Transcriptomic Analysis of Papillary Thyroid Cancer: A Focus on Immune-Subtyping, Oncogenic Fusion, and Recurrence.

Epidemiology of Thyroid Cancer.

Simple SummaryRecently, selpercatinib, a highly selective inhibitor of RET receptor tyrosine kinase, has been used for RET-altered thyroid cancer. However, real-world data of its effectiveness and safety in various clinical situations are lacking. We present four cases of patients with advanced thyroid cancer who were treated with selpercatinib with variable clinical situations. Selpercatinib showed good efficacy in all four patients without significant side effects. Despite facing varying clinical obstacles of the real world, selpercatinib safely proved remarkable therapeutic efficacy, although drug safety and durability through long-term use should be further validated.Recently, selpercatinib, a highly selective inhibitor of RET receptor tyrosine kinase, has been used for RET-altered thyroid cancer. We present four cases of patients with advanced thyroid cancer who were treated with selpercatinib. The first patient was a 63-year-old male with advanced medullary thyroid cancer (MTC) treated with vandetanib. Six months ago, he had an intracranial hemorrhage and swallowing difficulty. He started selpercatinib with percutaneous endoscopic gastrostomy (PEG). For 11 months, a partial response (PR) was observed stably with PEG administration without any more cardiovascular events. The second patient was a 67-year-old female with advanced MTC treated with vandetatib. After selpercatinib treatment, a PR was observed for most metastatic sites, including choroidal metastasis. The third patient was a 32-year-old female with advanced papillary thyroid cancer (PTC) without history of systematic treatment. For six months, a PR was observed at her metastatic site with manageable adverse events. The last patient was a 59-year-old female with advanced PTC treated with lenvatinib. She suffered from a panic disorder and pleural pain due to metastasis during lenvatinib treatment. After selpercatinib treatment, her pain and panic symptoms were improved. Facing varying clinical obstacles of the real world, selpercatinib safely proved remarkable therapeutic efficacy regardless of previous treatment or metastatic site.

To see whether prophylactic central compartment dissection is recommended for advanced papillary thyroid cancer or as part of selective neck node dissection. Central compartment dissection is a technically demanding surgical procedure and carries a higher incidence of complications. The present retrospective case-control study analysed the impact of prophylactic central compartment dissection on the long-term outcome of advanced (N0-T3/T4) papillary thyroid cancer. Case records of patients operated on for papillary thyroid cancer from 2005 to 2010 were reviewed and patients with Tumour stage 3-4 and N0 nodal status were included in the study. The institutional protocol was to perform total thyroidectomy with central compartment dissection during the early phase of the study period (2005 to 2008) but this strategy was shifted to total thyroidectomy alone during the latter phase. Fifty-five patients were included in the study and 29 of the cohort had total thyroidectomy with prophylactic central compartment dissection as the primary surgery and the remaining 26 had a total thyroidectomy as the primary surgical procedure. Patients were followed up for a median duration of 115 months and found to have no significant difference in the incidences of loco-regional recurrences between the groups. (n:4 (14%) Vs n: 3 (12%) p = .463). The disease-free survival and overall survival were not significantly different in the groups. There was a trend to an increase in the incidence of permanent hypoparathyroidism in patients who had central compartment dissection. Prophylactic central compartment dissection did not influence the 10-year outcome of advanced node-negative papillary thyroid cancers.

BackgroundThere is a high unmet need for effective systemic treatment for patients with metastatic radioactive iodine refractory (RAI-R) differentiated thyroid cancer (DTC) and anaplastic thyroid cancer (ATC). Immunotherapy may be...

