Dual targeting of MAPK and PI3K pathways unlocks redifferentiation of Braf-mutated thyroid cancer organoids.

Thyroid cancer is the most common endocrine malignancy and several genetic events have been described to promote the development of thyroid carcinogenesis. Besides the effects of specific mutations on thyroid cancer development, the molecular mechanisms controlling tumorigenesis, tumor behavior, and drug resistance are still largely unknown. Cancer organoids have been proposed as a powerful tool to study aspects related to tumor development and progression and appear promising to test individual responses to therapies. Here, using mESC-derived thyroid organoids, we developed a BrafV637E-inducible model able to recapitulate the features of papillary thyroid cancer in vitro. Overexpression of the murine BrafV637E mutation, equivalent to BrafV600E in humans, rapidly triggers to MAPK activation, cell dedifferentiation, and disruption of follicular organization. BrafV637E-expressing organoids show a transcriptomic signature for p53, focal adhesion, ECM-receptor interactions, EMT, and inflammatory signaling pathways. Finally, PTC-like thyroid organoids were used for drug screening assays. The combination of MAPK and PI3K inhibitors reversed BrafV637E oncogene-promoted cell dedifferentiation while restoring thyroid follicle organization and function in vitro. Our results demonstrate that pluripotent stem cells-derived thyroid cancer organoids can mimic tumor development and features while providing an efficient tool for testing novel targeted therapies.

Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism.

The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the generation of transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone in athyreotic mice as a proof of concept for future therapeutic development.

Dynamic Cancer Cell Heterogeneity: Diagnostic and Therapeutic Implications.

Though heterogeneity of cancers is recognized and has been much discussed in recent years, the concept often remains overlooked in different routine examinations. Indeed, in clinical or biological articles, reviews, and textbooks, cancers and cancer cells are generally presented as evolving distinct entities rather than as an independent heterogeneous cooperative cell population with its self-oriented biology. There are, therefore, conceptual gaps which can mislead the interpretations/diagnostic and therapeutic approaches. In this short review, we wish to summarize and discuss various aspects of this dynamic evolving heterogeneity and its biological, pathological, clinical, diagnostic, and therapeutic implications, using thyroid carcinoma as an illustrative example.

Thyroid cancer under the scope of emerging technologies.

The vast majority of thyroid cancers originate from follicular cells. We outline outstanding issues at each step along the path of cancer patient care, from prevention to post-treatment follow-up and highlight how emerging technologies will help address them in the coming years. Three directions will dominate the coming technological landscape. Genomics will reveal tumoral evolutionary history and shed light on how these cancers arise from the normal epithelium and the genomics alteration driving their progression. Transcriptomics will gain cellular and spatial resolution providing a full account of intra-tumor heterogeneity and opening a window on the microenvironment supporting thyroid tumor growth. Artificial intelligence will set morphological analysis on an objective quantitative ground laying the foundations of a systematic thyroid tumor classification system. It will also integrate into unified representations the molecular and morphological perspectives on thyroid cancer.

Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment.

Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures.

Single-cell transcriptome analysis reveals thyrocyte diversity in the zebrafish thyroid gland.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line that monitors pax2a expression in the thyrocytes. A population of pax2a-low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a-high and pax2a-low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2.

BACKGROUND: The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. METHODS: Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (ß radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. RESULTS: Overall, the thyrocyte responses following exposure to ß or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. ß and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to ß/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. CONCLUSIONS: TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

BRCA gene mutations do not shape the extent and organization of tumor infiltrating lymphocytes in triple negative breast cancer.

This study investigated the prevalence of TIL subpopulations, TLS, PD-1 and PD-L1 in tumors from TNBC patients harboring wild-type or mutated BRCA1 or BRCA2 germline genes. This TNBC cohort included 85% TIL-positive (≥10%) tumors with 21% classified as TILhi (≥50%). Interestingly, the BRCAmut group had a significantly higher incidence of TILpos tumors compared to the BRCAwt group (P = 0.037). T cells were dominant in the infiltrate but no statistically significant differences were detected between BRCAwt and BRCAmut for CD3+, CD4+ and CD8+ T cells or CD20+ B cells. TLS were detected in 74% of tumors but again no significant differences between the BRCA groups. PD-1 expression was observed in 33% and PD-L1 in 53% (any cell, cut-off ≥1%) tumors for the entire TNBC cohort. PD-1 expression correlated with PD-L1 and both with TIL and TLS but was not associated with BRCA mutational status. Our analyses reveal that BRCAwt and BRCAmut TNBC are similar except for a significant increase of TILpos tumors in the BRCAmut group. While BRCA gene mutations may not directly drive immune infiltration, the greater number of TILpos tumors could signal greater immunogenicity in this group.

