Building a vertically integrated genomic learning health system: The biobank at the Colorado Center for Personalized Medicine.

Precision medicine initiatives across the globe have led to a revolution of repositories linking large-scale genomic data with electronic health records, enabling genomic analyses across the entire phenome. Many of these initiatives focus solely on research insights, leading to limited direct benefit to patients. We describe the biobank at the Colorado Center for Personalized Medicine (CCPM Biobank) that was jointly developed by the University of Colorado Anschutz Medical Campus and UCHealth to serve as a unique, dual-purpose research and clinical resource accelerating personalized medicine. This living resource currently has more than 200,000 participants with ongoing recruitment. We highlight the clinical, laboratory, regulatory, and HIPAA-compliant informatics infrastructure along with our stakeholder engagement, consent, recontact, and participant engagement strategies. We characterize aspects of genetic and geographic diversity unique to the Rocky Mountain region, the primary catchment area for CCPM Biobank participants. We leverage linked health and demographic information of the CCPM Biobank participant population to demonstrate the utility of the CCPM Biobank to replicate complex trait associations in the first 33,674 genotyped individuals across multiple disease domains. Finally, we describe our current efforts toward return of clinical genetic test results, including high-impact pathogenic variants and pharmacogenetic information, and our broader goals as the CCPM Biobank continues to grow. Bringing clinical and research interests together fosters unique clinical and translational questions that can be addressed from the large EHR-linked CCPM Biobank resource within a HIPAA- and CLIA-certified environment.

Thyroid Cancer Polygenic Risk Score Improves Classification of Thyroid Nodules as Benign or Malignant.

CONTEXT: Thyroid nodule ultrasound-based risk stratification schemas rely on the presence of high-risk sonographic features. However, some malignant thyroid nodules have benign appearance on thyroid ultrasound. New methods for thyroid nodule risk assessment are needed. OBJECTIVE: We investigated polygenic risk score (PRS) accounting for inherited thyroid cancer risk combined with ultrasound-based analysis for improved thyroid nodule risk assessment. METHODS: The convolutional neural network classifier was trained on thyroid ultrasound still images and cine clips from 621 thyroid nodules. Phenome-wide association study (PheWAS) and PRS PheWAS were used to optimize PRS for distinguishing benign and malignant nodules. PRS was evaluated in 73 346 participants in the Colorado Center for Personalized Medicine Biobank. RESULTS: When the deep learning model output was combined with thyroid cancer PRS and genetic ancestry estimates, the area under the receiver operating characteristic curve (AUROC) of the benign vs malignant thyroid nodule classifier increased from 0.83 to 0.89 (DeLong, P value = .007). The combined deep learning and genetic classifier achieved a clinically relevant sensitivity of 0.95, 95% CI [0.88-0.99], specificity of 0.63 [0.55-0.70], and positive and negative predictive values of 0.47 [0.41-0.58] and 0.97 [0.92-0.99], respectively. AUROC improvement was consistent in European ancestry-stratified analysis (0.83 and 0.87 for deep learning and deep learning combined with PRS classifiers, respectively). Elevated PRS was associated with a greater risk of thyroid cancer structural disease recurrence (ordinal logistic regression, P value = .002). CONCLUSION: Augmenting ultrasound-based risk assessment with PRS improves diagnostic accuracy.

Fibronectin Contributes to a BRAF Inhibitor-driven Invasive Phenotype in Thyroid Cancer through EGR1, Which Can Be Blocked by Inhibition of ERK1/2.

