Medullary Thyroid Carcinoma Tumors Research Articles

Photosensitizing metal–organic framework nanoparticles combined with tumor-sensitization strategies can enhance the phototherapeutic effect upon medullary thyroid carcinoma

Photodynamic therapy (PDT) utilizing metal-organic frameworks (MOFs) has developed as a new and efficacious treatment for malignant tumors located on the surface of the human body. In order to achieve more effective PDT treatment outcomes, the traditional method has been to increase the intensity of the laser irradiation, but this approach can easily lead to tissue burns. In this study, we developed a new type of nanoparticle, F68-PKI@PCN224, aims to achieve effective PDT upon medullary thyroid carcinoma (MTC) which is an uncommon form of thyroid cancer that originates in the parafollicular cells of the thyroid and the therapeutic outlook for patients with MTC remains unsatisfactory. F68-PKI@PCN224 combines the antitumor features of PDT with mammalian target of rapamycin (mTOR) inhibitor PKI-587 (PKI). The tumor sensitization, slow release, and pH response features of F68-PKI@PCN224 was demonstrated by a series of in vitro and in vivo experiments / assays. F68-PKI@PCN224 achieved the long-term activation and slow releasing of PKI and TCPP in MTC tumor tissues. During the process of generating PDT effects, F68-PKI@PCN224 enhanced the tumor's sensitivity to PDT, direct laser irradiation of MTC cells or subcutaneous tumor tissues. As a result, low-dose phototherapy achieves a higher anti-tumor effect upon F68-PKI@PCN224 compared with TCPP. This study reveals the synergistic effect between tumor sensitization by mTOR inhibitor and PDT and initially unveils the mechanism of action of these nanoparticles.

6454 Identifying Gaps in Obtaining Germline RET Testing for Patients with Medullary Thyroid Cancer

Abstract Disclosure: A.N. Lim: None. J. Comeaux: None. C. Ricker: None. K.S. Wei: None. T.E. Angell: None. Background: Medullary thyroid carcinoma (MTC) occurs sporadically or from germline pathogenic variants of the RET proto-oncogene in multiple endocrine neoplasia type 2 (MEN2). Current ATA guidelines recommend germline RET testing in all patients with C-cell hyperplasia or MTC. Because disparities may exist in the implementation of this recommendation, we analyzed records of patients with MTC to identify variables associated with germline RET testing. Methods: Patients with MTC were identified from a cohort study of all adult patients undergoing thyroid surgery from 2015-2022 at two affiliated academic centers: a private hospital or a safety-net hospital. Patients were categorized as RET-tested or not RET-tested. Data collection included: patient age, gender, ethnicity, nodule and lymph nodule findings on ultrasound (US), calcitonin and CEA levels, and surgical pathology results. Medical reports were reviewed for reasons that RET testing was or was not obtained. Results: Of 24 MTC patients, 50% were female, mean age ± SD was 50.8 ± 12.3, and 15 (62.5%) had Hispanic ethnicity. RET testing was performed in 14 (58.3%) patients and 3 (21.4%) had a pathogenic variant. RET-tested patients had a median nodule size of 3.45 cm (IQR 2.40-4.53) vs. 2.40 cm (IQR 0.98-3.25) in not-RET tested patients (p<0.05). There were no significant differences between groups in age, gender, ethnicity, preoperative US findings, preoperative calcitonin or CEA levels, or histopathologic status. RET testing was performed in 11/14 (78.6%) patients at the private hospital vs. only 5/10 (50.0%) patients at the safety-net hospital (p=0.20). Considering this, we further investigated the use of RET testing in the initial management of MTC patients. RET testing was performed or planned during initial management in 13/14 (92.9%) patients at the private hospital vs. only 5/10 (50%) patients at the safety-net hospital (p=0.05). At the private hospital, RET testing was requested for apparent sporadic disease (n=11), family history of MTC (n=1), and coexistent pheochromocytoma (n=1). At the safety-net hospital, RET testing was requested for apparent sporadic disease (n=4) and family history of MTC (n=1), but reportedly was not pursued in 5 patients because MTC appeared to be sporadic. Discussion: In this cohort of patients with MTC, larger nodule size was significantly associated with the rate of germline RET testing. Larger MTC tumor size alone may have prompted greater attention to germline testing, but also may have been associated with other findings, which could be further assessed with a larger sample size by multivariable analysis. The disparity in RET testing as part of initial management at the safety-net hospital may be multifactorial, but future analyses should explore provider decision-making, as well as facility and patient variables. Presentation: 6/1/2024