Factors contributing to the resistance of the thyrocyte to hydrogen peroxide.

We studied the mechanism that may explain the relative resistance of thyrocytes to H2O2 compared to other cell types. Ability to degrade H2O2, glutathione peroxidase (GPx) activity, heme oxygenase-1 (HO-1) expression, cell survival and capacity to repair DNA damage after H2O2 exposure or irradiation were measured in human thyrocytes in primary culture and compared to the values obtained in human T-cells and different cell lines. Compared to other cell types, thyrocytes presented a low mortality rate after H2O2 exposure, rapidly degraded extracellular H2O2 and presented a high basal seleno-dependent GPx activity. Only in thyrocytes, H2O2 up-regulated GPx activity and expression of HO-1 mRNA. These effects were not reproduced by irradiation. DNA damage caused by H2O2 was more slowly repaired than that caused by irradiation and not repaired at all in T-cells. Our study demonstrates that the thyrocyte has specific protective mechanisms against H2O2 and its mutagenic effects.

Human-Specific NOTCH2NL Genes Expand Cortical Neurogenesis through Delta/Notch Regulation.

The cerebral cortex underwent rapid expansion and increased complexity during recent hominid evolution. Gene duplications constitute a major evolutionary force, but their impact on human brain development remains unclear. Using tailored RNA sequencing (RNA-seq), we profiled the spatial and temporal expression of hominid-specific duplicated (HS) genes in the human fetal cortex and identified a repertoire of 35 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among them NOTCH2NL, human-specific paralogs of the NOTCH2 receptor, stood out for their ability to promote cortical progenitor maintenance. NOTCH2NL promote the clonal expansion of human cortical progenitors, ultimately leading to higher neuronal output. At the molecular level, NOTCH2NL function by activating the Notch pathway through inhibition of cis Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes active during human corticogenesis and reveals how human-specific NOTCH paralogs may have contributed to the expansion of the human cortex.

Genomic hotspots but few recurrent fusion genes in breast cancer.

The advent of next generation sequencing technologies has boosted the interest in exploring the role of fusion genes in the development and progression of solid tumors. In breast cancer, most of the detected gene fusions seem to be "passenger" events while the presence of recurrent and driver fusions is still under study. We performed RNA sequencing in 55 well-characterized breast cancer samples and 10 adjacent normal breast tissues, complemented by an analysis of SNP array data. We explored the presence of fusion genes and defined their association with breast cancer subtypes, clinical-pathologic characteristics and copy number aberrations. Overall, 370 fusions were detected across the majority of the samples. HER2+ samples had significantly more fusions than triple negative and luminal subtypes. The number of fusions was correlated with histological grade, Ki67 and tumor size. Clusters of fusion genes were observed across the genome and a significant correlation of fusions with copy number aberrations and more specifically amplifications was also revealed. Despite the large number of fusion events, only a few were recurrent, while recurrent individual genes forming fusions with different partners were also detected including the estrogen receptor 1 gene in the previously detected ESR1-CCDC170 fusion. Overall we detected novel gene fusion events while we confirmed previously reported fusions. Genomic hotspots of fusion genes, differences between subtypes and small number of recurrent fusions are the most relevant characteristics of these events in breast cancer. Further investigation is necessary to comprehend the biological significance of these fusions.

Distinctive Desmoplastic 3D Morphology Associated With BRAFV600E in Papillary Thyroid Cancers.

