Assessing Adequacy: A Meta-Analysis of Rapid Onsite Evaluation of Thyroid Nodules.

INTRODUCTION: Fine-needle aspiration (FNA) is the standard form of preoperative evaluation of thyroid nodule cytological status. A significant number FNAs are classified as inadequate for interpretation, requiring a repeat FNA which is potentially avoidable, costly, and delays treatment. To address these concerns and maximize first-time FNA adequacy, rapid onsite evaluation (ROSE) of FNA specimens was introduced. Our study aims to determine the impact of ROSE on FNA adequacy. METHODS: PubMed, Embase, and Web of Science were searched for primary articles assessing the adequacy of ROSE in thyroid nodules. RESULTS: A total of 17 studies were included for a total of 24,649 thyroid nodes. Thirteen thousand two hundred fifteen (53.6%) thyroid nodules were assessed utilizing ROSE and 11,434 (46.4%) were not. Pooled adequacy increased significantly from 76% without ROSE to 92% with rose (P = 0.001). Use of ROSE increased the odds of adequate FNA by 22% (risk ratio (RR) = 1.22, 95% confidence interval (CI) = 1.12-1.32). At institutions with less than 85% effective diagnostic adequacy without ROSE, the risk for diagnostic adequacy increased by 28% with ROSE implementation (RR = 1.28, 95% CI = 1.20-1.37). In contrast, in studies reported from institutions with an effective diagnostic rate greater than 85% without the use of ROSE, the diagnostic adequacy only increased by 5% with ROSE implementation (RR = 1.05, 95% CI = 1.03-1.06). CONCLUSIONS: The use of ROSE during first-time FNA of thyroid nodules can significantly improve adequacy, especially at institutions with baseline high inadequacy rates. Implementation of ROSE can reduce repeat FNAs and its associated consequences.

Survival Outcomes of Medullary Thyroid Cancer With and Without Amyloid Deposition.

OBJECTIVE: Amyloid deposition within tumor stroma is a distinctive histologic feature of medullary thyroid cancer (MTC). However, its prognostic significance remains uncertain. We aimed to elucidate the impact of amyloid status on survival outcomes in a large cohort. METHODS: The Surveillance, Epidemiology, and End Results registry was queried to identify patients diagnosed with MTC from 2000 to 2019. Patients with amyloid-positive (International Classification of Diseases for Oncology, third edition code 8345/3) and amyloid negative (International Classification of Diseases for Oncology, third edition code 8510/3) tumors were analyzed. Overall and disease-specific survival were compared between matched cohorts using Kaplan-Meier and Cox proportional hazards analyses. RESULTS: Of the 2526 MTC patients, 511 of which were amyloid-positive and 2015 that were amyloid negative. Amyloid-positive patients displayed lower T stage (T3/4: 28% vs 85%, P < .001) and less extrathyroidal extension (11.3% vs 81.6%, P < .001). No difference in distant metastasis rate was observed between groups (14.5% vs 14.4%, P = .98). However, amyloid-positive patients showed a tendency for distal lymph node metastasis (1.2% vs 0.3%, P = .020). On univariate analysis, amyloid-positive status showed comparable overall survival times (mean 172.2 vs 177.8 months, P = .17), but a trend toward worse cancer-specific survival (hazard ratios [HR] = 1.31, 95% CI = 0.99-1.71, P = .051). After adjusting for covariates, amyloid deposition did not independently predict overall (HR = 1.15, 95% CI = 0.91-1.47, P = .25) or cancer-specific survival (HR = 1.30, 95% CI = 0.96-1.77, P = .09). Initiating therapy later than 1 month following diagnosis was associated with worse overall survival (HR = 1.25, 95% CI = 1.02-1.54, P = .029). CONCLUSIONS: The presence of amyloid in MTC paradoxically associates with lower T stage yet exhibits a trend toward worse cancer-specific mortality. Amyloid deposition alone does not independently influence prognosis. Delayed treatment adversely impacted overall survival.

CD4+ T helper 17 cell response of aged mice promotes prostate cancer cell migration and invasion.