Abstract 4665: USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase

Abstract Targeted therapy resistance is the main cause of disease progression in advanced medullary thyroid carcinoma (MTC). However, the key molecules and mechanisms remain unclear. We previously performed a genome-wide in vitro screen of Anlotinib resistance-related genes in human MTC cell line TT using CRISPR-dCas9-SAM system and identified ubiquitin-specific protease 18 (USP18) as a key molecule mediating Anlotinib resistance in MTC. However, its downstream mechanism needs further clarification. Based on the high-throughput DNA sequencing results of the control group and the Anlotinib screening group, a group of key genes related to Anlotinib resistance in MTC was screened through the MAGeCK algorithm and the enrichment ratio of sgRNA in the Anlotinib screening group. Among them, USP18 ranked first in the enrichment results. Analysis of the correlation between the expression level of USP18 in MTC patient specimens and survival revealed that patients with high USP18 expression in tumor cells had poorer prognosis. Overexpression of USP18 in MTC tumor cells significantly promoted the growth of subcutaneous xenograft tumors in NOD/SCID mice treated with Anlotinib. In vitro cell experiments also showed that overexpression of USP18 promoted the proliferation of MTC tumor cells and significantly inhibited the proportion of cell apoptosis after Anlotinib treatment. Further research found that USP18, as a deubiquitinating enzyme that can specifically remove ISG15-like ubiquitin-like proteins from substrate proteins, can improve the kinase stability of Aurora B by removing ISG15-like ubiquitin modification and promote MTC tumor cell proliferation through the activation of PI3K-Akt signaling regulated by Aurora B kinase. Application of Aurora B kinase inhibitor in Anlotinib-resistant MTC cell lines significantly inhibited tumor cell proliferation, and Aurora B kinase inhibitor significantly increased the proportion of cell apoptosis in MTC tumor cells overexpressing USP18. In the subcutaneous xenograft tumor model of MTC in NOD/SCID mice, the growth of xenograft tumors in the Anlotinib combined with Aurora B kinase inhibitor treatment group was also significantly inhibited. In summary, overexpressed USP18 can activate PI3K-Akt signaling and promote tumor cell proliferation by increasing the stability of Aurora B kinase, ultimately leading to the formation of Anlotinib resistance in MTC patients. Combined use of Aurora B kinase inhibitor is a potentially effective combined treatment strategy for Anlotinib-resistant MTC patients. Citation Format: Xuan Qin, Xing Wan, Dapeng Li, Yimeng Liu, Xianhui Ruan, Fangkun Luan, Kejia Xu, Jia Li, Rong Xiang, Yongjun Piao, Yi Shi, Xiangqian Zheng. USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4665.

The role of PEDF, VEGF, and Ki-67 in tissue invasion and tumor angiogenesis of medullary thyroid carcinoma

Objective: This study investigated the role of pigment epithelium-derived factor (PEDF), vascular endothelial growth factor (VEGF), nuclear factor kappa B (NF-kB), and Ki-67 proliferation index (PI) factors in Medullary thyroid carcinoma (MTC) tissue progression and their relationship with tumor angiogenesis. Immunohistochemical (IHC) analyses of PEDF expression in various human tumor samples and healthy tissues have shown that high levels of PEDF expression are associated with a positive prognosis, and low levels of PEDF suggest a poorer prognosis. With the completion of further studies showing the antitumor effects of PEDF on different types of cancer, PEDF’s potential as a therapeutic agent has become increasingly promising. To the best of our knowledge, the role of PEDF in tumor angiogenesis in MTC has not been previously investigated. Material and Methods: Thirty-seven tissue samples were retrospectively analyzed and sent/removed for pathology after total or subtotal thyroidectomy and archived between 01.01.2000 and 31.12.2015 in the Pathology Department of Hacettepe University. Relationships between categorical variables are analyzed using Pearson Chi-squared or Fisher’s Exact tests.The correlation between two numerical variables is analyzed using Spearman’s Rho correlation coefficient, and a comparison of numerical variables between two groups is made using the Mann-Whitney U test. Results: The clinicopathological features of the patients were evaluated as sporadic or hereditary, tumor node metastasis (TNM) staging, single or multifocal focus, recurrence or regional or distant metastasis, progression-free survival time (PFS), and appropriate pathology tissue samples were determined as PEDF, VEGF, NF-kB, Ki-67 staining was compared. As a result of this comparison, a moderate negative correlation was found between VEGF expression level and PFS (rho = - 0,407, p = 0,019). No statistically significant correlation was found between single or multifocal groups and Ki-67 proliferation index (PI) expression levels (p = 0.070), but this is a small p value, a variable that is likely to find significant differences in studies with larger sample groups.The relationship between PEDF, VEGF, NF- kB, Ki-67 PI factors and tumor angiogenesis as reflected by microvessel density (MVD) was also investigated. To this end, we correlated the expression levels of PEDF, VEGF, NF-kB, and Ki-67 staining of suitable pathology tissue samples of the patients with the results obtained from the staining with CD 31. As a result of this comparison, no statistically significant relationship was found. Conclusions: We demonstrated and described for the first time PEDF expression patterns in MTC tumor tissues. A direct relationship between PEDF expression level and angiogenesis, angiogenetic and clinicopathological factors does not seem to exist, at least as it pertains to the group studied. Further studies are required to fully elucidate mechanisms for tumor angiogenesis and invasion in MTC.

