Multicenter phase II trial of Camrelizumab combined with Apatinib and Eribulin in heavily pretreated patients with advanced triple-negative breast cancer.

In the later-line setting or for patients with PD-L1-negative tumors, immunotherapy-based regimens remain ineffective against advanced triple-negative breast cancer (TNBC). In this multicentered phase II trial (NCT04303741), 46 patients with pretreated advanced TNBC were enrolled to receive camrelizumab 200 mg (day 1), and apatinib 250 mg daily, plus eribulin 1.4 mg/m2 (day 1 and 8) on a 21-day cycle until progression, or unacceptable toxicity. Primary endpoint was objective response rate (ORR) according to RECIST 1.1. Secondary endpoints included toxicities, disease control rate (DCR), clinical benefit rate, progression-free survival (PFS), and 1-year overall survival. With a median of 3 lines of prior chemotherapy in the advanced setting, 17.4% had received PD-1/PD-L1 blockade plus chemotherapy for advanced disease. The ORR was 37.0% (17/46, 95% CI 23.2-52.5). The DCR was 87.0% (40/46, 95% CI 73.7-95.1). Median PFS was 8.1 (95% CI 4.6-10.3) months. Tertiary lymphoid structure was associated with higher ORR. Patients with lower tumor PML or PLOD3 expression had favorable ORR and PFS. PD-L1 status was not associated with ORR/PFS. Grade 3/4 treatment-related adverse events occurred in 19 (41.3%) of 46 patients. Camrelizumab plus apatinib and eribulin shows promising efficacy with a measurable safety profile in patients with heavily pretreated advanced TNBC.

An Audit to Evaluate Vancomycin Therapeutic Drug Monitoring in a Neonatal Intensive Care Unit.

BACKGROUND: Therapeutic drug monitoring (TDM) is routinely used for optimization of vancomycin therapy, because of exposure-related efficacy and toxicity, in addition to significant variability in pharmacokinetics, which leads to unpredictable drug exposure. OBJECTIVE: The aim of this study was to evaluate target attainment and TDM of vancomycin in neonates. METHODS: The authors conducted a retrospective study and collected data from medical records of all neonates who received vancomycin therapy in the neonatal intensive care unit between January 2019 and December 2019. The primary outcome was the proportion of vancomycin courses that reached target trough concentrations of 10-20 mg/L based on appropriate TDM samples collection. Secondary outcomes included proportion of courses with appropriate dose and dose frequency, and proportion of patients who achieved target concentrations after the first dose adjustment. RESULTS: In total, 69 patients were included, with 129 vancomycin courses. The median initial vancomycin trough concentration was 12 (range: 4-36) mg/L. The target trough concentration was achieved in 75% of courses after the initial dose with appropriate TDM, and 84% of courses after TDM-guided dose adjustments. Patients were dosed appropriately in 121/129 courses and TDM was performed correctly according to protocol in 51/93 courses. A dose adjustment was performed in 18/29 courses, to increase target attainment. CONCLUSIONS: This study showed that there is a need for an increase in dose to improve target attainment. There is also a need to explore more effective TDM strategies to increase the proportion of neonatal patients attaining vancomycin target trough concentrations.

Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

The ceRNA Network Has Potential Prognostic Value in Clear Cell Renal Cell Carcinoma: A Study Based on TCGA Database.

Clear cell renal cell carcinoma (ccRCC) is a very common cancer in urology. Many evidences suggest that complex changed pathways take a nonnegligible part in the occurrence and development of ccRCC. Nevertheless, the underlying mechanism is not clear. In this study, the expression data between ccRCC and normal tissue samples in TCGA database were compared to distinguish differentially expressed genes (DEGs: mRNAs, miRNAs, and lncRNAs). Afterwards, we used GO enrichment and KEGG pathway enrichment analyses to explore the functions of these DEGs. We also found the correlation between three RNAs and created a competing endogenous RNA (ceRNA) network. Moreover, we used univariate Cox regression analysis to select DEGs that are connected with overall survival (OS) of ccRCC patients. We found 1652 mRNAs, 1534 lncRNAs, and 173 miRNAs that were distinguished in ccRCC compared with normal tissues. According to GO analysis, the maladjusted mRNAs are mainly concentrated in immune cell activation and kidney development, while according to KEGG, they are mainly concentrated in pathways related to cancer. A total of 5 mRNAs, 1 miRNA, and 4 lncRNAs were connected with patients' OS. In this article, a network of lncRNA-miRNA-mRNA was established; it is expected to be able to indicate possible molecular mechanisms for initial of ccRCC and provide a new viewpoint for diagnosis of ccRCC.

