Emerging evidence shows that the efficacy of chemotherapeutic drugs are reliant on their capability to induce immunogenic cell death (ICD), thus transforming dying tumor cells into antitumor vaccines. We wanted to uncover potential therapeutic strategies that target ovarian cancer by having a better understanding of the standard-of-care chemotherapy treatment. Here, we showed in ovarian cancer that paclitaxel induced ICD-associated DAMPs (i.e. damage-associated molecular patterns, such as CALR exposure, ATP secretion and HMGB1 release) in vitro and elicited significant antitumor responses in tumor vaccination assays in vivo. Paclitaxel-induced TLR4 signaling was essential to the release of DAMPs, which lead to the activation of NF-κB-mediated CCL2 transcription and IKK2-mediated SNARE-dependent vesicle exocytosis, thus exposing CALR on the cell surface. Paclitaxel induced ER stress, which triggered PERK activation and eIF2α phosphorylation independent of TLR4. Paclitaxel chemotherapy induced T cell infiltration in ovarian tumors of the responsive patients; CALR expression in primary ovarian tumors also correlated with patients' survival and patient response to chemotherapy. These findings suggest that the effectiveness of paclitaxel relied upon the activation of antitumor immunity through ICD via TLR4 and highlighted the importance of CALR expression in cancer cells as an indicator of response to paclitaxel chemotherapy in ovarian cancer.

Any perturbation in the normal functioning of endoplasmic reticulum (ER), such as due to hypoxia, triggers the unfolded protein response (UPR). We studied the temporal variation in gene expression in murine kidney exposed to acute hypobaric hypoxia. Molecular chaperones like Grp78, Grp94, Canx and Calr in the ER were transcriptionally downregulated. Further, the splicing of Xbp1 mRNA decreased, whereas transcription of the unspliced mRNA increased. This step produces Xbp1 protein, which is negatively regulated by the unspliced protein. Hence, the decreased splicing of Xbp1 along with decreased transcription of ER chaperones in kidney is a definite indication of reduced stress.

Blood stasis syndrome (BSS) has been considered to be the major type of syndromes in unstable angina (UA) patients. The aim of this study was to find the systems biology-based microRNA (miRNA) and mRNA expression biomarkers for BSS of UA. We identified 1081 mRNAs and 25 miRNAs differentially expressed between BSS of UA patients and healthy controls by microarrays. We used DAVID, miRTrail, and the protein-protein interactions method to explore the related pathways and networks of differentially expressed miRNAs and mRNAs. By combining the results of pathways and networks, we found that the upregulation of miR-146b-5p may induce the downregulation of CALR to attenuate inflammation and the upregulation of miR-199a-5p may induce the downregulation of TP53 to inhibit apoptosis in BSS of UA patients. The expression patterns of miR-146b-5p, miR-199a-5p, CALR, and TP53 were confirmed by qRT-PCR in an independent validation cohort including BBS of UA patients, non-BBS of UA patients, and healthy controls. miR-146b-5p, miR-199a-5p, CALR, and TP53 could be significant biomarkers of BSS of UA patients. The systems biology-based miRNA and mRNA expression biomarkers for the BSS of UA may be helpful for the further stratification of UA patients when deciding on interventions or clinical trials.