RREB1 promotes the development of parafollicular carcinogenesis through the Ras-Raf-1-ELK3 signaling pathway

Thyroid cancer (TC) is the most common endocrine malignancy. Medullary thyroid carcinoma (MTC) is derived from parathyroid follicle cells (C cells) secrete calcitonin, accounting for approximately 5-10% of all thyroid cancers. The malignancy is between differentiated and undifferentiated thyroid cancer and undifferentiated thyroid cancer and has a relatively poor prognosis. In MTC tumor cells, RREB1 regulates the differentiation of parathyroid cells via RAS-Raf-1-ELK3 signaling and induce calcitonin secretion. Therefore, it is easy to induce parathyroid parafollicular cells canceration and medullary thyroid carcinoma. Here, we investigated the correlation between RREB1, RAS-Raf-1-ELK3 signaling pathway and medullary thyroid carcinoma with various phases. Our results suggest that RREB1 promotes parafollicular carcinoma through the Ras-Raf1-elk3 signaling pathway, providing a rationale to further investigate the role of RREB1 in parafollicular carcinoma. It provides theoretical guidance for the clinical treatment of medullary thyroid cancer.

Radiomics Features of Different Sizes of Medullary Thyroid Carcinoma (MTC) and Papillary Thyroid Carcinoma (PTC) Tumors: A Comparative Study.

Background:Radiomics strategies exhibit great promise in the context of thyroid nodule diagnosis. This study aimed to compare radiomics features of different sizes of medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC) tumors and to compare the efficiency of radiomics approaches as a means of differentiating between these tumor types.Methods:In total, 86 MTC and 330 PTC nodules were divided into the macronodular (>10 mm) and micronodular (⩽10 mm) categories. The radiomics features of these nodules were analyzed to identify independent prognosis factors and evaluate the efficacy of individual and combined indicators as predictors of tumor type.Results:In total, 12 radiomics features were found to differ significantly between MTC and PTC macronodules, while 6 differed significantly between MTC and PTC micronodules. Shape 2D_Sphericity, firstorder_Skewness, glrlm_RunLengthNonUniformity, glszm_GrayLevelNonUniformity, and glszm_SizeZoneNonUniformity were features that were independently associated with the differential diagnoses of MTC and PTC macronodules. Receiver operating characteristic (ROC) curve analyses of the efficacy of these 5 single indicators and a combined indicator composed thereof yielded area under the curve (AUC) values of 0.621, 0.678, 0.704, 0.762, 0.747, and 0.824, respectively, with respective sensitivities of 55.3%, 43.0%, 53.1%, 56.3%, 46.9%, and 65.6%, and respective specificity values of 65.6%, 89.1%, 81.6%, 88.8%, 95.0%, and 91.1%. The glrlm_RunEntropy and glszm_SizeZoneNonUniformity features were identified as independent factors associated with the differential diagnoses of MTC and PTC micronodules. Receiver operating characteristic curve analyses of the efficacy of these 2 single indicators and a combined indicator composed thereof yielded respective AUC values of 0.678, 0.678, and 0.771; Sensitivities of 57.0%, 72.7%, and 72.7%; and specificities of 77.3%, 64.2%, and 77.5%.Conclusions:A range of different radiomics features can enable effective differentiation between MTC and PTC nodules of different sizes. Moreover, analyses of combinations of radiomics features yielded diagnostic efficiency values higher than those associated with single radiomics features, highlighting a more reliable approach to diagnosing MTC and PTC tumors.

Procalcitonin as an Alternative Tumor Marker of Medullary Thyroid Carcinoma.