Metalloprotoporphyrin Inhibition of HCV NS3-4A Protease: Structure-Activity Relationships.

BACKGROUND: Antiviral actions of tetrapyrroles have been described in a number of systems. Our goal was to evaluate antagonism of the HCV NS3-4A protease by a variety of common porphyrins and characterize structure-activity relationships that may be useful for future drug design of HCV and related Flaviviruses. METHODS: Using fluorometric assays, common metalloprotoporphyrins (MPP) all inhibited NS3-4A protease with IC50 values in low micromolar ranges [CoPP (1.4 µM) < ZnPP = MnPP = SnPP < CuPP < FePP (6.5 µM) = protoporphyrin]. RESULTS: Lineweaver-Burk plots confirmed that MPP: NS3 inhibition was basically competitive. All tested MPPs inhibited HCV genotype 1A, 1B, 2A and 3A recombinant proteases with the same fidelity suggesting wide antagonistic capabilities. However, when the MPPs were tested in cellular incubations with HCV replicons only Zn, Fe and free-base protoporphyrin showed comparable EC50 and IC50 values suggesting that there may be critical differences in MPP uptake and intracellular availability. Meso, deutero, and isohematoporphyrin derivatives, with or without metal substitution, all showed less anti-protease and antiviral activities as compared to protoporphyrins, suggesting that the planar, vinyl side chains are important for protease active site binding. MPPs were also active against three common protease mutants (T54A, A156T, and V36M) with equivalent or better IC50 values as compared to wild type enzyme. CONCLUSION: These findings document the versatility of MPPs as antiviral agents with an expanded sensitivity for HCV genotypes and resistance to some common viral mutations. The results also suggest that further study of MPP structure and function will be useful for the development of new antiviral agents.

HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes.

INTRODUCTION: Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. RESULTS: Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2-4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase 'holoenzyme' complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. CONCLUSIONS: HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.

Restoration of type I interferon expression by heme and related tetrapyrroles through inhibition of NS3/4A protease.

BACKGROUND: Tetrapyrrole substrates and products of heme oxygenase are potent inhibitors of hepatitis C virus (HCV) replication. It is not clear whether this occurs through primary induction of type I interferon (IFN), inhibition of viral NS3/4A protease, or a combination of these mechanisms. We studied the antiviral actions of tetrapyrroles and their potential influence on type I IFN induction. METHODS: The effects of tetrapyrrole on NS3/4A protease activity and type I IFN induction were assessed in HCV-permissive cells, replicons, or human embryonic kidney (HEK) 293 cells transfected with NS3/4A protease. Activation of innate immune signaling was determined after transfection of double-strand surrogate nucleic acid antigens or infection with defined sequence HCV cell culture (HCVcc) RNA. RESULTS: Tetrapyrroles failed to directly induce IFN expression at concentrations that inhibited HCV replication and NS3/4A protease activity. However, they potently restored IFN induction after attenuation with NS3/4A protease, a process accompanied by preservation of the adapter protein, mitochondrial antiviral signaling protein, nuclear localization of IFN regulatory factor 3, and augmentation of IFN-stimulated gene products. CONCLUSIONS: Tetrapyrroles do not directly induce IFN, but they dramatically restore type I IFN signaling pathway after attenuation with NS3/4A protease. They show immunomodulatory as well as antiprotease activity and may be useful for treatment of HCV infection.

Heme and HO-1 Inhibition of HCV, HBV, and HIV.

Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system inhibit replication of all 3 viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections.

Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: mechanism for the antiviral effects of heme oxygenase?

UNLABELLED: Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 µM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 µM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons. CONCLUSION: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.

Hepatitis C virus core protein enhances Telomerase activity in Huh7 cells.

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.

Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules.

CONTEXT: Thyroid nodules are common in adults, but only a small fraction of them are malignant. Fine-needle aspiration (FNA) with cytological evaluation is the most reliable tool for cancer diagnosis in thyroid nodules. However, 10-40% of nodules are diagnosed as indeterminate by cytology, making it difficult to optimally manage these patients. OBJECTIVE: The aim of this study was to establish the feasibility and role of testing for tumor-specific mutations in improving the FNA diagnosis of thyroid nodules. DESIGN: The prospective study included 470 FNA samples of thyroid nodules from 328 patients. At the time of aspiration, a small portion of the material was collected and tested for BRAF, RAS, RET/PTC, and PAX8/PPARgamma mutations. The mutational status was correlated with cytology and either surgical pathology diagnosis or follow-up (mean, 34 months). RESULTS: A sufficient amount of nucleic acids were isolated in 98% of samples. Thirty-two mutations were found, including 18 BRAF, eight RAS, five RET/PTC, and one PAX8/PPARgamma. The presence of any mutation was a strong indicator of cancer because 31 (97%) of mutation-positive nodules had a malignant diagnosis after surgery. A combination of cytology and molecular testing showed significant improvement in the diagnostic accuracy and allowed better prediction of malignancy in the nodules with indeterminate cytology. CONCLUSIONS: These results indicate that molecular testing of thyroid nodules for a panel of mutations can be effectively performed in a clinical setting. It enhances the accuracy of FNA cytology and is of particular value for thyroid nodules with indeterminate cytology.

Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury.

UNLABELLED: Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.

Prevalence of RET/PTC rearrangements in thyroid papillary carcinomas: effects of the detection methods and genetic heterogeneity.

CONTEXT: RET/PTC rearrangements have been reported in papillary thyroid carcinomas with variable frequency in studies that used different detection methods. OBJECTIVE: Our objective was to determine the role of different detection methods and tumor genetic heterogeneity on RET/PTC detection. DESIGN: Sixty-five papillary carcinomas were analyzed for RET/PTC1 and RET/PTC3 using five detection methods: standard-sensitivity RT-PCR, high-sensitivity RT-PCR, real-time LightCycler RT-PCR, Southern blot analysis, and fluorescence in situ hybridization. RESULTS: RET/PTC rearrangements were detected by standard-sensitivity RT-PCR in 14 tumors. High-sensitivity RT-PCR detected RET/PTC in all of these and in 12 additional cases, where the levels of expression corresponded to one to five positive cells. Real-time LightCycler RT-PCR detected RET/PTC in 12 and Southern blot analysis in 11 tumors. By fluorescence in situ hybridization, 14 tumors were positive, including nine cases with 50-86% positive cells and five cases with 17-35% positive cells. Overall, nine (14%) tumors harbored clonal rearrangements, which were present in the majority of tumor cells and detected by all five methods. Five (8%) cases had subclonal rearrangements present in a smaller portion of tumor cells and detected by most methods. Twelve (18%) tumors had nonclonal RET/PTC that were detected only by high-sensitivity RT-PCR. No other mutations were found in tumors harboring clonal RET/PTC, whereas 60% of tumors with subclonal and 42% of tumors with nonclonal RET/PTC harbored additional mutations. CONCLUSIONS: Our data suggest that broad variability in the reported prevalence of RET/PTC rearrangement is at least in part a result of the use of different detection methods and tumor genetic heterogeneity.

Delineation, functional validation, and bioinformatic evaluation of gene expression in thyroid follicular carcinomas with the PAX8-PPARG translocation.