BACKGROUND: Aging is the most important risk factor for prostate cancer (PCa), but how age contributes to PCa is poorly understood. Aging is characterized by low-grade systemic inflammation (i.e., inflammaging) that is often attributed to the progressive activation of immune cells over time, which may play an important role in prostate carcinogenesis. Th17 response is elevated in aging humans and mice, but it remains unknown whether it is increased in prostate tissue or contributes to prostate carcinogenesis during aging. In this study, we aimed to determine the role of age-related Th17 response in PCa cell growth, migration, and invasion. METHODS: C57BL/6J (B6) mouse was used as an aging animal model and the prostate histopathology during aging was analyzed. Splenic CD4+ T cells were isolated from young (16-20 weeks old) and aged (96-104 weeks old) mice, and cultured in the presence of plate-bound anti-CD3/anti-CD28, with or without Th17 differentiation conditions. The cells were collected and used for subsequent flow cytometry or quantitative reverse transcription polymerase chain reaction. The supernatant was collected and used to treat PCa cell lines. The treated PCa cells were analyzed for cell viability, migration, invasion, and nuclear factor kappa B (NF-κB) signaling. RESULTS: Aged mice had enlarged prostate glands and increased morphological alterations, with not only increased inflammatory cell infiltration but also increased Th17 cytokines in prostate tissue, compared to young mice. Naïve CD4+ T cells from aged mice differentiated increased interleukin (IL)-17-expressing cells. CD4+ T cells from aged mice spleen had increased Th17 cells, Th17 cytokines and Th17/Treg ratio compared to young mice. Factors secreted from aged CD4+ T cells, especially from ex vivo differentiated Th17 cells, not only promoted PCa cell viability, migration, and invasion but also activated the NF-κB signaling in PCa cells compared to young mice. CONCLUSIONS: These results indicate that age-related CD4+ T cells, especially Th17 cells-secreted factors have the potential to contribute to prostate carcinogenesis. Our work could prompt further research using autochthonous PCa mouse models at different ages to elucidate the functional role of Th17 response in prostate carcinogenesis during aging.

Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model.

BACKGROUND: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking. METHODS: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining. RESULTS: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres. CONCLUSIONS: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Lack of mutation-histopathology correlation in a patient with Proteus syndrome.

Proteus syndrome (PS) is characterized by progressive, disproportionate, segmental overgrowth, and tumor susceptibility caused by a somatic mosaic AKT1 activating mutation. Each individual has unique manifestations making this disorder extremely heterogeneous. We correlated three variables in 38 tissue samples from a patient who died with PS: the gross affection status, the microscopic affection status, and the mutation level. The AKT1 mutation was measured using a PCR-based RFLP assay. Thirteen samples were grossly normal; six had detectable mutation (2-29%) although four of these six were histopathologically normal. Of the seven grossly normal samples that had no mutation, only four were histologically normal. The mutation level in the grossly abnormal samples was 3-35% and all but the right and left kidneys, skull, and left knee bone, with mutation levels of 19%, 15%, 26%, and 17%, respectively, had abnormal histopathology. The highest mutation level was in a toe bone sample whereas the lowest levels were in the soft tissue surrounding that toe, and an omental fat nodule. We also show here that PS overgrowth can be caused by cellular proliferation or by extracellular matrix expansion. Additionally, papillary thyroid carcinoma was identified, a tumor not previously associated with PS. We conclude that gross pathology and histopathology correlate poorly with mutation levels in PS, that overgrowth can be mediated by cellular proliferation or extracellular matrix expansion, and that papillary thyroid carcinoma is part of the tumor susceptibility of PS. New methods need to be developed to facilitate genotype-phenotype correlation in mosaic disorders. © 2016 Wiley Periodicals, Inc.

Construction and Validation of a Multi-Institutional Tissue Microarray of Invasive Ductal Carcinoma From Racially and Ethnically Diverse Populations.

BACKGROUND: The scarcity of tissues from racial and ethnic minorities at biobanks poses a scientific constraint to research addressing health disparities in minority populations. METHODS: To address this gap, the Minority Biospecimen/Biobanking Geographic Management Program for region 3 (BMaP-3) established a working infrastructure for a "biobanking" hub in the southeastern United States and Puerto Rico. Herein we describe the steps taken to build this infrastructure, evaluate the feasibility of collecting formalin-fixed, paraffin-embedded tissue blocks and associated data from a single cancer type (breast), and create a web-based database and tissue microarrays (TMAs). RESULTS: Cancer registry data from 6 partner institutions were collected, representing 12,408 entries from 8,279 unique patients with breast cancer (years 2001-2011). Data were harmonized and merged, and deidentified information was made available online. A TMA was constructed from formalin-fixed, paraffin-embedded samples of invasive ductal carcinoma (IDC) representing 427 patients with breast cancer (147 African Americans, 168 Hispanics, and 112 non-Hispanic whites) and was annotated according to biomarker status and race/ethnicity. Biomarker analysis of the TMA was consistent with the literature. CONCLUSIONS: Contributions from participating institutions have facilitated a robust research tool. TMAs of IDC have now been released for 5 projects at 5 different institutions.