Mutations in BRAF are common in advanced papillary and anaplastic thyroid cancer (PTC and ATC). However, patients with BRAF-mutant PTC currently lack therapies targeting this pathway. Despite the approved combination of BRAF and MEK1/2 inhibition for patients with BRAF-mutant ATC, these patients often progress. Thus, we screened a panel of BRAF-mutant thyroid cancer cell lines to identify new therapeutic strategies. We showed that thyroid cancer cells resistant to BRAF inhibition (BRAFi) exhibit an increase in invasion and a proinvasive secretome in response to BRAFi. Using reverse-phase protein array (RPPA), we identified a nearly 2-fold increase in expression of the extracellular matrix protein, fibronectin, in response to BRAFi treatment, and a corresponding 1.8- to 3.0-fold increase in fibronectin secretion. Accordingly, the addition of exogenous fibronectin phenocopied the BRAFi-induced increase in invasion while depletion of fibronectin in resistant cells resulted in loss of increased invasion. We further showed that BRAFi-induced invasion can be blocked by inhibition of ERK1/2. In a BRAFi-resistant patient-derived xenograft model, we found that dual inhibition of BRAF and ERK1/2 slowed tumor growth and decreased circulating fibronectin. Using RNA sequencing, we identified EGR1 as a top downregulated gene in response to combined BRAF/ERK1/2 inhibition, and we further showed that EGR1 is necessary for a BRAFi-induced increase in invasion and for induction of fibronectin in response to BRAFi. IMPLICATIONS: Together, these data show that increased invasion represents a new mechanism of resistance to BRAF inhibition in thyroid cancer that can be targeted with an ERK1/2 inhibitor.

Computer-Assisted Levothyroxine Dose Selection for the Treatment of Postoperative Hypothyroidism.

Background: Thyroid hormone replacement with levothyroxine (LT4) is a recommended treatment for patients undergoing thyroidectomy. The starting LT4 dose is frequently calculated based on the patient's weight. However, the weight-based LT4 dosing performs poorly in clinical practice, with only ∼30% of patients achieving target thyrotropin (TSH) levels at the first thyroid function testing after treatment initiation. A better way to calculate the LT4 dose for patients with postoperative hypothyroidism is needed. Methods: In this retrospective cohort study we used demographic, clinical, and laboratory data for 951 patients after thyroidectomy and several regression and classification machine learning methods to develop an LT4 dose calculator for treating postoperative hypothyroidism targeting the desired TSH level. We compared the accuracy with the current standard-of-care practice and other published algorithms and evaluated generalizability with fivefold cross-validation and out-of-sample testing. Results: The retrospective clinical chart review showed that only 285/951 (30%) patients met their postoperative TSH goal. Obese patients were overtreated with LT4. An ordinary least squares regression based on weight, height, age, sex, calcium supplementation, and height:sex interaction predicted prescribed LT4 dose in 43.5% of all patients and 45.3% of patients with normal postoperative TSH (0.45-4.5 mIU/L). The ordinal logistic regression, artificial neural networks regression/classification, and random forest methods achieved comparable performance. LT4 calculator recommended lower LT4 doses to obese patients. Conclusions: The standard-of-care LT4 dosing does not achieve the target TSH in most thyroidectomy patients. Computer-assisted LT4 dose calculation performs better by considering multiple relevant patient characteristics and providing personalized and equitable care to patients with postoperative hypothyroidism. Prospective validation of LT4 calculator performance in patients with various TSH goals is needed.

BCL2L11 Induction Mediates Sensitivity to Src and MEK1/2 Inhibition in Thyroid Cancer.

Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response.

Migrating a research data warehouse to a public cloud: challenges and opportunities.

OBJECTIVE: Clinical research data warehouses (RDWs) linked to genomic pipelines and open data archives are being created to support innovative, complex data-driven discoveries. The computing and storage needs of these research environments may quickly exceed the capacity of on-premises systems. New RDWs are migrating to cloud platforms for the scalability and flexibility needed to meet these challenges. We describe our experience in migrating a multi-institutional RDW to a public cloud. MATERIALS AND METHODS: This study is descriptive. Primary materials included internal and public presentations before and after the transition, analysis documents, and actual billing records. Findings were aggregated into topical categories. RESULTS: Eight categories of migration issues were identified. Unanticipated challenges included legacy system limitations; network, computing, and storage architectures that realize performance and cost benefits in the face of hyper-innovation, complex security reviews and approvals, and limited cloud consulting expertise. DISCUSSION: Cloud architectures enable previously unavailable capabilities, but numerous pitfalls can impede realizing the full benefits of a cloud environment. Rapid changes in cloud capabilities can quickly obsolete existing architectures and associated institutional policies. Touchpoints with on-premise networks and systems can add unforeseen complexity. Governance, resource management, and cost oversight are critical to allow rapid innovation while minimizing wasted resources and unnecessary costs. CONCLUSIONS: Migrating our RDW to the cloud has enabled capabilities and innovations that would not have been possible with an on-premises environment. Notwithstanding the challenges of managing cloud resources, the resulting RDW capabilities have been highly positive to our institution, research community, and partners.