Calcitonin (CT) measurement is pivotal in the management of medullary thyroid carcinoma (MTC), but several pitfalls can affect its reliability. Procalcitonin (ProCT) has been reported as a promising alternative MTC tumor marker. This study aimed to determine the ProCT diagnostic accuracy in prediction and treatment monitoring of MTC. Electronic databases were searched for observational studies published until May 2021 without language or time restrictions. Studies comparing ProCT and calcitonin accuracy were included. After removing duplicates and exclusion of not-eligible articles, relevant articles were screened independently by 2 reviewers. Eleven studies (4.5% of the identified studies) met the selection criteria. Two reviewers independently extracted data and assessed data quality and validity through QUADAS-2. A meta-analysis was performed on 11 sufficiently clinically and statistically homogeneous studies (n = 5817 patients, 335 MTC patients). Hierarchical summary receiver operating characteristics and bivariate methods were applied. Serum ProCT was found to be a highly accurate test for MTC diagnosis and monitoring. The pooled sensitivity, specificity, positive and negative likelihood ratios, area under the curve, and positive and negative predictive values for ProCT were 0.90 (95% CI: 0.71-0.97), 1.00 (95% CI: 0.85-1.00), 288 (95% CI: 5.6-14 929.3), 0.10 (95% CI: 0.03-0.33), 0.97 (95% CI: 0.95-0.98), 99%, and 2%, respectively. The high accuracy, compounded with favorable analytical characteristics, give ProCT great potential to replace calcitonin as a new standard of care in the management of MTC.

Expression of miR-127, miR-154, and miR-183 in Medullary Thyroid Carcinoma Tumors.

Background:Medullary thyroid cancer (MTC) accounts for 5%–10% of all thyroid cancers, but causes 13% of all thyroid cancer related deaths. MicroRNAs (miRs) have key functions in the development and progression of MTC. Altered expression of some miRs has been reported in many human cancers, including Thyroid cancer. Therefore, we aimed to analyze the expression of miR-154, miR-183 and miR-127 in MTC tumor tissues.Methods:In this case-control study, 15 MTC Formalin-fixed, paraffin-embedded (FFPE) tissue samples and 15 adjacent normal thyroid FFPE tissues, as a control group, were collected from Taleghani, and Loghman Hakim Hospitals, Tehran, Iran since 2005 till 2015. After RNA extraction and cDNA synthesis, the expression of miR-127, miR-154 and miR-183 was measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results:Our data showed a significant increase in the expression of miR-127 in MTC samples in comparison with the control group (P<0.05). Although miR-154 and miR-183 expression levels had increase expression in MTC tumors, this change was not statistically significant.Conclusion:The miR-127 could be considered as a prognostic, diagnostic and therapeutic marker for the management of MTC, and it is proposed for further investigation to fully establish the role of this miRNA in MTC.

Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Medullary Thyroid Carcinoma: An Update on Imaging.

Medullary thyroid carcinoma (MTC), arising from the parafollicular C cells of the thyroid, accounts for 1–2% of thyroid cancers. MTC is frequently aggressive and metastasizes to cervical and mediastinal lymph nodes, lungs, liver, and bones. Although a number of new imaging modalities for directing the management of oncologic patients evolved over the last two decades, the clinical application of these novel techniques is limited in MTC. In this article, we review the biology and molecular aspects of MTC as an important background for the use of current imaging modalities and approaches for this tumor. We discuss the modern and currently available imaging techniques—advanced magnetic resonance imaging (MRI)-based techniques such as whole-body MRI, dynamic contrast-enhanced (DCE) technique, diffusion-weighted imaging (DWI), positron emission tomography/computed tomography (PET/CT) with 18F-FDOPA and 18F-FDG, and integrated positron emission tomography/magnetic resonance (PET/MR) hybrid imaging—for primary as well as metastatic MTC tumor, including its metastatic spread to lymph nodes and the most common sites of distant metastases: lungs, liver, and bones.

MiR-182 promotes cancer invasion by linking RET oncogene activated NF-\u03baB to loss of the HES1/Notch1 regulatory circuit

BackgroundDominant-activating mutations in the RET proto-oncogene, a receptor tyrosine kinase, are responsible for the development of medullary thyroid carcinoma (MTC) and causative for multiple endocrine neoplasia (MEN) type 2A and 2B. These tumors are highly aggressive with a high propensity for early metastasis and chemoresistance. This attribute makes this neoplasia an excellent model for probing mechanisms underlying cancer progression.MethodsThe expression level of miR-182 was measured in MTC tumor specimens and in TT cells by real-time RT-PCR. TT cells and modified NThy-ori 3.1 that stably express RETM918T were used to investigate RET-dependent regulation of miR-182. Identification and validation of miR-182 targets and pathways was accomplished with luciferase assays, qRT-PCR, Western blotting and immunofluorescence. In vitro, overexpression and knockdown experiments were carried out to examine the impact of miR-182 and HES1 on invasion and migration.ResultsWe found that miR-182 expression is significantly upregulated in MTC patient samples and tumor-derived cell lines harboring mutated RET. Inhibition of RET oncogenic signaling through a dominant-negative RET∆TK mutant in TT cells reduces miR-182, whereas overexpression of RETM918T in NThy-ori 3.1 cells increases miR-182 levels. We further show that overexpression of this miRNA in NThy.miR-182 cells promotes the invasive and migratory properties without affecting cell proliferation. MiR-182 is upregulated after RET induced NF-κB translocation into the nucleus via binding of NF-κB to the miR-182 promoter. Database analysis revealed that HES1, a repressor of the Notch pathway, is a target of miR-182, whose upregulation correlates with loss of HES1 transcription in MTC tissue samples and mutant RET cell lines. Moreover, we demonstrated that the 3′UTR of the HES1 mRNA bearing the targeting sequence for miR-182 clearly reduced luciferase reporter activity in cells expressing miR-182. Decreased expression of HES1 promotes migration by upregulating Notch1 inhibitor Deltex1 and consequent repression of Notch1.ConclusionWe demonstrate a novel mechanism for MTC aggressiveness in which mutated RET/NF-κB-driven expression of miR-182 impedes HES1 activation in a negative feedback loop. This observation might open new possibilities to treat RET oncogene associated metastatic cancer.