Context: Although 60% of papillary thyroid carcinomas are BRAFV600E mutant (PTCV600E), the increased aggressiveness of these cancers is still debated. Objective: For PTCV600E we aimed to further characterize the extent of the stroma and its activation, the three-dimensional (3D) tumor-stroma interface, and the proliferation rates of tumor and stromal fibroblasts. Design: We analyzed exomes, transcriptomes, and images of 364 papillary thyroid carcinoma (PTCs) from The Cancer Genome Atlas (TCGA), including 211 PTCV600E; stained 22 independent PTCs for BRAFV600E and Ki67; sequenced the exomes and stained BRAFV600E in 5 primary tumor blocks and 4 nodal metastases from one patient with PTCV600E; and reconstructed the 3D volumes of one tumor and one metastatic block at histological resolution. Results: In TCGA, BRAFV600E was associated with higher expression of proliferation markers and lower expression of thyroid differentiation markers, independently of tumor purity. Moreover, PTCV600E, in line with their overall lower purity, also had higher expression of fibroblast- and T cell-associated genes and presented more fibrosis. Tumor cells that appeared disconnected on two-dimensional histological slices were revealed to be part of a unique tumor component in the 3D reconstructed microvolumes, and they formed a surprisingly complex connected space, infiltrating a proliferative stroma. Finally, in our PTC set, both stromal fibroblasts and tumor cells presented higher proliferation rates in PTCV600E. Conclusions: Our results support the increased aggressiveness associated with BRAFV600E in PTC and shed light on the important role of the stroma in tumor expansion. The greater and more active fibrotic component predicts better efficiency of combined targeted treatments, as previously proposed for melanomaV600E.

Immune Checkpoint Molecules on Tumor-Infiltrating Lymphocytes and Their Association with Tertiary Lymphoid Structures in Human Breast Cancer.

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4+ or CD8+ T cells and CTLA-4 is expressed on CD4+ T cells. The global proportion of PD-L1+, PD-L2+, LAG3+, and TIM3+ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1+, PD-L2+, LAG3+, and TIM3+ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC.

A research note regarding "Variation in cancer risk among tissues can be explained by the number of stem cell divisions".

Tomasetti and Vogelstein argued that 2/3 of human cancers are due to 'bad luck' and that "primary prevention measures [against cancer] are not likely to be very effective". We demonstrate that their calculations for hepatocellular carcinomas overlooked a major subset of these cancers proven to be preventable through vaccination. The problem, which is not limited to hepatocellular carcinoma, arises from the general reliance of their analysis on average incidences in the United States and the omission of incidences in specific risk groups.

miRNA expression and function in thyroid carcinomas: a comparative and critical analysis and a model for other cancers.

As in many cancer types, miRNA expression profiles and functions have become an important field of research on non-medullary thyroid carcinomas, the most common endocrine cancers. This could lead to the establishment of new diagnostic tests and new cancer therapies. However, different studies showed important variations in their research strategies and results. In addition, the action of miRNAs is poorly considered as a whole because of the use of underlying dogmatic truncated concepts. These lead to discrepancies and limits rarely considered. Recently, this field has been enlarged by new miRNA functional and expression studies. Moreover, studies using next generation sequencing give a new view of general miRNA differential expression profiles of papillary thyroid carcinoma. We analyzed in detail this literature from both physiological and differential expression points of view. Based on explicit examples, we reviewed the progresses but also the discrepancies and limits trying to provide a critical approach of where this literature may lead. We also provide recommendations for future studies. The conclusions of this systematic analysis could be extended to other cancer types.

Gene signature of the post-Chernobyl papillary thyroid cancer.

PURPOSE: Following the nuclear accidents in Chernobyl and later in Fukushima, the nuclear community has been faced with important issues concerning how to search for and diagnose biological consequences of low-dose internal radiation contamination. Although after the Chernobyl accident an increase in childhood papillary thyroid cancer (PTC) was observed, it is still not clear whether the molecular biology of PTCs associated with low-dose radiation exposure differs from that of sporadic PTC. METHODS: We investigated tissue samples from 65 children/young adults with PTC using DNA microarray (Affymetrix, Human Genome U133 2.0 Plus) with the aim of identifying molecular differences between radiation-induced (exposed to Chernobyl radiation, ECR) and sporadic PTC. All participants were resident in the same region so that confounding factors related to genetics or environment were minimized. RESULTS: There were small but significant differences in the gene expression profiles between ECR and non-ECR PTC (global test, p < 0.01), with 300 differently expressed probe sets (p < 0.001) corresponding to 239 genes. Multifactorial analysis of variance showed that besides radiation exposure history, the BRAF mutation exhibited independent effects on the PTC expression profile; the histological subset and patient age at diagnosis had negligible effects. Ten genes (PPME1, HDAC11, SOCS7, CIC, THRA, ERBB2, PPP1R9A, HDGF, RAD51AP1, and CDK1) from the 19 investigated with quantitative RT-PCR were confirmed as being associated with radiation exposure in an independent, validation set of samples. CONCLUSION: Significant, but subtle, differences in gene expression in the post-Chernobyl PTC are associated with previous low-dose radiation exposure.