Targeted genomic analysis of 364 adrenocortical carcinomas.

Despite recent advances in elucidating molecular pathways underlying adrenocortical carcinoma (ACC), this orphan malignancy is associated with poor survival. Identification of targetable genomic alterations is critical to improve outcomes. The objective of this study was to characterize the genomic profile of a large cohort of patient ACC samples to identify actionable genomic alterations. Three hundred sixty-four individual patient ACC tumors were analyzed. The median age of the cohort was 52 years and 60.9% (n = 222) were female. ACC samples had common alterations in epigenetic pathways with 38% of tumors carrying alterations in genes involved in histone modification, 21% in telomere lengthening, and 21% in SWI/SNF complex. Tumor suppressor genes and WNT signaling pathway were each mutated in 51% of tumors. Fifty (13.7%) ACC tumors had a genomic alteration in genes involved in the DNA mismatch repair (MMR) pathway with many tumors also displaying an unusually high number of mutations and a corresponding MMR mutation signature. In addition, genomic alterations in several genes not previously associated with ACC were observed, including IL7R, LRP1B, FRS2 mutated in 6, 8 and 4% of tumors, respectively. In total, 58.5% of ACC (n = 213) had at least one potentially actionable genomic alteration in 46 different genes. As more than half of ACC have one or more potentially actionable genomic alterations, this highlights the value of targeted sequencing for this orphan cancer with a poor prognosis. In addition, significant incidence of MMR gene alterations suggests that immunotherapy is a promising therapeutic for a considerable subset of ACC patients.

Mastermind Like Transcriptional Coactivator 3 (MAML3) Drives Neuroendocrine Tumor Progression.

Metastatic disease in pheochromocytomas and paragangliomas (PCC/PGL) is not well-understood. The Cancer Genome Atlas discovered recurrent MAML3 fusion genes in a subset of tumors that lacked known germline or somatic driver mutations and were associated with aggressive disease. Here, we aimed to investigate the role of MAML3 in tumorigenesis. Human PCC/PGLs were used for IHC and genetic analysis. Three neuroendocrine tumor cell lines, SK-N-SH, QGP-1, and BON-1, were transiently transfected with MAML3 (FL) or exon 1 deleted MAML3 (dEx1; mimicking the fusion), and biologic effects of overexpression were examined in vitro. We found 7% (4/55) of human PCC/PGL have UBTF∼MAML3 fusions and all were sporadic cases with metastatic disease. Fusion-positive tumors had intense MAML3 nuclear staining and increased ß-catenin by IHC and showed increased WNT4 expression. In vitro, overexpression of FL and dEx1 MAML3 increased invasion in SK-N-SH, QGP-1, and BON-1 (all P < 0.05) and increased soft-agar colony formation in QGP-1 and BON-1 (all P < 0.05). Cotransfection with FL or dEx1 MAML3 and ß-catenin increased TCF/LEF promoter activation by luciferase activity and coimmunoprecipitation confirmed interaction between MAML3 and ß-catenin. These data suggest MAML3 is involved in WNT signaling pathway activation. In summary, UBTF∼MAML3 fusions are present in a subset of PCC/PGL and associated with metastatic disease without other known drivers. MAML3 overexpression led to increased tumorigenicity in neuroendocrine tumor cells and the mechanism of action may involve WNT signaling pathways. IMPLICATIONS: MAML3 increases tumorigenicity and invasion in neuroendocrine tumor cells and may be a prognostic marker for aggressive disease.