Neoplastic reprogramming of patient-derived adipose stem cells by prostate cancer cell-associated exosomes.

Emerging evidence suggests that mesenchymal stem cells (MSCs) are often recruited to tumor sites but their functional significance in tumor growth and disease progression remains elusive. Herein we report that prostate cancer (PC) cell microenvironment subverts PC patient adipose-derived stem cells (pASCs) to undergo neoplastic transformation. Unlike normal ASCs, the pASCs primed with PC cell conditioned media (CM) formed prostate-like neoplastic lesions in vivo and reproduced aggressive tumors in secondary recipients. The pASC tumors acquired cytogenetic aberrations and mesenchymal-to-epithelial transition and expressed epithelial, neoplastic, and vasculogenic markers reminiscent of molecular features of PC tumor xenografts. Our mechanistic studies revealed that PC cell-derived exosomes are sufficient to recapitulate formation of prostate tumorigenic mimicry generated by CM-primed pASCs in vivo. In addition to downregulation of the large tumor suppressor homolog2 and the programmed cell death protein 4, a neoplastic transformation inhibitor, the tumorigenic reprogramming of pASCs was associated with trafficking by PC cell-derived exosomes of oncogenic factors, including H-ras and K-ras transcripts, oncomiRNAs miR-125b, miR-130b, and miR-155 as well as the Ras superfamily of GTPases Rab1a, Rab1b, and Rab11a. Our findings implicate a new role for PC cell-derived exosomes in clonal expansion of tumors through neoplastic reprogramming of tumor tropic ASCs in cancer patients.

In vivo anticancer synergy mechanism of doxorubicin and verapamil combination treatment is impaired in BALB/c mice with metastatic breast cancer.

The development of resistance to anticancer drugs is a major unsolved problem in the chemotherapy treatment of metastatic breast cancer. We have shown that increased expression of P-glycoprotein (P-gp) prevented nuclear entry of the doxorubicin molecules into murine breast cancer cells (4T1-R) leading to doxorubicin chemoresistance. This study was performed to test whether inhibition of P-gp using verapamil could overcome doxorubicin chemoresistance and eliminate multiorgan metastasis 4T1-R cells in BALB/c mouse. The 4T1-R cells were treated with doxorubicin alone, verapamil alone, and a combination of both. Multiorgan metastasis of 4T1-R cells in the presence and in the absence of combination treatment was determined in the BALB/c mouse model. Verapamil induced nuclear translocation of doxorubicin, G2-phase growth arrest and synergistically induced 100% cytotoxicity in 4T1-R cells in culture. However, the combination treatment using verapamil and doxorubicin did not improve the overall survival of BALB/c mice with metastatic breast cancer. Our results indicate that the combination treatment of verapamil and doxorubicin did not inhibit tumor growth in the lungs and liver indicating that the anticancer synergy mechanism of verapamil and doxorubicin is impaired in vivo in BALB/c mouse model with metastatic breast cancer. We propose that understanding the mechanisms as to why the combination of doxorubicin and verapamil treatment was impaired in the mouse model should allow novel approaches to improve chemotherapy response of metastatic breast cancer.

Impaired autophagy response in human hepatocellular carcinoma.

BACKGROUND: Autophagy is a cellular lysosomal degradation mechanism that has been implicated in chronic liver diseases and hepatocellular carcinoma (HCC). Association of autophagy defect with the development of human HCC has been shown in transgenic mouse model. AIM: We performed this study to verify whether a defect in autophagy would play a role in human hepatocellular carcinoma (HCC). METHODS: Archival tissue sections of 20 patients with HCC with or without hepatitis C virus (HCV) infection were studied. All slides were immunostained using monoclonal antibodies to p62 and glypican-3 with appropriate positive and negative controls. The expression of p62 and glycican-3 in the HCC and the surrounding non-tumor was semiquantitated. The cytoplasmic staining was graded as negative, weak or strong. RESULTS: Positive p62 staining was found in 20 out of 20 (100%) HCCs and negative staining was observed in 20 out of 20 non-tumor areas and cirrhotic nodules. Positive glypican-3 staining was found in 70% of HCCs and negative staining was seen in all non-tumor areas. An autophagy defect leading to increased expression of p62 and glypican-3 was also seen in the HCC cell line (Huh-7.5), but not in the primary human hepatocytes. Activation of cellular autophagy in Huh-7.5 cells efficiently cleared p62 and glypican-3 expression and inhibition of autophagy induced the expression of p62 and glypican-3. CONCLUSIONS: This study shows that p62 is increased in HCC compared to the surrounding non-tumorous liver tissue suggesting that human HCCs are autophagy defective. We provide further evidence that glypican-3 expression in HCC may also be related to defective autophagy. Our study indicates that p62 immunostain may represent a novel marker for HCC.

Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Introduction: Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. Aim: This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. Methods: HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. Results: HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10µM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8µM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Conclusion: Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

High circulating estrogens and selective expression of ERß in prostate tumors of Americans: implications for racial disparity of prostate cancer.

Although estrogen receptor beta (ERß) has been implicated in prostate cancer (PCa) progression, its potential role in health disparity of PCa remains elusive. The objective of this study was to examine serum estrogens and prostate tumor ERß expression and examine their correlation with clinical and pathological parameters in African American (AA) versus Caucasian American (CA) men. The circulating 17ß-estradiol (E2) was measured by enzyme immunoassay in blood procured from racially stratified normal subjects and PCa patients. Differential expression profile analysis of ERß was analyzed by quantitative immunohistochemistry using ethnicity-based tissue microarray encompassing 300 PCa tissue cores. In situ ERß expression was validated by quantitative reverse transcription-PCR in matched microdissected normal prostate epithelium and tumor cells and datasets extracted from independent cohorts. In comparison with normal age-matched subjects, circulating E2 levels were significantly elevated in all PCa patients. Further analysis demonstrates an increase in blood E2 levels in AA men in both normal and PCa in comparison with age- and stage-matched counterparts of CA decent. Histochemical score analysis reveals intense nuclear immunoreactivity for ERß in tumor cores of AA men than in CA men. Gene expression analysis in microdissected tumors corroborated the biracial differences in ERß expression. Gene expression analysis from independent cohort datasets revealed correlation between ERß expression and PCa progression. However, unlike in CA men, adjusted multivariate analysis showed that ERß expression correlates with age at diagnosis and low prostate-specific antigen recurrence-free survival in AA men. Taken together, our results suggest that E2-ERß axis may have potential clinical utility in PCa diagnosis and clinical outcome among AA men.

Increased expression of P-glycoprotein and doxorubicin chemoresistance of metastatic breast cancer is regulated by miR-298.

MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3' untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer.

A comparison of clinical and pathologic assessments for the prediction of occult nipple involvement in nipple-sparing mastectomies.

BACKGROUND: Nipple-sparing mastectomy (NSM) for both risk reduction and cancer is increasing. In the cancer setting, most studies suggest the use of both clinical and intraoperative biopsy criteria in patient selection. This study examines the use of both biopsy and clinical criteria in women undergoing total nipple-removing mastectomy. METHODS: The study consisted of 58 patients undergoing total mastectomy without nipple sparing. Biopsies of the subareola tissue (SA), proximal nipple (NC) contents and radial sections of the residual nipple (NR) were examined microscopically. Tumor size and distance from the nipple were also noted. RESULTS: Using clinical criteria alone, the false negative rate was 53.8% and a false positive rate of 44.4%. When adding subareola and nipple core biopsies to clinical criteria the false negative rate fell to 7.7% but the false positive rate remained at 44.4%. When using only SA and NC biopsies to predict occult nipple involvement, the false negative rate was 11.8%. In 4 cases the NC was positive while the SA was negative for cancer and in 6 cases the SA was positive and NC negative. In 2 cases both the NC and SA biopsies were negative while the NR was positive. CONCLUSIONS: This study supports a more limited role in the use of clinical criteria for evaluating patients for NSM. This maximizes the number of patients who are candidates for NSM with minimal risk of nipple involvement. It was also noted that intraoperative biopsies are not totally reliable in predicting occult nipple involvement.

Clinical significance of CD146 and latexin during different stages of thyroid cancer.