Heat shock proteins HSP90, HSP70 and GRP78 expression in medullary thyroid carcinoma

Medullary thyroid carcinoma management consists mainly of surgical resection and is largely chemoresistant. There is ongoing effort to discover novel therapies for medullary thyroid carcinoma. Increased levels of heat shock proteins have been associated with multiple cancers and are being studied as potential therapeutic targets. The purpose of this study was to determine the expression levels of heat shock proteins 90 and 70 and of glucose related protein 78 in medullary thyroid carcinoma tissues compared with normal thyroid tissues. 20 tissue specimens of medullary thyroid carcinoma and 10 specimens of thyroids without malignancy were analyzed by immunohistochemistry. Medullary thyroid carcinoma specimens showed 27% higher expression level of heat shock protein 90 immunostaining, and a 43% higher expression level of heat shock protein 70 immunostaining versus normal controls. These differences, however, were not statistically significant. A significantly higher expression level was noted for glucose related protein 78 in the medullary thyroid carcinoma specimens than in the controls. This study indicates increased expression levels of heat shock proteins 90 and 70 and glucose related protein 78 levels in medullary thyroid carcinoma. These findings, though preliminary imply that these proteins may have a role in medullary thyroid carcinoma's tumor biology and may have and future therapeutic options. Larger cohorts are needed to corroborate these results.

Abstract A33: Biomarkers of Cdk5 driven neuroendocrine tumors

Abstract Background: Neuroendocrine (NE) cancers originate from secretory cells of the body's various endocrine structures. Surgical resection is the primary treatment for these tumors. However, the majority of NE cancers are metastatic at the time of detection and no effective adjuvant therapies exist for primary NE tumors or their metastases. Patients often develop two or more NE cancers, a syndrome termed multiple endocrine neoplasia (MEN). DNA sequencing has lead to breakthroughs in the identification of genes mutated in four types of familial MEN syndromes: Menin, RET, and CDNK1B. Unfortunately, a large percentage of NE cancers are sporadic, resulting from somatic mutations, and a high percentage of these do not harbor menin, RET, or CDNK1B mutations. Our lab has determined that Cdk5 promotes growth in multiple types of NE cancer. Phosphoproteomic studies revealed a selective set of proteins potentially phosphorylated downstream of Cdk5 activation. We hypothesize that these phosphoproteins will serve as biomarkers to identify tumors that will be responsive to anti-Cdk5 therapies. Methods: Phosphorylation state-specific antibodies were generated to novel phosphoprotein sites by inoculation of New Zealand White rabbits with phosphopeptides specific to each site. Antibodies were isolated from rabbit sera by affinity purification using phosphopeptide columns. Human NE cell lines were treated with Cdk5 inhibitors and monitored for effects on phosphoprotein levels. Phosphoprotein levels were examined in growing and arrested medullary thyroid carcinoma tumors isolated from an inducible/arrestable (NSE-tTA; tetO-p25GFP) mouse model and in normal lung versus small cell lung cancer tumors isolated from a triple conditional knockout (p130 f/f; p53 f/f; Rb f/f) mouse model. Phosphoprotein levels were examined in control tissue and human medullary thyroid carcinoma, gastrointestinal NETs, and pancreatic NETs. Results: Phosphorylation state-specific antibodies were successfully generated to 10 novel phosphoproteins. Cdk5 inhibition decreases phosphorylation levels of 5 of these 10 proteins in several types of NE cell lines. Levels of 4 of these phosphoproteins are elevated in human NE tumors. Conclusion: Four novel phosphoproteins downstream of Cdk5 comprise a set of biomarkers for Cdk5-dependent human NE tumors. The combination of these biomarkers with pathway specific therapies currently under development will constitute a coupled diagnostic-therapeutic regimen for personalized treatment of NE cancer patients. Citation Format: Angela M. Carter, Rahul Telange, Sarah Oltmann, Fiemu Nwariaku, Bruce Robinson, Elizabeth Grubbs, James Bibb. Biomarkers of Cdk5 driven neuroendocrine tumors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A33.