Inhibition of BRAF and ERK1/2 has synergistic effects on thyroid cancer growth in vitro and in vivo.

Mutations in the BRAF gene are highly prevalent in thyroid cancer. However, the response rate of thyroid tumors to BRAF-directed therapies has been mixed. Increasingly, combination therapies inhibiting the MAPK pathway at multiple nodes have shown promise. Recently developed ERK1/2 inhibitors are of interest for use in combination therapies as they have the advantage of inhibiting the most downstream node of the MAPK pathway, therefore preventing pathway reactivation. Here, we examined the effect of combined BRAF inhibition (dabrafenib) and ERK1/2 inhibition (SCH772984) on the growth and survival of a panel of BRAF-mutant thyroid cancer cell lines using in vitro and in vivo approaches. We found that resistance due to MAPK pathway reactivation occurs quickly with single-agent BRAF inhibition, but can be prevented with combined BRAF and ERK1/2 inhibition. Combined inhibition also results in synergistic growth inhibition, decreased clonogenic survival, and enhanced induction of apoptosis in a subset of BRAF-mutant thyroid cancer cells. Finally, combined inhibition of BRAF and ERK1/2 results in enhanced inhibition of tumor growth in an anaplastic thyroid cancer in vivo model. These results provide key rationale to pursue combined BRAF and ERK1/2 inhibition as an alternative therapeutic strategy for BRAF-mutant advanced thyroid cancer patients.

Comprehensive Immune Profiling of Medullary Thyroid Cancer.

Background: Despite advances in targeted kinase inhibitor development for patients with medullary thyroid cancer (MTC), most patients develop resistance and would benefit from alternative approaches. Immune-based therapies are now considered for patients with progressive MTC. This study is the first comprehensive assessment of the immune milieu, immune-suppressive molecules, and potential tumor antigens in patients with MTC. Methods: Primary and/or regionally metastatic tumor tissues from 46 patients with MTC were screened for immune infiltrates by using standard immunohistochemistry (IHC) and further analyzed by multispectral imaging for T cell and myeloid markers. RNASeq expression profiling was performed in parallel. RNASeq, targeted sequencing, and IHC techniques identified cancer-associated mutations and MTC-enriched proteins. Results: Organized immune infiltration was observed in 49% and 90% of primary and metastatic tumors, respectively. CD8+ cells were the dominant T cell subtype in most samples, while CD163+ macrophages were most frequent among myeloid infiltrates. PD-1+ T cells were evident in 24% of patients. Myeloid subsets were largely major histocompatibility complex II (MHCII-), suggesting a dysfunctional phenotype. Expression profiling confirmed enrichment in T cell, macrophage, and inflammatory profiles in a subset of samples. PD-L1 was expressed at low levels in a small subset of patients, while the immune regulatory molecules CD155 and CD47 were broadly expressed. Calcitonin, GRP, HIST1H4E, NOMO3, and NPIPA2 were highly and specifically expressed in MTC. Mutations in tumor suppressors, PTEN and p53, and mismatch repair genes, MSH2 and MSH6, may be relevant to disease progression and antigenicity. Conclusions: This study suggests that MTC is a more immunologically active tumor that has been previously reported. Patients with advanced MTC should be screened for targetable antigens and immune checkpoints to determine their eligibility for current clinical trials. Additional studies are necessary to fully characterize the antigenic potential of MTC and may encourage the development of adoptive T cells therapies for this rare tumor.

Leptomeningeal Metastasis from Adrenocortical Carcinoma: A Case Report.