Thyroid hyperplasia/multinodular goiter, characterized by cellular and follicular overgrowth, is a very common endocrine condition, resulting in various functional and structural consequences and certain malignant potential. Its genetic background is completely unknown. Given previous genetic and epigenetic data suggesting that RASAL1 might be a thyroid tumor suppressor gene, particularly in follicular thyroid neoplasm, we hypothesized that the RASAL1 gene could play a fundamental role in the development of thyroid hyperplasia/multinodular goiter. To explore the role of the RASAL1 gene in the development of thyroid hyperplasia/multinodular goiter, we developed a novel Rasal1 knockout mouse model using the CRISPR/Cas9 approach and the C57BL/6J zygotes, followed by observing the pathological changes in the thyroid gland of the knockout mice in comparison with the wildtype mice. We successfully created a novel Rasal1 knockout mouse model with establishment of several generations of mice carrying the heterozygous and homozygous knockout of the Ra-sal1 gene at exon 2, which was confirmed by genetic testing and by Western blotting of the Rasal1 protein. All the mice with Rasal1 knockout, whether heterozygous or homozygous, developed grossly visible multinodular goiter with microscopic confirmation of follicular cell hyperplasia, with occasional case showing thyroid cancer. In contrast, only occasional case of aged wild-type mice developed thyroid hyperplasia and no wild-type mice developed thyroid malignancy. These data demonstrate that defect of Rasal1 can cause full and complete development of thyroid hyperplasia/multinodular goiter in mice. Given the striking similarity of thyroid hyperplasia/multinodular goiter in these Rasal1 knockout mice with that in humans, it is plausible to suggest that RASAL1 is a master gene involved in the development and pathogenesis of human thyroid hyperplasia/ multinodular goiter. This project also provides a new genetic knockout mouse model for future study of thyroid neoplasm.

Disclosure: S. Toda: None. A. Takahashi: None. K. Masudo: None. H. Iwasaki: None. Background: Thyroid cancer has a high incidence of actionable genetic alterations, but the frequency of mutations in advanced thyroid cancer that requires drug therapy is unknown. Therefore, we examined the outcomes of genetic testing performed in a real clinical setting for the purpose of pharmacotherapy. Methods: In Japan, Oncomine Dx is approved for thyroid cancer that requires drug therapy, and comprehensive genomic profiling is approved for anaplastic thyroid carcinoma and other thyroid carcinomas after the induction of tyrosine kinase inhibitors. We retrospectively reviewed the cases of those who had their tests performed between 2020 and 2022. Results: The study included 65 patients, and the testing methods used were Oncomine Dx in 36 cases, FoundationOne in 25 cases, and FoundationOne Liquid in 4 cases. With a median age of 74 years (range 17–86), 42 were female and 23 were male. Moreover, 13 cases had anaplastic thyroid carcinoma (ATC), 4 follicular thyroid carcinoma (FTC), 42 papillary thyroid carcinoma (PTC), and 6 poorly differentiated thyroid carcinoma (PDTC). Of the PTC examined by Oncomine Dx, 96.8% (30/31) were RAI-refractory; all comprehensive genomic profiling testing cases except ATC had previously been treated with tyrosine kinase inhibitors. In the following cases, actionable mutations were observed. Among 13 ATC cases, 46.2% (6) had BRAF mutation and 7.7% (1) had RET fusion. Of 42 PTC cases, 85.7% (36) had BRAF mutation and 7.1% (3) had RET fusion. Of 6 PDTC cases, 33.3% (2) had BRAF mutation and 16.7% (1) had RET fusion. No actionable mutations were observed in FTC (0/4). We found one case each of ATC and PTC with tumor mutation burden of 10 or higher, both with BRAF mutation. NTRK fusion was not observed in any of the 65 patients studied. There were four PTC cases (9.5%) with iodine accumulation in metastases, two with BRAF mutations, one with RET fusion, and one without detectable mutations. Comparing mutations in ATC and non-ATC, TERT promoter mutations were found in 84.6% of ATC (11/13) vs. 56.2% of non-ATC (9/16), whereas TP53 mutations were found in 76.9% of ATC (10/13) vs. 12.5% of non-ATC (2/16). ATC has a significantly higher prevalence of TP53 mutations. Conclusion: Advanced thyroid cancer is more likely to have actionable mutations. BRAF mutations in advanced PTC may be more common than those previously reported in standard PTC. Moreover, ATC has a higher rate of TP53 mutation than other thyroid carcinomas. Presentation: Thursday, June 15, 2023