A subset of follicular thyroid carcinomas contains a balanced translocation, t(2;3)(q13;p25), that results in fusion of the paired box gene 8 (PAX8) and peroxisome proliferator-activated receptor gamma (PPARG) genes with concomitant expression of a PAX8-PPARgamma fusion protein, PPFP. PPFP is thought to contribute to neoplasia through a mechanism in which it acts as a dominant-negative inhibitor of wild-type PPARgamma. To better understand this type of follicular carcinoma, we generated global gene expression profiles using DNA microarrays of a cohort of follicular carcinomas along with other common thyroid tumors and used the data to derive a gene expression profile characteristic of PPFP-positive tumors. Transient transfection assays using promoters of four genes whose expression was highly associated with the translocation showed that each can be activated by PPFP. PPFP had unique transcriptional activities when compared with PAX8 or PPARgamma, although it had the potential to function in ways qualitatively similar to PAX8 or PPARgamma depending on the promoter and cellular environment. Bioinformatics analyses revealed that genes with increased expression in PPFP-positive follicular carcinomas include known PPAR target genes; genes involved in fatty acid, amino acid, and carbohydrate metabolism; micro-RNA target genes; and genes on chromosome 3p. These results have implications for the neoplastic mechanism of these follicular carcinomas.

Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas.

Papillary carcinoma is the most common type of thyroid malignancy. It has been recently shown that these tumors commonly have one of three genetic alterations: BRAF point mutations, RET/PTC rearrangements, or RAS point mutations. In this study, we analyze the relationship between these alterations and the microscopic features of papillary carcinomas, their clinical features, and prognostic characteristics. Ninety-seven papillary carcinomas were studied; in all cases, frozen tissue was available for nucleic acid extraction. Of 96 unselected cases, 42% were positive for BRAF, 18% for RET/PTC, and 15% for RAS mutations. Morphologic features were evaluated in detail in 61 cases and 6 characteristic nuclear features and 3 additional microscopic features were assessed quantitatively. At least 4 nuclear features were found in each tumor, with nuclear pseudoinclusions being the least frequent finding in all mutation groups. BRAF mutations were associated with older patient age, typical papillary appearance or the tall cell variant, a higher rate of extrathyroidal extension, and more advanced tumor stage at presentation. RET/PTC rearrangements presented at younger age and had predominantly typical papillary histology, frequent psammoma bodies, and a high rate of lymph node metastases. Tumors with RAS mutations were exclusively the follicular variant of papillary carcinoma and correlated with significantly less prominent nuclear features and low rate of lymph node metastases. These findings demonstrate that BRAF, RET/PTC, and RAS mutations are associated with distinct microscopic, clinical, and biologic features of thyroid papillary carcinomas.

BRAF copy number gains in thyroid tumors detected by fluorescence in situ hybridization.

Point mutation of the BRAF gene is a common genetic event in papillary thyroid carcinomas. More recently, it has been found that BRAF can also participate in chromosomal rearrangement. In this study, we explore yet another possible mechanism of BRAF alteration, which involves copy number gain. Using fluorescence in situ hybridization with BRAF specific and chromosome 7 centromeric probes, we studied 62 follicular thyroid tumors and 32 papillary carcinomas. We found that numerical changes in BRAF copy number were rare in papillary thyroid carcinomas, while they occurred in 16-45% of follicular tumors of conventional and oncocytic (Hürthle cell) types. They were due to amplification of the gene or gain of one or more copies of chromosome 7. Tetrasomy for chromosome 7 was overall the most common finding. The changes in BRAF copy number did not overlap with RAS mutations in follicular tumors. In a group of follicular carcinomas, tumors with BRAF copy number gain were significantly more often widely invasive (67%) compared to tumors with no copy number change (18%). By Western blotting, the tumors carrying four copies of the gene revealed higher expression of BRAF protein, suggesting that copy number gain may represent another mechanism of BRAF activation in thyroid tumors.

Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation-specific gene expression profiles discovered by DNA microarray analysis.