This study aimed at estimating the proportion of human myenteric Dogiel type II neurons, putative intrinsic primary afferent neurons (IPANs), in relation to the entire myenteric neuron population. Since, at present, there is no known single marker, which specifically labels these neurons, we tried to identify the most appropriate marker combination based on the results of an earlier study. For this purpose, 10 wholemounts derived from human small intestinal segments were immunohistochemically triple-stained for calretinin (CALR), somatostatin (SOM) and neurofilaments (NF) and 9 were stained for substance P (SP), SOM and NF. In each wholemount, 15 ganglia selected randomly were evaluated. On the basis of their NF-reactivity, neurons reactive for one or co-reative for both of the other two markers, respectively, were morphologically classified as type II or non-type II neurons. We found that the majorities of neurons co-reactive for CALR/SOM and SP/SOM, respectively, were type II neurons whereas this was not the case for neurons, which were reactive for only one of the two markers. One of the statistical parameters estimated was the positive predictive value, the probability that a neuron displaying CALR/SOM- or SP/SOM-co-reactivity, respectively, is a type II neuron. This value was 97% in case of CALR/SOM- and 95% in case of SP/SOM-co-staining. Although the difference of the statistical parameters between the two stainings was not significant, CALR and SOM were used to estimate indirectly the proportion of type II neurons, in wholemounts co-stained with the pan-neuronal marker neuronal protein HuC/HuD (HU). In these wholemounts, altogether 9.1% of neurons were coreactive for CALR/SOM. We suggest that the proportion of myenteric type II neurons in the human small intestine is related to the proportion of CALR/SOM-co-reactive neurons and may be near to one tenth of the total myenteric neuronal population.

Calretinin (CALR) is often used as an immunohistochemical marker for the histopathological diagnosis of human intestinal neuropathies. However, little is known about its distribution pattern with respect to specific human enteric neuron types. Prior studies revealed CALR in both myenteric and submucosal neurons, most of which colabel with choline acetyl transferase (ChAT). Here, we specified the chemical code of CALR-positive neurons in small and large intestinal wholemounts in a series of 28 patients. Besides other markers, we evaluated the labeling pattern of CALR in combination with vasoactive intestinal peptide (VIP). In colonic submucosa, CALR and VIP were almost completely colocalized in about three-quarters of all submucosal neurons. In the small intestinal submucosa, both the colocalization rate of CALR and VIP as well as the proportion of these neurons were lower (about one-third). In the myenteric plexus of both small intestine and colon, CALR amounted to 11 and 10 %, respectively, whereas VIP to 5 and 4 % of the whole neuron population, respectively. Colocalization of both markers was found in only 2 and 3 % of myenteric neurons, respectively. In section specimens, nerve fibers coreactive for CALR and VIP were found in the mucosa but not in the muscle coat. Summarizing the present and earlier results, CALR was found in at least one submucosal and two myenteric neuron populations. Submucosal CALR+/VIP+/ChAT± neurons innervate mucosal structures. Furthermore, CALR immunoreactivity in the myenteric plexus was observed in morphological type II (supposed primary afferent) and spiny type I (supposed inter- or motor-) neurons.

We screened 136 patients with myelofibrosis and a median age of 58 years who underwent allogeneic stem cell transplantation (AHSCT) for molecular residual disease for JAKV617F (n=101), thrombopoietin receptor gene (MPL) (n=4) or calreticulin (CALR) (n=31) mutation in peripheral blood on day +100 and +180 after AHSCT. After a median follow-up of 78 months, the 5-year estimated overall survival was 60% (95% confidence interval (CI): 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. The percentage of molecular clearance on day 100 was higher in CALR-mutated patients (92%) in comparison with MPL- (75%) and JAKV617F-mutated patients (67%). Patients with detectable mutation at day +100 or at day +180 had a significant higher risk of clinical relapse at 5 years than molecular-negative patients (62% vs 10%, P<0.001) and 70% vs 10%, P<0.001, respectively) irrespectively of the underlying mutation. In a multivariate analysis, high-risk diseases status (hazard ratio (HR) 2.5; 95% CI: 1.18-5.25, P=0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76-25.30, P<0.001) were significant factors for a higher risk of relapse.

Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30-40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR (p = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR- as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity.

OBJECTIVE

To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility.

METHODS

The study examined the distribution of the P2X₂ receptor (P2X₂R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X₂R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm²) and area profile (μm²) of P2X₂R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area.

RESULTS

In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X₂R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X₂R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm²) of P2X₂R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CalR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (μm²) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups.

CONCLUSIONS

We demonstrate increases in P2X₂R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.

BACKGROUND

Primary hyperparathyroidism (PHPT) is most frequently present in postmenopausal women. Although the involvement of estrogen has been suggested, current literature indicates that parathyroid tumors are estrogen receptor (ER) α negative.