Solid tumors contain not only malignant cells, but also extracellular matrix and many other nonmalignant cells including fibroblasts, endothelial cells, and inflammatory cells such as macrophages, neutrophils, and lymphocytes. The presence of inflammatory cells within solid tumors suggests that the inflammatory microenvironment could play a major role in promoting tumorigenesis and progression. Macrophage infiltrates in the context of or surrounding a variety of malignancies represent the host's immune response to the tumor [1,2]. It was recently reported that tumor-associated macrophages (TAMs) had important roles in the tumor progression and metastasis of various cancers, including advanced thyroid cancer [3-9]. However, the role of TAMs in papillary thyroid carcinoma (PTC) has not been fully elucidated. Fiumara et al. [10] studied the tissue distribution and prognostic significance of TAMs in a retrospective series of 121 PTCs. They found tumor-infiltrating macrophages in approximately 70% of cases. Phagocytosis of neoplastic cells by macrophages was observed in approximately 15% of tumors, and none of these tumors developed distant metastases. These data suggest that neoplastic cell phagocytosis by macrophages and lymphocytic infiltration play a protective role in the development of distant metastases in patients with PTC. Conversely, emerging data suggest that TAMs promote tumor progression and metastasis in PTC. Ryder et al. [8] reported that an increased density of TAMs was associated with tumor progression in advanced thyroid cancers and that there was a significant correlation between increased TAMs and histological grade, tumor invasiveness, and decreased cancerrelated survival in PTC. Very recently, they also reported that TAMs promoted PTC progression in BRAF-induced PTC mouse models, and that targeting CCR2-expressing cells during BRAF induction reduced TAM density and impaired PTC development [11]. These results suggested that therapeutic strategies targeting TAMs may be beneficial in the treatment of advanced PTC. Qing et al. [9] investigated TAMs density in both benign thyroid lesions and PTC tumors by CD68 immunostaining. They found that the overall density of TAMs was significantly higher in PTC tumors compared with thyroid goiter and follicular adenoma. In addition, the density of TAMs was positively associated with lymph node (LN) metastasis in TNM stages III/VI compared with stages I/II. However, no association was observed with other common tumor features, including BRAF mutation. In this issue, Kim et al. [12] investigated the expression of TAMs in 36 PTC patients with LN metastasis using immunohistochemical staining with anti-CD68 antibody. They reported that a higher density of TAMs was correlated with larger tumor size, suggesting a protumorigenic role of TAMs in PTCs. Comparing clinicopathologic characteristics among low (<25%) and high (25% to 70%) TAM density groups, primary tumor size was larger in the high density group compared to the low density group (2.0±0.1 vs. 1.5±0.1; P=0.009). However, there was no significant association between high TAM density and poorer clinicopathologic characteristics including multifocality, LN metastasis, and extrathyroidal extension. These data were not in agreement with those of previous studies, which reported that high TAM density was correlated with tumor invasiveness and poor clinicopathologic characteristics, excepting tumor size [8,9,11]. Kim et al. [12] also analyzed the morphological characteristics of TAMs in PTC. They found that TAMs had thin, elongated cytoplasmic extensions, forming a canopy structure over tumor cells, and that the morphological characteristics of thyroid cancer tissue were well maintained irrespective of the presence of TAMs. In view of this, they inferred that TAMs do not play key role in tumor development in PTC. However, Caillou et al. [13] reported that anaplastic thyroid cancer displayed a very dense network of interconnected ramified TAMs in direct contact with intermingled cancer cells, and that this TAM network was directly related to the aggressiveness of thyroid cancer. Unfortunately, Kim et al. [12] conducted this study using anti-CD68 antibody on only a small sample of PTCs with LN metastasis. A major limitation of this study was the lack of follow-up data with which to evaluate long-term outcomes. Therefore, further studies using a more specific antibody and larger sample sizes of various stages of PTC are warranted to investigate the role of TAMs in tumor development, tumor invasiveness, and metastasis of PTC. Finally, TAM-targeted pharmacologic therapy in patients with advanced thyroid cancer should also be investigated.

Molecular Rearrangements and Morphology in Thyroid Cancer

Advanced human thyroid cancers are densely infiltrated with tumor-associated macrophages (TAMs) and this correlates with a poor prognosis. We used BRAF-induced papillary thyroid cancer (PTC) mouse models to examine the role of TAMs in PTC progression. Following conditional activation of BRAFV600E in murine thyroids there is an increased expression of the TAM chemoattractants Csf-1 and Ccl-2. This is followed by the development of PTCs that are densely infiltrated with TAMs that express Csf-1r and Ccr2. Targeting CCR2-expressing cells during BRAF-induction reduced TAM density and impaired PTC development. This strategy also induced smaller tumors, decreased proliferation and restored a thyroid follicular architecture in established PTCs. In PTCs from mice that lacked CSF-1 or that received a c-FMS/CSF-1R kinase inhibitor, TAM recruitment and PTC progression was impaired, recapitulating the effects of targeting CCR2-expressing cells. Our data demonstrate that TAMs are pro-tumorigenic in advanced PTCs and that they can be targeted pharmacologically, which may be potentially useful for patients with advanced thyroid cancers.