Adrenocortical carcinoma (ACC) is an uncommon endocrine malignancy with limited treatment options. While the overall 5-year survival rate in patients with ACC is 35%, the disease is often rapidly progressive with long-term survival in only 5% of patients. Although tumor stage, grade, and excess hormonal activity predict unfavorable prognosis, additional biomarkers are needed to identify patients with aggressive disease. A 23-year-old woman presented with rapidly progressing signs and symptoms of Cushing's syndrome, with associated abdominal pain and fullness. Evaluation revealed a large left adrenal mass which had developed over 8 months. En bloc surgical resection was performed by an endocrine surgeon, and pathology revealed adrenocortical carcinoma with Ki67 of 60%. Despite adjuvant treatment with mitotane and etoposide-doxorubicin-carboplatin chemotherapy, the patient had rapid disease progression with metastatic spread to liver, lung, bone, brain, and leptomeningies, and she died 11 months after the initial diagnosis. Subsequent analysis of the patient's tumor revealed mutations in TP53 and MEN1. RNA sequencing was compared against the the Cancer Genome Atlas data set and clustered with the high steroid, proliferative subtype, associated with the worst prognosis. The tumor also demonstrated a low BUB1B/PINK1 ratio and G0S2 hypermethylation, both predictive of very aggressive ACC. This case represents a subset of ACC characterized by rapid and fatal progression. Clinically available predictors as well as recently reported molecular signatures and biomarkers correlated with this tumor's aggressiveness, suggesting that development and validation of combinations of biomarkers may be useful in guiding personalized approaches to patients with ACC.

Molecular therapeutics for anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) represents one of the most lethal human cancers and although this tumor type is rare, ATC accounts for the majority of deaths from thyroid cancer. Due to the rarity of ATC, a comprehensive genomic characterization of this tumor type has been challenging, and thus the development of new therapies has been lacking. To date, there is only one mutation-driven targeted therapy for BRAF-mutant ATC. Recent genomic studies have used next generation sequencing to define the genetic landscape of ATC in order to identify new therapeutic targets. Together, these studies have confirmed the role of oncogenic mutations of MAPK pathway as key drivers of differentiated thyroid cancer (BRAF, RAS), and that additional genetic alterations in the PI3K pathway, TP53, and the TERT promoter are necessary for anaplastic transformation. Recent novel findings have linked the high mutational burden associated with ATC with mutations in the Mismatch Repair (MMR) pathway and overactivity of the AID/APOBEC family of cytidine deaminases. Additional novel mutations include cell cycle genes, SWI/SNF chromatin remodeling complex, and histone modification genes. Mutations in RAC1 were also identified in ATC, which have important implications for BRAF-directed therapies. In this review, we summarize these novel findings and the new genetic landscape of ATC. We further discuss the development of therapies targeting these pathways that are being tested in clinical and preclinical studies.

Adherence to Recommended Post-Splenectomy Immunizations to Reduce the Risk of Sepsis: The University of Washington Experience.

Immunizations against Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b are recommended for patients undergoing splenectomy to decrease the risk of developing overwhelming infections. The authors sought to determine adherence to these recommendations by providers at UW Medicine. Regional immunization records for patients who underwent splenectomy between October 2015 and January 2019 were analyzed to measure compliance with immunization guidelines from the US Centers for Disease Control and Prevention (CDC). Among 253 patients who underwent splenectomy, 38 (15%) received all 7 immunizations against S pneumoniae, N meningitidis, and H influenzae type b recommended by the CDC; 95% of patients received at least 1 pneumococcal vaccine; 26% percent of patients did not receive MenB-4C vaccine. Many patients (3% to 10%) received redundant immunizations not in accordance with CDC recommendations. Development of state and national immunization registries and systems to improve adherence with post-splenectomy immunization guidelines may reduce risk for life-threatening infections.

Comprehensive Genetic Characterization of Human Thyroid Cancer Cell Lines: A Validated Panel for Preclinical Studies.

PURPOSE: Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers. EXPERIMENTAL DESIGN: We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers. RESULTS: Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAFV600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization. CONCLUSIONS: This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.

Establishment and Characterization of Four Novel Thyroid Cancer Cell Lines and PDX Models Expressing the RET/PTC1 Rearrangement, BRAFV600E, or RASQ61R as Drivers.