Molecular mechanisms underlying thyroid tumorigenesis and identifying new therapeutic targets are still under investigation. We aim to investigate the role of CD146 and latexin (Lxn) and examine whether they have any clinical significance in thyroid cancer. Human thyroid papillary (PTC), follicular (FTC), anaplastic (ATC) cancer cells, and other control cells were used in this study. Western blot, cell proliferation, invasion assay, and shRNA were applied to study the expression levels and functional significances of CD146 and Lxn in thyroid cells. The protein expression was evaluated by immunohistochemistry using human tissue microarray (TMA) slides. Multivariate analysis was used to examine whether these proteins have any clinical significance in patients with thyroid cancer. The protein expressions of CD146 and Lxn were detected in most thyroid cancer cell lines when compared with normal cells. Notably, knockdown of CD146 reduced the migration and invasion in K1 (PTC) and OCUT-1 (ATC) cells. TMAs showed more immunoreactivity against CD146 and Lxn in PTC cores compared with FTC, ATC, and normal tissues. A positive correlation was established between CD146 and both Lxn (r = 0.421, p = 0.045) and age (r = 0.566, p = 0.012); however, it showed a negative correlation with tumor stage (r = -0.231, p = 0.010). In conclusion, CD146 and Lxn increased tumor migration and invasion in vitro and showed a high expression in PTC compared to those in ATC and normal human tissues demonstrating their role in early stage of thyroid tumorigenesis. CD146 was positively correlated with age, but negatively correlated with tumor stage.

Chromatin-Accessible miRNA Regulons Driving Thyroid Tumorigenesis and Progression.

BACKGROUND: Although papillary thyroid cancer can remain indolent, associated lymph node metastases and recurrence rates are approximately 50% and 20%, respectively. Omics-based medicine has led to the discovery of predictive biomarkers that can be used to predict tumor progression and clinical outcomes. We aimed to develop a noninvasive omics-driven blood test to allow accurate risk stratification and help tailor individual patient treatment plans. STUDY DESIGN: RNA sequencing (seq) and microRNA analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets were employed to identify an epigenetic prognostic panel. Integrated bulk assay for transposase-accessible chromatin-seq and RNA-seq experiments confirmed the results. Sixty-two paired tumor and adjacent control thyroid tissues and 67 blood samples (62 papillary thyroid cancer and 5 controls) were analyzed for validation using sequencing and real-time polymerase chain reaction and correlated to clinical outcomes. A liposome-exosome fusion clustered regularly interspaced short palindromic repeats (CRISPR)-fluorescent detection system miRNA assay was developed. A predictive risk nomogram was generated and tested for performance. RESULTS: Our miRNA panel (miR-146b-5p and miR-221-3p) from tissue and blood was associated with aggressive features and was located within accessible chromatin regions. The miRNA risk score and prognostic nomogram showed higher accuracy in predicting lymph node metastases (miR-146b: area under the curve [AUC] 0.816, sensitivity 76.9%; miR-221: AUC 0.740, sensitivity 79.5%) and recurrence (miR-146b: AUC 0.921, sensitivity 75.0%; miR-221: AUC 0.756, sensitivity 70.0%; p < 0.001) than staging and American Thyroid Association risk stratification. CRISPR-based miRNA assays showed upregulation in the blood of cancer cohorts. CONCLUSIONS: CRISPR-based detection of miR-146b and miR-221 in the blood of thyroid cancer patients is a reliable and noninvasive tool for real-time assessment and prognostication that has great potential to provide a direct impact on the care of these patients.

Genomic Breakpoint Characterization and Transcriptome Analysis of Metastatic, Recurrent Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion oncogene. Despite initial response to chemotherapy, DSRCT has a recurrence rate of over 80% leading to poor patient prognosis with a 5-year survival rate of only 15-25%. Owing to the rarity of DSRCT, sample scarcity is a barrier in understanding DSRCT biology and developing effective therapies. Utilizing a novel pair of primary and recurrent DSRCTs, we present the first map of DSRCT genomic breakpoints and the first comparison of gene expression alterations between primary and recurrent DSRCT. Our genomic breakpoint map includes the lone previously published DSRCT genomic breakpoint, the breakpoint from our novel primary/recurrent DSRCT pair, as well as the breakpoints of five available DSRCT cell lines and five additional DSRCTs. All mapped breakpoints were unique and most breakpoints included a 1-3 base pair microhomology suggesting microhomology-mediated end-joining as the mechanism of translocation fusion and providing novel insights into the etiology of DSRCT. Through RNA-sequencing analysis, we identified altered genes and pathways between primary and recurrent DSRCTs. Upregulated pathways in the recurrent tumor included several DNA repair and mRNA splicing-related pathways, while downregulated pathways included immune system function and focal adhesion. We further found higher expression of the EWSR1-WT1 upregulated gene set in the recurrent tumor as compared to the primary tumor and lower expression of the EWSR1-WT1 downregulated gene set, suggesting the EWSR1-WT1 fusion continues to play a prominent role in recurrent tumors. The identified pathways including upregulation of DNA repair and downregulation of immune system function may help explain DSRCT's high rate of recurrence and can be utilized to improve the understanding of DSRCT biology and identify novel therapies to both help prevent recurrence and treat recurrent tumors.