Purpose: To determine whether the selective BRAF inhibitor dabrafenib can stimulate radioactive iodine uptake in BRAF V600E mutated advanced papillary thyroid cancer (PTC) that is unable take up iodine. Patients and Methods: Ten patients with unresectable or metastatic PTC were enrolled. All had tumors with a BRAF V600E mutation and complete absence of radioiodine uptake on iodine-131 whole body scan obtained within 14 months of study entry. Each patient received dabrafenib (150 mg twice daily) for 3 weeks followed by thyrotropin alfa-stimulated iodine-131 whole body scan. If new iodine uptake occurred, dabrafenib was continued for an additional two weeks, followed by the administration of a therapeutic dose of iodine-131. Results: All ten patients completed the study. Dabrafenib resulted in new iodine-131 uptake in six of the ten patients. All six were treated with radioioactive iodine, leading to a complete response in one patient, a partial response in a second patient and stable disease in three patients. Four of six treated patients had decreases in serum thyroglobulin. No toxic effects of grade 2 or higher were attributable to dabrafenib. One patient developed a new squamous cell carcinoma of the skin which was successfully excised with clear margins. Conclusion: Dabrafenib produces increases in iodine uptake in patients with iodine-refractory advanced BRAF V600E mutant PTC and is well tolerated. Additional studies to determine efficacy are warranted. Citation Format: Stephen M. Rothenberg, David G. McFadden, Edwin L. Palmer, Gilbert H. Daniels, Lori J. Wirth. Dabrafenib stimulates radioiodine uptake in BRAF V600E mutant advanced papillary thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT234. doi:10.1158/1538-7445.AM2014-CT234

The treatment options for advanced papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) refractory to standard therapies are limited. Although anti-PD-1 therapy has a manageable safety profile and has been effective in a small percentage of patients with advanced PTC and refractory ATC, the majority of the patients either do not respond or develop resistance to anti-PD-1 therapy. N6-methyladenosine (m6A) modification is a critical determinant of the complexity of the tumor microenvironment (TME). However, it is unclear whether and how m6A modification in tumor cells shapes the immune landscape of PTC and ATC. In this study, we performed bulk and single cell RNA sequencing analysis of PTC and ATC tissues, and found that low METTL3 expression not only correlated to poor response to immune checkpoint blockade (ICB) but was also associated with increased TNF family-related ligand-receptor interactions in the immunosuppressive Tregs and exhausted T cells. Furthermore, overexpression of METTL3 in PTC and ATC cells enhanced the efficacy of anti-PD-1 therapy in a peripheral blood mononuclear cell humanized NCG (huPBMC-NCG) mouse model. Mechanistically, M2 macrophage-derived extracellular vesicles (M2 EVs) inhibited METTL3 expression in PTC and ATC cells via miR-21-5p. Downregulation of METTL3 promoted demethylation of CD70 mRNA, which prevented YTHDF2-mediated degradation of the transcripts. The stabilization of CD70 mRNA, and the subsequent upregulation in CD70 protein levels increased the abundance of the immunosuppressive Tregs and terminally exhausted T cells, thereby inducing resistance to anti-PD-1 therapy. Furthermore, blocking CD70 using cusatuzumab, a high-affinity monoclonal antibody, reversed the anti-PD-1 therapy resistance induced by M2 EVs in vivo. Finally, we demonstrated that METTL3 expression negatively correlated with CD70 expression and M2 macrophages and Tregs infiltration in PTC and ATC tissues. Our findings provide new insights into developing novel therapies for advanced PTC and ATC.

The new 4th edition World Health Organization classification for thyroid tumors, Asian perspectives.

BackgroundTreatment options for advanced thyroid cancer refractory to standard therapies are limited. The safety and efficacy of pembrolizumab were evaluated in patients with advanced differentiated thyroid cancer expressing programmed death ligand 1 (PD-L1).MethodsPatients with advanced thyroid cancer were enrolled in the nonrandomized, phase Ib KEYNOTE-028 trial conducted to evaluate safety and antitumor activity of the anti–programmed death 1 (PD-1) antibody pembrolizumab in advanced solid tumors. Key eligibility criteria were advanced papillary or follicular thyroid cancer, failure of standard therapy, and PD-L1 expression in tumor or stroma cells (assessed by immunohistochemistry). Pembrolizumab 10 mg/kg was administered every 2 weeks up to 24 months or until confirmed progression or intolerable toxicity. The primary endpoint was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors, version 1.1.ResultsTwenty-two patients were enrolled: median age was 61 years; 59% were women; and 68% had papillary carcinoma. Median follow-up was 31 months (range, 7–34 months). Treatment-related adverse events were observed in 18 (82%) patients; those occurring in ≥15% of patients were diarrhea (n = 7) and fatigue (n = 4). One grade ≥ 3 treatment-related adverse event occurred (colitis, grade 3); no treatment-related discontinuations or deaths occurred. Two patients had confirmed partial response, for an ORR of 9% (95% confidence interval [CI], 1–29%); response duration was 8 and 20 months. Median progression-free survival was 7 months (95% CI, 2–14 months); median overall survival was not reached (95% CI, 22 months to not reached).ConclusionsResults of this phase Ib proof-of-concept study suggest that pembrolizumab has a manageable safety profile and demonstrate evidence of antitumor activity in advanced differentiated thyroid cancer in a minority of patients treated. Further analyses are necessary to confirm these findings.Trial registrationClinicaltrials.gov identifier: NCT02054806. Registered 4 February 2014.