Thyroid cancer poses a significant clinical challenge, and our understanding of its pathogenesis is incomplete. To gain insight into the pathogenesis of papillary thyroid carcinoma, transcriptional profiles of four normal thyroids and 51 papillary carcinomas (PCs) were generated using DNA microarrays. The tumors were genotyped for their common activating mutations: BRAF V600E point mutation, RET/PTC1 and 3 rearrangement and point mutations of KRAS, HRAS and NRAS. Principal component analysis based on the entire expression data set separated the PCs into three groups that were found to reflect tumor morphology and mutational status. By combining expression profiles with mutational status, we defined distinct expression profiles for the BRAF, RET/PTC and RAS mutation groups. Using small numbers of genes, a simple classifier was able to classify correctly the mutational status of all 40 tumors with known mutations. One tumor without a detectable mutation was predicted by the classifier to have a RET/PTC rearrangement and was shown to contain one by fluorescence in situ hybridization analysis. Among the mutation-specific expression signatures were genes whose differential expression was a direct consequence of the mutation, as well as genes involved in a variety of biological processes including immune response and signal transduction. Expression of one mutation-specific differentially expressed gene, TPO, was validated at the protein level using immunohistochemistry and tissue arrays containing an independent set of tumors. The results demonstrate that mutational status is the primary determinant of gene expression variation within these tumors, a finding that may have clinical and diagnostic significance and predicts success for therapies designed to prevent the consequences of these mutations.

Dose-dependent generation of RET/PTC in human thyroid cells after in vitro exposure to gamma-radiation: a model of carcinogenic chromosomal rearrangement induced by ionizing radiation.

Ionizing radiation is a well-known risk factor for thyroid cancer in human populations. Chromosomal rearrangements involving the RET gene, known as RET/PTC, are prevalent in thyroid papillary carcinomas from patients with radiation history. We studied the generation of RET/PTC in HTori-3 immortalized human thyroid cells exposed to a range of doses of gamma-radiation and harvested 2, 5-6, and 9 d later. RET/PTC1 and RET/PTC3 were detected by RT-PCR followed by Southern blotting and hybridization with internal oligonucleotide probes. No RET/PTC was found in cells harvested 2 and 5-6 d after irradiation, whereas 59 RET/PTC events were detected in cells collected 9 d after exposure. The average rate of RET/PTC induction was 0.1 x 10(-6) after exposure to 0.1 Gy, 1.6 x 10(-6) after 1 Gy, 3.0 x 10(-6) after 5 Gy, and 0.9 x 10(-6) after 10 Gy. When adjusted for cell survival, the rate after 10 Gy was comparable with those after 5 Gy. RET/PTC1 was more common than RET/PTC3 after each dose, comprising 80% of all rearrangements. In this study, we demonstrate a dose-dependent induction of RET/PTC rearrangements in human thyroid cells after exposure to 0.1-10 Gy gamma-radiation. This provides additional evidence for a direct link between this genetic event and radiation exposure and offers a powerful experimental system for studying radiation-induced carcinogenesis in the thyroid gland.

Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer.

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.

BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas.

Activating point mutations of the BRAF gene have been recently reported in papillary thyroid carcinomas. In this study, we analyzed 320 thyroid tumors and six anaplastic carcinoma cell lines and detected BRAF mutations in 45 (38%) papillary carcinomas, two (13%) poorly-differentiated carcinomas, three (10%) anaplastic carcinomas, and five (83%) thyroid anaplastic carcinoma cell lines but not in follicular, Hürthle cell, and medullary carcinomas, follicular and Hürthle cell adenomas, or benign hyperplastic nodules. All mutations involved a T-->A transversion at nucleotide 1796. In papillary carcinomas, BRAF mutations were associated with older age, classic papillary carcinoma or tall cell variant histology, extrathyroidal extension, and more frequent presentation at stages III and IV. All BRAF-positive poorly differentiated and anaplastic carcinomas contained areas of preexisting papillary carcinoma, and mutation was present in both the well-differentiated and dedifferentiated components. These data indicate that BRAF mutations are restricted to papillary carcinomas and poorly differentiated and anaplastic carcinomas arising from papillary carcinomas. They are associated with distinct phenotypical and biological properties of papillary carcinomas and may participate in progression to poorly differentiated and anaplastic carcinomas.