New global analysis of the microRNA transcriptome of primary tumors and lymph node metastases of papillary thyroid cancer.

BACKGROUND: Papillary Thyroid Cancer (PTC) is the most prevalent type of endocrine cancer. Its incidence has rapidly increased in recent decades but little is known regarding its complete microRNA transcriptome (miRNome). In addition, there is a need for molecular biomarkers allowing improved PTC diagnosis. METHODS: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deepsequencing of 59 normal samples, 495 PTC, and 8 LNM). RESULTS: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deep-sequencing of 59 normal samples, 495 PTC, and 8 LNM). We confirmed already described up-regulations of microRNAs in PTC, such as miR-146b-5p or miR-222-3p, but we also identified down-regulated microRNAs, such as miR-7-5p or miR-30c-2-3p. We showed that these down-regulations are linked to the tumorigenesis process of thyrocytes. We selected the 14 most down-regulated microRNAs in PTC and we showed that they are potential biomarkers of PTC samples. Nevertheless, they can distinguish histological classical variants and follicular variants of PTC in the TCGA dataset. In addition, 12 of the 14 down-regulated microRNAs are significantly less expressed in aggressive PTC compared to non-aggressive PTC. We showed that the associated aggressive expression profile is mainly due to the presence of the BRAF V600E mutation. In general, primary tumors and LNM presented similar microRNA expression profiles but specific variations like the down-regulation of miR-7-2-3p and miR-30c-2-3p in LNM were observed. Investigations of the 5p-to-3p arm expression ratios, non-templated additions or isomiRs distributions revealed no major implication in PTC tumorigenesis process or LNM appearance. CONCLUSIONS: Our results showed that down-regulated microRNAs can be used as new potential common biomarkers of PTC and to distinguish main subtypes of PTC. MicroRNA expressions can be linked to the development of LNM of PTC. The bioinformatics framework that we have developed can be used as a starting point for the global analysis of any microRNA deep-sequencing data in an unbiased way.

Principles Governing A-to-I RNA Editing in the Breast Cancer Transcriptome.

Little is known about how RNA editing operates in cancer. Transcriptome analysis of 68 normal and cancerous breast tissues revealed that the editing enzyme ADAR acts uniformly, on the same loci, across tissues. In controlled ADAR expression experiments, the editing frequency increased at all loci with ADAR expression levels according to the logistic model. Loci-specific "editabilities," i.e., propensities to be edited by ADAR, were quantifiable by fitting the logistic function to dose-response data. The editing frequency was increased in tumor cells in comparison to normal controls. Type I interferon response and ADAR DNA copy number together explained 53% of ADAR expression variance in breast cancers. ADAR silencing using small hairpin RNA lentivirus transduction in breast cancer cell lines led to less cell proliferation and more apoptosis. A-to-I editing is a pervasive, yet reproducible, source of variation that is globally controlled by 1q amplification and inflammation, both of which are highly prevalent among human cancers.

No significant viral transcription detected in whole breast cancer transcriptomes.

BACKGROUND: Studies evaluating the presence of viral sequences in breast cancer (BC), including various strains of human papillomavirus and human herpes virus, have yielded conflicting results. Most were based on RT-PCR and in situ hybridization. METHODS: In this report we searched for expressed viral sequences in 58 BC transcriptomes using five distinct in silico methods. In addition, we complemented our RNA sequencing results with exome sequencing, PCR and immunohistochemistry (IHC) analyses. A control sample was used to test our in silico methods. RESULTS: All of the computational methods correctly detected viral sequences in the control sample. We identified a small number of viral sequences belonging to human herpesvirus 4 and 6 and Merkel cell polyomavirus. The extremely low expression levels-two orders of magnitude lower than in a typical hepatitis B virus infection in hepatocellular carcinoma-did not suggest active infections. The presence of viral elements was confirmed in sample-matched exome sequences, but could not be confirmed by PCR or IHC. CONCLUSIONS: Our results show that no viral sequences are expressed in significant amounts in the BC investigated. The presence of non-transcribed viral DNA cannot be excluded.