI) of neck and chest, whole body positron emission tomography using 18-fluoro-2-deoxy-D-glucose (FDGPET) were done, as also was determination of the tumour markers, serum thyroglobulin (TG) and calcitonin. Patients were followed for one to 108 months after the initial presentation. Intraluminal tracheal obstruction was severe in eight patients; five had near-total-occlusion. Paralysis of the vocal folds was present in five. Evidence of metastatic disease was present in most patients. Dissociation between iodine uptake and TG synthesis was evident in five patients during follow-up. Four patients died of cancer. Of the nine living patients; cancer persisted in six, recurred in two patients, and remitted in one. This study has identified obstructive endotracheal lesion of thyroid cancer as a distinct entity apart from tracheal wall disease. These data provide evidence that intraluminal tracheal invasion of thyroid cancer is an ominous sign and a frequent cause of morbidity.

The mammalian target of rapamycin (mTOR) signaling cascade is a key regulatory pathway controlling initiation of messenger RNA in mammalian cells. Although dysregulation of mTOR signaling has been reported earlier in cancers, there is paucity of data about mTOR expression in papillary thyroid carcinoma (PTC). Therefore, in this study, we investigated the presence of mTORC2 and mTORC1 complexes in a large cohort of >500 PTC samples. Our clinical data showed the presence of active mTORC1 and mTORC2 in 81 and 39% of PTC samples, respectively. Interestingly, coexpression of mTORC1 and mTORC2 activity was seen in a 32.5% (164/504) of the PTC studied and this association was statistically significant (P = 0.0244). mTOR signaling complex was also found to be associated with activated AKT and 4E-BP1. In vitro, using Torin2, a second-generation mTOR inhibitor or gene silencing of mTOR expression prevented mTORC1 and mTORC2 activity leading to inactivation of P70S6, 4E-BP1, AKT and Bad. Inhibition of mTOR activity led to downregulation of cyclin D1, a gene regulated by messenger RNA translation via phosphorylation of 4E-BP1. Torin2 treatment also inhibited cell viability and induced caspase-dependent apoptosis via activation of mitochondrial apoptotic pathway in PTC cells. Finally, Torin2 treatment induces anticancer effect on PTC xenograft tumor growth in nude mice via inhibition of mTORC1 and mTORC2 and its associated pathways. Our results suggest that coexpression of mTORC1 and mTORC2 is seen frequently in the clinical PTC samples and dual targeting of mTORC1 and mTORC2 activity may be an attractive therapeutic target for treatment of PTC.