Fine needle aspiration (FNA) biopsy is an established procedure by which to sample thyroid nodules to ascertain etiology and produce a diagnosis conveying risk of malignancy with recommended patient follow-up. This procedure is well-tolerated and endorsed given the accessibility and vascularity of the thyroid gland. FNA cytopathology has proven efficacious for the primary assessment of thyroid nodules. Well-differentiated papillary thyroid carcinoma (PTC) and benign lymphocytic (Hashimoto) thyroiditis (HT) are distinct thyroid lesions that may be reported with diagnostic confidence based on their characteristic cytomorphologic features. However depending on the adequacy of FNA sampling and the morphology of aspirated cellular material, thyroid nodules with coexisting PTC and HT may pose diagnostic pitfalls. This may be dependent upon: (a) the architectural nature of the coexisting lesions in-vivo; (b) whether both lesions are adequately sampled through FNA; and (c) which of the cell types and cytomorpholo...

Background: Papillary thyroid cancer (PTC) and lymphocytic thyroiditis (LT) co-occur with a prevalence of about 30%. PTC harboring BRAFV600E (PTC-BRAF) confers a worse prognosis, but it is unclear if LT alters prognostic features and recurrence of PTC. Objective: We compared the prevalence of PTC-BRAF with and without LT. The risk of adverse pathological features in (i) PTC in the presence and absence of BRAF mutation, irrespective of LT status, was compared to (ii) PTC in the presence and absence of LT, irrespective of BRAF status. Methods: We searched PubMed, Embase, and Web of Science Core Collection for observational studies published from 2010 to June 2023 on adult patients with PTC. The search strategy yielded 47 studies with relevant data. Data of baseline characteristics, clinicopathological features, and the quality assessment tool were extracted by two reviewers. The study was registered with PROSPERO (CRD42023437492). Results: Of the 47 studies, 39 studies with a total cohort of 28 143, demonstrated that the odds of PTC-BRAF were significantly lower in the presence of LT compared to its absence (odds ratio [OR] 0.53, 95% confidence interval [CI]: 0.48-0.58, p < 0.00001). In PTC-BRAF patients, there was a positive association of central neck nodal disease (CNND), PTC > 1 cm, extra-thyroidal extension, American Joint Committee on Cancer (AJCC) Stage 3-4, and multifocality with pooled ORs of 1.54 (95% CI: 1.16-2.04), 1.14 (95% CI: 0.82-1.58), 1.66 (95% CI: 1.40-1.97), 1.53 (95% CI: 1.35-1.75), and 1.24 (95% CI: 1.11-1.40) respectively, compared to wild-type PTC, irrespective of LT status. In the same studies, PTC with LT patients had lower pooled ORs of 0.64 (95% CI: 0.51-0.81) for CNND, 0.83 (95% CI: 0.73-0.95) for PTC > 1 cm, 0.71 (95% CI: 0.58-0.86) for ETE, 0.84 (95% CI: 0.75-0.94) for AJCC Stage 3-4 compared to PTC without LT, irrespective of BRAF status. PTC recurrence was not affected by BRAF or LT, with pooled ORs of 1.12 (95% CI: 0.66-1.90, p = 0.67) and 0.60 (95% CI: 0.28-1.30, p = 0.20) respectively. Similar results were seen with recurrence expressed as hazard ratio in this limited data-set. Conclusion: The odds of PTC-BRAF are significantly lower in the presence of LT than without. PTC with LT, irrespective of BRAF status, was significantly associated with better prognostic factors. Further studies are required to evaluate if LT inhibits PTC-BRAF, and whether this is relevant to the role of immunotherapy in advanced thyroid cancer.