Abstract 3567: Identification of potential biomarkers and novel therapeutic candidates for neuroendocrine cancer

Abstract Neuroendocrine (NE) cancers originate from secretory cells of the body's various endocrine structures. Surgical resection is the primary treatment for these tumors. However, the majority of NE cancers are metastatic at the time of detection and no effective adjuvant therapies exist for primary NE tumors or their metastases. Patients often develop two or more NE cancers, a syndrome termed multiple endocrine neoplasia (MEN). DNA sequencing has lead to breakthroughs in the identification of the causes of familial forms of the four types of MEN syndromes. Mutations in a protein of unknown function, menin, are associated with MEN1. Mutations in the receptor tyrosine kinase RET are linked to MEN2 and MEN3. The cyclin-dependent kinase inhibitor, CDNK1B, is mutated in MEN4. Unfortunately, a large percentage of NE cancers are sporadic, resulting from somatic mutations, and a high percentage of these do not harbor menin, RET, or CDNK1B mutations. Due to the lack of development of successful adjuvant therapies there has been little improvement in survival rates of NE cancer patients in the past several decades. Thus, there is a great need for the elucidation of signaling molecules that promote NE tumorigenesis in order to guide the development of novel and effective treatments for this family of cancers. Our laboratory has found that cyclin-dependent kinase 5 (Cdk5) is an important tumorigenic signaling molecule in sporadic medullary thyroid carcinoma (MTC), a type of NE cancer that originates from calcitonin-producing C cells in the thyroid. New data supports a role for Cdk5 in multiple types of NE cancer and provides a new prospective from which to explore their causes. Cdk5 is expressed in many NE cancers and inhibition of Cdk5 activity blocks growth of NE cancer cell lines. Phosphoproteomic analysis of growing versus arrested MTC tumors, induced by expression of the Cdk5 activator p25 in our transgenic mouse model of MTC, revealed elevation of over 200 phosphorylation sites. We have used short interfering peptides (SIPs) to selectively target a set of these phosphorylation sites and found that 15 of these SIPs blocked growth of NE cancer cell lines. Phosphorylation state specific antibodies were generated to several of these sites and phosphorylation levels were confirmed to be decreased in arrested MTC tumors. In conclusion, 1) multiple types of NE cancer cells are dependent on Cdk5 activity for growth, 2) phosphorylation of multiple signaling proteins is elevated downstream of Cdk5, and 3) select inhibition of these pathways blocks growth of NE cancer cells. Future studies will focus on analysis of these phosphoproteins in human NE tumors to validate of them as biomarkers for targeted cancer therapies as well as exploration of these phosphoproteins as direct targets for developing new therapeutics that are effective against NE cancers. Citation Format: Angela Carter, Karine Pozo, Chun-Feng Tan, Fiemu Nwariaku, James Bibb. Identification of potential biomarkers and novel therapeutic candidates for neuroendocrine cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2015-3567

Abstract 3923: Molecular characterization of an animal model for neuroendocrine thyroid cancer

Abstract Neuroendocrine tumors are rare forms of cancers originating from hormone-producing cells that are scattered throughout the body. Medullary thyroid carcinoma (MTC) develops from calcitonin-secreting, C cells, in the thyroid gland. Surgical removal of the thyroid is the best treatment currently available for early stage cancer patients but advanced forms of MTC are often incurable and fatal. Thus, better therapies are needed. A lack of relevant animal models for this disease has slowed the development of new treatments. We have recently reported a novel conditional mouse model for MTC. In these mice, overexpression of the cyclin-dependent kinase 5 (Cdk 5), activator, p25 in C cells induces MTC tumors. Furthermore, interruption of p25 overexpression results in growth arrest of these tumors. Here we take advantage of our conditional mouse model to investigate the molecular pathways underlying MTC tumorigenesis. We used Illumina Mouse WG-6 v2.0 Expression BeadChip to compare the gene expression profile of proliferating versus arrested mouse MTC tumors. We found that a total of 920 genes encoding transcription factors, cell cycle proteins, motor proteins and angiogenesis factors were differentially regulated in growing versus arrested tumors. Particularly the genes encoding cell cycle proteins that are involved in the regulation of Rb pathway were significantly up-regulated in the proliferating tumors. Selected genes with higher degrees of change were evaluated by quantitative PCR and immunoblotting analyses. Taken together these findings suggests that the Rb signaling pathways is involved in MTC tumorigenesis and that Cdk5 may act as a critical regulator of the Rb signaling pathway in this cancer. Thus, targeting Cdk5 and/or the Rb pathway may be a valid strategy to stop the progression of neuroendocrine thyroid cancer. Citation Format: Karine Pozo, Antje Hillmann, Alexander Augustyn, Tanvir Singh, Florian Plattner, John Minna, Herbert Chen, Gilbert J. Cote, James A. Bibb, Fiemu E. Nwariaku. Molecular characterization of an animal model for neuroendocrine 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 3923. doi:10.1158/1538-7445.AM2014-3923

VEGF, VEGFR3, and PDGFRB protein expression is influenced by RAS mutations in medullary thyroid carcinoma.