OBJECTIVE

The aim of the study was to evaluate the expression of ERs and their putative function in parathyroid tumors.

METHODS

A panel of 37 parathyroid tumors was analyzed for expression and promoter methylation of the ESR1 and ESR2 genes as well as expression of the ERα and ERβ1/ERβ2 proteins. Transcriptome changes in primary cultures of parathyroid adenoma cells after treatment with the selective ERβ1 agonist diarylpropionitrile (DPN) and 4-hydroxytamoxifen were identified using next-generation RNA sequencing.

RESULTS

Immunohistochemistry revealed very low expression of ERα, whereas all informative tumors expressed ERβ1 (n = 35) and ERβ2 (n = 34). Decreased nuclear staining intensity and mosaic pattern of positive and negative nuclei of ERβ1 were significantly associated with larger tumor size. Tumor ESR2 levels were significantly higher in female vs. male cases. In cultured cells, significantly increased numbers of genes with modified expression were detected after 48 h, compared to 24-h treatments with DPN or 4-hydroxytamoxifen, including the parathyroid-related genes CASR, VDR, JUN, CALR, and ORAI2. Bioinformatic analysis of transcriptome changes after DPN treatment revealed significant enrichment in gene sets coupled to ER activation, and a highly significant similarity to tumor cells undergoing apoptosis.

CONCLUSIONS

Parathyroid tumors express ERβ1 and ERβ2. Transcriptional changes after ERβ1 activation and correlation to clinical features point to a role of estrogen signaling in parathyroid function and disease.

Loss of expression of calreticulin (CALR) has been detected by immunohistochemistry in a fraction of non-small cell lung cancers (NSCLC) and has been demonstrated to have a major negative prognostic impact on overall patient survival. Here, we analyzed the impact of CALR expression levels detected by microarray finding a positive correlation between CALR and the expression of a metagene indicating the presence of cytotoxic T lymphocytes (CTL) in NSCLC and ovarian cancer. In addition, we detected a positive correlation with a metagene suggestive of activated dendritic cell (aDC) infiltration in ovarian cancer. Combination of two parameters (CALR + DC (dendritic cell) in NSCL and CALR + aDC in ovarian cancer) or three parameters (CALR + CTL + DC in NSCL and CALR + CTL + aDC in ovarian cancer) had a significant impact on overall patient survival in NSCL (Adenoconsortium) and ovarian cancer (TCGA collection), allowing the stratification of patients in high-risk and low-risk groups. In addition, CALR and aDC alone have a significant impact on overall survival in ovarian cancer. In contrast, in mammary, colorectal and prostate cancer, CALR had no impact on patient survival if analyzed alone or in combination with the immune infiltrate. In addition, CALR correlates with CTL infiltrate in three cancer types (colorectal, breast, ovarian). Altogether, these results support the contention that, at least in some cancers, loss of CALR expression may negatively affect immunosurveillance, thereby reducing patient survival.

Mutations in the calreticulin gene, CALR, have recently been discovered in subsets of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). We investigated Korean patients with ET and PMF to determine the prevalence, and clinical and laboratory correlations of CALR/JAK2/MPL mutations. Among 84 ET patients, CALR mutations were detected in 23 (27.4%) and were associated with higher platelet counts (P=0.006) and lower leukocyte counts (P=0.035) than the JAK2 V617F mutation. Among 50 PMF patients, CALR mutations were detected in 11 (22.0%) and were also associated with higher platelet counts (P=0.035) and trended to a lower rate of cytogenetic abnormalities (P=0.059) than the JAK2 V617F mutation. By multivariate analysis, triple-negative status was associated with shorter overall survival (HR, 7.0; 95% CI, 1.6-31.1, P=0.01) and leukemia-free survival (HR, 6.3; 95% CI, 1.8-22.0, P=0.004) in patients with PMF. The type 1 mutation was the most common (61.1%) type among all patients with CALR mutations, and tended toward statistical predominance in PMF patients. All 3 mutations were mutually exclusive and were never detected in patients with other myeloid neoplasms showing thrombocytosis. CALR mutations characterize a distinct group of Korean ET and PMF patients. Triple-negative PMF patients in particular have an unfavorable prognosis, which supports the idea that triple-negative PMF is a molecularly high-risk disease.