Cancer cell lines are critical models to study tumor progression and response to therapy. In 2008, we showed that approximately 50% of thyroid cancer cell lines were redundant or not of thyroid cancer origin. We therefore generated new authenticated thyroid cancer cell lines and patient-derived xenograft (PDX) models using in vitro and feeder cell approaches, and characterized these models in vitro and in vivo. We developed four thyroid cancer cell lines, two derived from 2 different patients with papillary thyroid cancer (PTC) pleural effusions, CUTC5, and CUTC48; one derived from a patient with anaplastic thyroid cancer (ATC), CUTC60; and one derived from a patient with follicular thyroid cancer (FTC), CUTC61. One PDX model (CUTC60-PDX) was also developed. Short tandem repeat (STR) genotyping showed that each cell line and PDX is unique and match the original patient tissue. The CUTC5 and CUTC60 cells harbor the BRAF (V600E) mutation, the CUTC48 cell line expresses the RET/PTC1 rearrangement, and the CUTC61 cells have the HRAS (Q61R) mutation. Moderate to high levels of PAX8 and variable levels of NKX2-1 were detected in each cell line and PDX. The CUTC5 and CUTC60 cell lines form tumors in orthotopic and flank xenograft mouse models. IMPLICATIONS: We have developed the second RET/PTC1-expressing PTC-derived cell line in existence, which is a major advance in studying RET signaling. We have further linked all cell lines to the originating patients, providing a set of novel, authenticated thyroid cancer cell lines and PDX models to study advanced thyroid cancer.

Resistance to Src inhibition alters the BRAF-mutant tumor secretome to promote an invasive phenotype and therapeutic escape through a FAK>p130Cas>c-Jun signaling axis.

Few therapy options exist for patients with advanced papillary and anaplastic thyroid cancer. We and others have previously identified c-Src as a key mediator of thyroid cancer pro-tumorigenic processes and a promising therapeutic target for thyroid cancer. To increase the efficacy of targeting Src in the clinic, we sought to define mechanisms of resistance to the Src inhibitor, dasatinib, to identify key pathways to target in combination. Using a panel of thyroid cancer cell lines expressing clinically relevant mutations in BRAF or RAS, which were previously developed to be resistant to dasatinib, we identified a switch to a more invasive phenotype in the BRAF-mutant cells as a potential therapy escape mechanism. This phenotype switch is driven by FAK kinase activity, and signaling through the p130Cas>c-Jun signaling axis. We have further shown this more invasive phenotype is accompanied by alterations in the secretome through the increased expression of pro-inflammatory cytokines, including IL-1ß, and the pro-invasive metalloprotease, MMP-9. Furthermore, IL-1ß signals via a feedforward autocrine loop to promote invasion through a FAK>p130Cas>c-Jun>MMP-9 signaling axis. We further demonstrate that upfront combined inhibition of FAK and Src synergistically inhibits growth and invasion, and induces apoptosis in a panel of BRAF- and RAS-mutant thyroid cancer cell lines. Together our data demonstrate that acquired resistance to single-agent Src inhibition promotes a more invasive phenotype through an IL-1ß>FAK>p130Cas>c-Jun >MMP signaling axis, and that combined inhibition of FAK and Src has the potential to block this inhibitor-induced phenotype switch.

Hypofractionated Radiotherapy Is Superior to Conventional Fractionation in an Orthotopic Model of Anaplastic Thyroid Cancer.

BACKGROUND: Anaplastic thyroid cancer (ATC) is an aggressive and highly lethal disease with poor outcomes and resistance to therapy. Despite multimodality treatment, including radiation therapy and chemotherapy, response rates remain <15%, with a median time to progression of less than three months. Recent advances in radiotherapy (RT) delivery and gene-expression profiling may help guide patient selection for personalized therapy. The purpose of this study was to characterize the response to radiation in a panel of ATC cell lines and to test alternative RT fractionation schedules for overcoming radioresistance. MATERIALS AND METHODS: The cellular response to radiation was characterized based on clonogenic assays. Radiation response was correlated with microarray gene-expression data. Hypofractionated and conventional RT was tested in an orthotopic ATC tumor model, and tumor growth was assayed locally and distantly with in vivo and ex vivo bioluminescence imaging. RESULTS: A spectrum of radiosensitivities was observed in ATC cell lines. Radioresistant cell lines had higher levels of CXCR4 compared to radiosensitive cell lines. Compared to conventionally fractionated RT, hypofractionated RT resulted in significantly improved tumor growth delay, decreased regional and distant metastases, and improved overall survival. CONCLUSIONS: The findings demonstrate the heterogeneity of response to radiation in ATC tumors and the superiority of hypofractionated RT in improving local control, metastatic spread, and survival in preclinical models. These data support the design of clinical trials targeting radioresistant pathways in combination with hypofractionated RT.