Tyrosine kinase inhibitors (TKIs) have achieved remarkable clinical results in medullary thyroid carcinoma (MTC) patients. However, the considerable variability in patient response to treatment with TKIs remains largely unexplained. There is evidence that it could be due, at least in part, to alterations in genes associated with the disease via their effect on the expression of TKI targets. The objective of this study was to evaluate the influence of RAS mutations on the expression levels in MTC tumors of eight key TKI target proteins. We assessed by immunohistochemistry the expression of EGFR, KIT, MET, PDGFRB, VEGF, VEGFR1, VEGFR2, and VEGFR3 in a series of 84 primary MTC tumors that had previously been molecularly characterized, including 14 RAS-positive, 18 RET(M918T)-positive, and 24 RET(C634)-positive tumors, as well as 15 wild-type tumors with no mutations in the RET or RAS genes. In contrast to RET-positive tumors, RAS-positive tumors expressed neither PDGFRB nor MET (p=0.0060 and 0.047, respectively). Similarly, fewer RAS-positive than RET-related tumors expressed VEGFR3 (p=0.00062). Finally, wild-type tumors expressed VEGF more often than both RAS- and RET-positive tumors (p=0.0082 and 0.011, respectively). This is the first study identifying that the expression of TKI targets differs according to the presence of RAS mutations in MTC. This information could potentially be used to select the most beneficial TKI treatment for these patients.

Carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4) is specifically expressed in medullary thyroid carcinoma cells.

Carcinoembryonic antigen (CEA), an oncofetal cell surface glycoprotein, has been widely used as a human tumor marker due to its high expression in tumors and secretion to serum. It belongs to the immunoglobulin superfamily named CEA-related cell adhesion molecule (CEACAM) family. Members of this family are detected in various cancers and have been shown to be involved in cancer growth and invasion. In this study, we examined the mRNA expression profiles of CEACAM family members including CEACAM1, CEACAM3, CEACAM4, CEACAM5 (CEA), CEACAM6, CEACAM7, and CEACAM8 in various tumor cell lines. Our screening data indicated that the mRNA expression patterns of CEACAMs in TT cells, which are derived from medullary thyroid carcinoma (MTC), were distinct from other tumor cell lines. Additionally, CEACAM4 was only expressed in TT cells, in which two novel splice variants of CEACAM4 were expressed. These findings suggested that production of CEA and CEA-related molecules in MTC may be distinct from other tumor-based production of those molecules and that the specific expression of CEACAM4 would make possible to differentiate between MTC and other CEA-producing tumors.

Abstract 346: p53 mutation in Rb deficient neuroendocrine lung and thyroid cells results in high grade neuroendocrine carcinomas promoted by mTOR pathway activation .

Abstract Small cell lung carcinoma (SCLC) and medullary thyroid carcinoma (MTC) are aggressive, rapidly metastatic neuroendocrine carcinomas that portend a dismal prognosis. Rb loss is detected in ≥ 90% of SCLC and p53 is mutated in ∼80% of cases providing evidence that Rb and p53 suppress SCLC. Extensive genetic evidence in mouse models also supports a critical role for Rb in suppressing MTC: however, the mechanisms promoting tumorigenesis in Rb and p53 deficient cells and their relation to therapeutic response remain unclear. In this study, a conditional SCLC and MTC mouse model was generated by targeting Rb and p53 ablation or mutant protein expression to lung and thyroid epithelial cells. Rb loss alone resulted in slowly progressing MTC and lung neuroendocrine hyperplasia. While p53 alteration alone was not sufficient for tumor initiation, p53 loss or mutation in Rb deficient cells led to MTC and SCLC with metastases, phenotypically mimicking human disease. Mice expressing p53 R172H (R175H in human), but not R270H (R273H in human), mutant protein had a gain-of-function phenotype as evidenced by decreased survival of Rb/p53R172H as compared to Rb/p53 ablated mice. No dominant negative effect was detected by comparing Rb/p53+/mutant to Rb/p53+/− mice. Mechanistic studies revealed p53 dependent mammalian target of rapamycin (mTOR) pathway activation in MTC without coincident AKT or MAPK pathway activation. Similar mTOR pathway activation was detected in Rb/p53 deficient and mutant lungs. Rapamycin dramatically inhibited mTOR pathway signaling in Rb/p53 deficient or mutant thyroid tumors and lungs in vivo as well as in MTC and SCLC tumor cells in culture. Interestingly, mTORC1 inhibition was not associated with feedback AKT or MAPK pathway activation in vitro or in vivo as previously reported for breast and prostate cancers. Rapamycin inhibited MTC and SCLC tumor cell growth demonstrating a functional role for mTOR activation in tumor progression. These studies identify distinct, cooperative Rb and p53 tumor suppressive functions. The oncogenic stimulus provided by Rb loss induces aberrant cell growth and leads to a p53 tumor suppressive response that inhibits tumor progression and mTOR dependent cell growth demonstrating that mTOR pathway inhibition is a critical p53 tumor suppressive function. Notably, rapamycin did not result in feedback MAPK or AKT pathway activation providing evidence this is not a general mechanism limiting therapeutic response to mTORC1 inhibition. Together these studies provide mechanistic links between genetic alterations and aberrant signaling pathways critical in carcinogenesis. In addition to providing mechanistic insights, this genetically engineered mouse model provides a robust preclinical platform to explore novel therapeutic strategies and define mechanisms underlying treatment response and resistance. Citation Format: Nagako Akeno, Ashley Miller, Ncik Levinsky, Kathryn A. Wikenheiser-Brokamp. p53 mutation in Rb deficient neuroendocrine lung and thyroid cells results in high grade neuroendocrine carcinomas promoted by mTOR pathway activation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2013-346