Sporadic essential thrombocythaemia (ET) is rare in paediatrics, and the diagnostic and clinical approach to paediatric cases cannot be simply copied from experience with adults. Here, we assessed 89 children with a clinical diagnosis of ET and found that 23 patients (25·8%) had a clonal disease. The JAK2 V617F mutation was identified in 14 children, 1 child had the MPL W515L mutation, and 6 had CALR mutations. The monoclonal X-chromosome inactivation pattern was seen in six patients (two with JAK2 V617F and two with CALR mutations). The other 66 patients (74·2%) had persistent thrombocytosis with no clonality. There were no clinical or haematological differences between the clonal and non-clonal patients. The relative proportion of ET-specific mutations in the clonal children was much the same as in adults. The higher prevalence of non-clonal cases suggests that some patients may not have myeloproliferative neoplasms, with significant implications for their treatment.

Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.

To detect drug candidates for dengue haemorrhagic fever (DHF), we employed a computational drug repositioning method to perform an integrated multiple omics analysis based on transcriptomic, proteomic, and interactomic data. We identified 3,892 significant genes, 389 proteins, and 221 human proteins by transcriptomic analysis, proteomic analysis, and human-dengue virus protein-protein interactions, respectively. The drug candidates were selected using gene expression profiles for inverse drug-disease relationships compared with DHF patients and healthy controls as well as interactomic relationships between the signature proteins and chemical compounds. Integrating the results of the multiple omics analysis, we identified eight candidates for drug repositioning to treat DHF that targeted five proteins (ACTG1, CALR, ERC1, HSPA5, SYNE2) involved in human-dengue virus protein-protein interactions, and the signature proteins in the proteomic analysis mapped to significant pathways. Interestingly, five of these drug candidates, valparoic acid, sirolimus, resveratrol, vorinostat, and Y-27632, have been reported previously as effective treatments for flavivirus-induced diseases. The computational approach using multiple omics data for drug repositioning described in this study can be used effectively to identify novel drug candidates.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by ineffective clonal hematopoiesis, splenomegaly, bone marrow fibrosis, and the propensity for transformation to acute myeloid leukemia. The discovery of mutations in JAK2, CALR, and MPL have uncovered activated JAK-STAT signaling as a primary driver of MF, supporting a rationale for JAK inhibition. However, JAK inhibition alone is insufficient for long-term remission and offers modest, if any, disease-modifying effects. Given this, there is great interest in identifying mechanisms that cooperate with JAK-STAT signaling to predict disease progression and rationally guide the development of novel therapies. This review outlines the latest discoveries in the biology of MF, discusses current clinical management of patients with MF, and summarizes the ongoing clinical trials that hope to change the landscape of MF treatment.

An important pathogenetic distinction in the classification of myeloproliferative neoplasms (MPNs) is the presence or absence of the BCR-ABL fusion gene, which encodes a unique oncogenic tyrosine kinase. The BCR-ABL fusion, caused by the formation of the Philadelphia chromosome (Ph) through translocation, constitutes the disease-initiating event in chronic myeloid leukemia. The development of successive BCR-ABL-targeted tyrosine-kinase inhibitors has led to greatly improved outcomes in patients with chronic myeloid leukemia, including high rates of complete hematologic, cytogenetic, and molecular responses. Such levels of treatment success have long been elusive for patients with Ph-negative MPNs, because of the difficulties in identifying specific driver proteins suitable as drug targets. However, in recent years an improved understanding of the complex pathobiology of classic Ph-negative MPNs, characterized by variable, overlapping multimutation profiles, has prompted the development of better and more broadly targeted (to pathway rather than protein) treatment options, particularly JAK inhibitors. In classic Ph-negative MPNs, overactivation of JAK-dependent signaling pathways is a central pathogenic mechanism, and mutually exclusive mutations in JAK2, MPL, and CALR linked to aberrant JAK activation are now recognized as key drivers of disease progression in myelofibrosis (MF). In clinical trials, the JAK1/JAK2 inhibitor ruxolitinib - the first therapy approved for MF worldwide - improved disease-related splenomegaly and symptoms independent of JAK2 (V617F) mutational status, and prolonged survival compared with placebo or standard therapy in patients with advanced MF. In separate trials, ruxolitinib also provided comprehensive hematologic control in patients with another Ph-negative MPN - polycythemia vera. However, complete cytogenetic or molecular responses with JAK inhibitors alone are normally not observed, underscoring the need for novel combination therapies of JAK inhibitors and complementary agents that better address the complexity of the pathobiology of classic Ph-negative MPNs. Here, we discuss the role of tyrosine-kinase inhibitors in the current MPN-treatment landscape.