Matrix Metalloproteinase 9/microRNA-145 Ratio: Bridging Genomic and Immunological Variabilities in Thyroid Cancer.

Matrix metalloproteinase 9 (MMP9) and microRNA-145 (miR-145) have emerged as essential biomarkers in thyroid cancer progression and metastasis. However, their combined evaluation and clinical utility as a unified prognostic marker across diverse thyroid cancer subgroups remain unexplored. We investigated the diagnostic and prognostic value of the MMP9/miR-145 ratio in thyroid cancer, hypothesizing it may overcome inter-patient heterogeneity and serve as a versatile biomarker regardless of genetic mutations or autoimmune status. MMP9 and miR-145 expressions were analyzed in 175 paired papillary thyroid cancer (PTC) and normal tissues. Plasma levels were assessed perioperatively and longitudinally over 12-18 months in 86 matched PTC patients. The associations with clinicopathological parameters and patient outcomes were evaluated. MMP9 was upregulated, and miR-145 downregulated in cancer tissues, with a median MMP9/miR-145 ratio 17.6-fold higher versus controls. The tissue ratio accurately diagnosed thyroid malignancy regardless of BRAF mutation or Hashimoto's thyroiditis status, overcoming genetic and autoimmune heterogeneity. A high preoperative circulating ratio predicted aggressive disease features, including lymph node metastasis, extrathyroidal extension, progression/relapse, and recurrence. Although the preoperative plasma ratio was elevated in patients with unfavorable outcomes, it had limited utility for post-surgical monitoring. In conclusion, the MMP9/miR-145 ratio is a promising biomarker in PTC that bridges genetic and immunological variabilities, enhancing preoperative diagnosis and prognostication across diverse patient subgroups. It accurately stratifies heterogenous cases by aggressiveness. The longitudinal trends indicate decreasing applicability for post-thyroidectomy surveillance. Further large-scale validation and protocol standardization can facilitate clinical translation of the MMP9/miR-145 ratio to guide personalized thyroid cancer management.

Diagnostic Performance of Afirma and Interpace Diagnostics Genetic Testing in Indeterminate Thyroid Nodules: A Single Center Study.

Indeterminate thyroid nodules (ITN) represent 20-30% of biopsied nodules, with a 10-60% risk of malignancy. Molecular testing can stratify the risk of malignancy among ITNs, and subsequently reduce the need for unnecessary diagnostic surgery. We aimed to assess the performance of these molecular tests at a single institution. Patients with Bethesda III, IV, and V nodules with Afirma and Interpace Diagnostics genetic testing data from November 2013 to November 2021 were included. Three cohorts were formed, including GSC + XA, ThyGeNEXT + ThyraMIR, and GSC + GEC. Statistical analysis determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy of each type of testing. The PPV of nodules undergoing genetic testing by ThyGeNEXT + ThyraMIR (45.00%, 95%CI: 28.28-62.93%, p = 0.032) and GSC + XA (57.14%, 95%CI: 29.32-81.08%, p < 0.001) were superior to that of GEC + GSC (30.72%, 95%CI: 26.83-34.90%). The NPV was above 85% in all cohorts, suggesting overall suitable rule-out tests. The Afirma platform (GSC + XA) had the highest NPV at 96.97%. The overall accuracy for nodules undergoing ThyGeNEXT + ThyraMIR was 81.42% (95%CI: 73.01-88.11%, p < 0.001). A total of 230 patients underwent thyroidectomy, including less than 60% of each of the ThyGeNEXT + ThyraMIR and GSC + XA cohorts. Specifically, only 25% of patients in the GSC + XA cohort underwent surgery, considerably decreasing the rate of unnecessary surgical intervention. Sub-group analysis, including only patients with surgical pathology, found that PPV tended to be higher in the GSC + XA cohort, at 66.67% (95%CI: 37.28-87.06%), as compared to the ThyGeNEXT + ThyraMIR cohort, at 52.94% (95%CI: 35.25-69.92%). The Afirma genetic testing platform GSC + XA outperformed the other platforms with regards to both PPV and NPV and decreased the rate of surgery in patients with ITNs by 75%, significantly preventing unnecessary surgical intervention.

Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model.

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.