Simultaneous Medullary and Differentiated Thyroid Cancer: A Population-Level Analysis of an Increasingly Common Entity

Simultaneous medullary thyroid carcinoma (MTC) and differentiated thyroid carcinoma (DTC) is a rare entity. This is the first population-level analysis of the characteristics and outcomes of simultaneous MTC/DTC. In the Surveillance, Epidemiology, and End Results (SEER) database (1988-2008), patients with simultaneous MTC/DTC were retrospectively compared with those with MTC alone using χ(2), ANOVA, log-rank tests, Cox multivariate regression, and Kaplan-Meier analyses. A total of 162 patients had simultaneous MTC/DTC; 1,699 had MTC alone. MTC was diagnosed first in 67.9 % of simultaneous MTC/DTC cases. Simultaneous MTC/DTC increased from 2.7 % of all MTCs in 1988-1997 to 12.3 % in 2003-2008. Compared with MTC alone, simultaneous MTC/DTC had smaller mean MTC tumor size (2.9 vs. 2.2 cm; p = 0.005) and lower rates of MTC extrathyroidal extension (25.4 vs. 16.8 %; p = 0.015) and distant metastases (15.7 vs. 9.3 %; p = 0.032). Patients diagnosed with DTC first had smaller mean MTC tumor sizes (p = 0.01), whereas patients diagnosed with MTC first had tumor sizes similar to those of MTC alone. Compared with MTC alone, patients with simultaneous MTC/DTC were more likely to receive thyroidectomy (84.7 vs. 93.2 %; p = 0.003) and radioisotopes (4.4 vs. 25 %; p < 0.001). On Kaplan-Meier analysis, disease-specific survival rates were higher for simultaneous MTC/DTC than for MTC alone (10-year survival rates 87 vs. 81 %; p = 0.056). Simultaneous MTC/DTC is diagnosed earlier in tumor development than MTC alone, with a trend toward better prognosis. This entity likely represents a primary tumor with an incidental pathologic finding of a second malignancy. Each malignancy should be treated according to its respective stage and current guidelines.

Metastasis to the breast from medullary thyroid carcinoma

ABSTRACTMetastasis to the breast from medullary thyroid carcinoma (MTC) is extremely rare and the associate cytological findings have not been reported in Korea until now. We report a case of metastatic MTC presented as palpable neck and breast nodules in a 39‐year‐old woman. Fine needle aspiration of breast nodules showed a cellular smear with relatively homogenous, poorly cohesive tumor cells in a relatively clean background. The tumor cells were round to oval, with hyperchromatic nuclei, abundant cytoplasm and plasmacytoid in appearance. A few amorphous materials, possibly amyloid, were also noted. The patient underwent radical thyroidectomy, cervical lymph node dissection and excision of breast nodules. The primary thyroid mass was typical of MTC but metastatic breast tumors showed more homogenous and smaller tumor cells. The cellular origin of the breast tumors was confirmed by their immunoreactivity for calcitonin, chromogranin, synaptophysin and TTF‐1. Distinguishing MTC from primary breast cancer via fine needle aspiration cytology of breast nodules is important in planning treatment. Detection of amyloid‐like materials and immunohistochemical staining for calcitonin or TTF‐1 is helpful in diagnosing metastatic MTC in breast nodules when clinically suspected.