The genetics underlying thyroid cancer dedifferentiation is only partly understood and has not yet been characterized using comprehensive pan-genomic analyses. We investigated a unique case with synchronous follicular thyroid carcinoma (FTC), poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC), as well as regional lymph node metastases from the PDTC and ATC from a single patient using whole-genome sequencing (WGS). The FTC displayed mutations in CALR, RB1 and MSH2 and the PDTC exhibited mutations in TP53, DROSHA, APC, TERT and additional DNA repair genes - associated with an immense increase in subclonal somatic mutations. All components displayed an overrepresentation of C>T transitions with associated microsatellite instability (MSI) in the PDTC and ATC, with borderline MSI in the FTC. Clonality analyses pinpointed a shared ancestral clone enriched for mutations in TP53-associated regulation of DNA repair and identified important sub-clones for each tumour component already present in the corresponding preceding lesion. This genomic characterization of the natural progression of thyroid cancer reveals several novel genes of interest for future studies. Moreover, the findings support the theory of a stepwise dedifferentiation process and suggest that defects in DNA repair could play an important role in the clonal evolution of thyroid cancer. This article is protected by copyright. All rights reserved.

The hallmark of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) is the presence of a driver mutation in JAK2, CALR, or MPL gene. These genetic alterations represent a key feature, useful for diagnostic, prognostic and therapeutical approaches. Molecular biology tests are now widely available with different specificity and sensitivity. Recently, the allele burden quantification of driver mutations has become a useful tool, both for prognostication and efficacy evaluation of therapies. Moreover, other sub-clonal mutations have been reported in MPN patients, which are associated with poorer prognosis. ASXL1 mutation appears to be the worst amongst them. Both driver and sub-clonal mutations are now taken into consideration in new prognostic scoring systems and may be better investigated using next generation sequence (NGS) technology. In this review we summarize the value of NGS and its contribution in providing a comprehensive picture of mutational landscape to guide treatment decisions. Finally, discussing the role that NGS has in defining the potential risk of disease development, we forecast NGS as the standard molecular biology technique for evaluating these patients.

The treatment of breast cancer largely depends on the utilization of immunogenic chemotherapeutics, which, as a common leitmotif, stimulate the exposure of calreticulin (CALR) on the surface of cancer cells, thereby facilitating their recognition by dendritic cells for the uptake of tumor-associated antigens and subsequent antigen cross-presentation to cytotoxic T cells. Breast cancer cells also express the calreticulin antagonist CD47, which inhibits tumor cell phagocytosis and consequently subverts anticancer immune responses. Here, we treated carcinogen-induced or transplantable mouse models of cancer by a CD47 blocking antibody that was at least as efficient as chemotherapy and that could be favorably combined with the anthracycline mitoxantrone in the context of carcinogen-induced orthotopic breast cancers. Monotherapy by CD47 blockade led to a reduction in tumor growth and an increase in overall survival. Of note, this treatment lead to a moderate depletion of M2 macrophages as well as close-to-complete elimination of regulatory T cells from the tumor bed, suggesting a strong favorable impact of CD47 blockade on the tumor microenvironment.