Outcomes of Bethesda categories III and IV thyroid nodules over 5 years and performance of the Afirma gene expression classifier: A single-institution study.

OBJECTIVE: The second edition Bethesda System for Reporting Thyroid Cytology estimates 6%-18% malignancy rate of category III (B3) and 10%-40% for category IV (B4) nodules; however, reported malignancy rates have considerable variability among institutions. Use of molecular classifiers (including Afirma Gene Expression Classifier, GEC) can be utilized in management of thyroid nodules. Our objective was to analyse malignancy rates of B3 and B4 nodules and determine clinical outcomes of GEC Benign nodules. METHODS: A retrospective analysis of 2019 thyroid FNAs was performed at the University of Colorado from 2011 to 2015, including molecular, surgical and clinical follow-up. RESULTS: Of 2019 FNAs analysed, 231 (11.4%) were diagnosed as B3 and 80 (4.0%) as B4. GEC was obtained in 54.1% of B3 cases, with nearly half (48.8%) having a Benign result. Surgery was performed in 40.7% B3 cases with a 24.5% malignancy rate, ranging 8%-38% by year. In the B4 group, 52.5% underwent molecular testing with 28.6% as GEC Benign. About 68.8% of B4 cases underwent surgery with a 20% malignancy rate, ranging 0%-42% by year. Seventy-three GEC Benign cases were reviewed: 5 (6.8%) underwent surgery, with none demonstrating malignancy in the target nodule. Size remained stable for most GEC Benign nodules: 75.9% (B3) and 71.4% (B4) with no malignancy on repeat FNA. CONCLUSIONS: Our 5-year review demonstrated that malignancy rates of B3 and B4 nodules showed year-to-year variability. We suggest that clinicians use a multi-year average of their institution's malignancy rates to optimally manage patients. Follow-up for GEC Benign cases thus far supports their indolent nature.

Genetic Analysis of 779 Advanced Differentiated and Anaplastic Thyroid Cancers.

Purpose: To define the genetic landscape of advanced differentiated and anaplastic thyroid cancer (ATC) and identify genetic alterations of potential diagnostic, prognostic, and therapeutic significance.Experimental Design: The genetic profiles of 583 advanced differentiated and 196 ATCs generated with targeted next-generation sequencing cancer-associated gene panels MSK-IMPACT and FoundationOne were analyzed.Results: ATC had more genetic alterations per tumor, and pediatric papillary thyroid cancer had fewer genetic alterations per tumor when compared with other thyroid cancer types. DNA mismatch repair deficit and activity of APOBEC cytidine deaminases were identified as mechanisms associated with high mutational burden in a subset of differentiated thyroid cancers and ATCs. Copy number losses and mutations of CDKN2A and CDKN2B, amplification of CCNE1, amplification of receptor tyrosine kinase genes KDR, KIT, and PDGFRA, amplification of immune evasion genes CD274, PDCD1LG2, and JAK2, and activating point mutations in small GTPase RAC1 were associated with ATC. An association of KDR, KIT, and PDGFRA amplification with the sensitivity of thyroid cancer cells to lenvatinib was shown in vitro Three genetically distinct types of ATCs are proposed.Conclusions: This large-scale analysis describes genetic alterations in a cohort of thyroid cancers enriched in advanced cases. Many novel genetic events previously not seen in thyroid cancer were found. Genetic alterations associated with anaplastic transformation were identified. An updated schematic of thyroid cancer genetic evolution is proposed. Clin Cancer Res; 24(13); 3059-68. ©2018 AACR.