Long-term hypothyroidism in patients started on levothyroxine during pregnancy.

Background: Current guidelines recommend different postpartum approaches for patients started on levothyroxine (LT4) during pregnancy. Objective: We studied the postpartum management of these patients and determined factors associated with long-term hypothyroidism. Methods: A retrospective study was conducted at a tertiary center between 2014 and 2020, with LT4 initiation according to 2014 ETA recommendations. We performed multivariate logistic regression (MVR) and a receiver operating characteristic curve analysis to determine variables associated with long-term hypothyroidism and their optimal cutoffs. Results: LT4 was initiated in 177 pregnant women, and 106/177 (60%) were followed at long-term (at least 6 months post partum) (28.5 (9.0-81.9) months). LT4 could have been stopped in 45% of patients who continued it immediately after delivery. Thirty-six out of 106 (34%) patients were long-term hypothyroid. In them, LT4 was initiated earlier during pregnancy than in euthyroid women (11.7 ± 4.7 vs 13.7 ± 6.5 weeks, P = 0.077), at a higher thyroid-stimulating hormone (TSH) level (4.1 (2.2-10.1) vs 3.5 (0.9-6.9) mU/L, P = 0.005), and reached a higher dose during pregnancy (62.8 ± 22.2 vs 50.7 ± 13.9 µg/day, P = 0.005). In the MVR, only the maximal LT4 dose during pregnancy was associated with long-term hypothyroidism (odds ratio (OR) = 1.03, 95% CI: 1.00-1.05, P = 0.003). The optimal cutoffs for predicting long-term hypothyroidism were an LT4 dose of 68.75 µg/day (87% specificity, 42% sensitivity; P = 0.013) and a TSH level ≥ 3.8 mU/L (68.5% specificity, 77% sensitivity; P = 0.019). Conclusion: One-third of the patients who started on LT4 during pregnancy had long-term hypothyroidism. The TSH level at treatment initiation and the LT4 dose during pregnancy could guide the decision for continuing long-term LT4.

Novel germline JAK2R715T mutation causing PV-like erythrocytosis in 3 generations. Amelioration by Ropeg-Interferon.

Polycythemia vera (PV) is a clonal disorder arising from the acquired somatic mutations of the JAK2 gene, including JAK2V617F or several others in exon 12. A 38-year-old female had a stroke at age 32 and found to have elevated hemoglobin, normal leukocytes, normal platelets, and tested negative for JAK2V617F and exon 12 mutations. Next generation sequencing revealed a novel mutation: JAK2R715T in the pseudokinase domain (JH2) at 47.5%. Its presence in her nail DNA confirmed a germline origin. Her mother and her son similarly had erythrocytosis and a JAK2R715T mutation. Computer modeling indicated gain-of-function JAK2 activity. The propositus and her mother had polyclonal myelopoiesis, ruling out another somatic mutation-derived clonal hematopoiesis. Some erythroid progenitors of all three generations grew without erythropoietin, a hallmark of PV. The in vitro reporter assay confirmed increased activity of the JAK2R715T kinase. Similar to PV, the JAK2R715T native cells have increased STAT5 phosphorylation, augmented transcripts of prothrombotic and inflammatory genes, and decreased KLF2 transcripts. The propositus was not controlled by hydroxyurea, and JAK2 inhibitors were not tolerated; however, Ropeginterferon-alfa-2b (Ropeg-IFN-α) induced a remission. Ropeg-IFN-α treatment also reduced JAK2 activity in the propositus, her mother and JAK2V617F PV subjects. We report dominantly inherited erythrocytosis secondary to a novel germline JAK2R715T gain-of-function mutation with many but not all comparable molecular features to JAK2V617F PV. We also document a previously unreported inhibitory mechanism of JAK2 signaling by Ropeg-IFN-α.

How often should we perform magnetic resonance imaging (MRI) for the follow-up of pituitary adenoma?

Magnetic resonance imaging (MRI) is the examination of choice for diagnosing and monitoring pituitary adenoma (also known as pituitary neuroendocrine tumor or PitNET), whether treated or not. However, repeating the examination too often (and sometimes unnecessarily) is costly, and worrying data on tissue accumulation (brain, bone, etc.) of gadolinium atoms dissociated from their carrier molecule (chelator) have led European authorities to ban contrast agents based on linear chelators of gadolinium, which are particularly susceptible to rapid dissociation, in favor of chemically more stable macrocyclic chelators. It is therefore important to determine the optimal frequency for pituitary MRI monitoring in order to safely assess the natural history or therapeutic response of pituitary adenomas. The aim of this article is to summarize the most recent data on optimal follow-up intervals depending on the type, size and location of the pituitary tumor and the clinical situation in general, in order to generate monitoring algorithms to guide clinicians.

Translating radiological research into practice-from discovery to clinical impact.

At the European Society of Radiology (ESR), we strive to provide evidence for radiological practices that improve patient outcomes and have a societal impact. Successful translation of radiological research into clinical practice requires multiple factors including tailored methodology, a multidisciplinary approach aiming beyond technical validation, and a focus on unmet clinical needs. Low levels of evidence are a threat to radiology, resulting in low visibility and credibility. Here, we provide the background and rationale for the thematic series Translating radiological research into practice-from discovery to clinical impact, inviting authors to describe their processes of achieving clinically impactful radiological research. We describe the challenges unique to radiological research. Additionally, a survey was sent to non-radiological clinical societies. The majority of respondents (6/11) were in the field of gastrointestinal/abdominal medicine. The implementation of CT/MRI techniques for disease characterisation, detection and staging of cancer, and treatment planning and radiological interventions were mentioned as the most important radiological developments in the past years. The perception was that patients are substantially unaware of the impact of these developments. Unmet clinical needs were mostly early diagnosis and staging of cancer, microstructural/functional assessment of tissues and organs, and implant assessment. All but one respondent considered radiology important for research in their discipline, but five indicated that radiology is currently not involved in their research. Radiology research holds the potential for being transformative to medical practice. It is our responsibility to take the lead in studies including radiology and strive towards the highest levels of evidence.Critical relevance statement For radiological research to make a clinical and societal impact, radiologists should take the lead in radiological studies, go beyond the assessment of technical feasibility and diagnostic accuracy, and-in a multidisciplinary approach-address clinical unmet needs.Key points• Multiple factors are essential for radiological research to make a clinical and societal impact.• Radiological research needs to go beyond diagnostic accuracy and address unmet clinical needs.• Radiologists should take the lead in radiological studies with a multidisciplinary approach.

Significance of thyroperoxidase and thyroglobulin antibodies in medically treated Graves' disease.

Background: Thyroperoxidase (TPOAb) and thyroglobulin (TgAb) antibodies are highly prevalent in Graves' disease (GD), but their significance is controversial. Methods: We retrospectively analyzed TPOAb and TgAb levels and evolution in 136 patients with newly diagnosed GD between 2000 and 2022, treated with anti-thyroid drugs (ATD) in a block-and-replace (B+R) regimen for at least 12 months and followed up for at least 1 year after ATD discontinuation or until disease relapse. Results: At diagnosis, 98 out of 136 (72%) patients were TPOAb positive and 73 out of 136 (54%) patients were TgAb positive. The presence of TPOAb or TgAb antibodies at diagnosis was generally not related to GD presentation and did not influence the risk of relapse (P = 0.304 and P = 0.348, respectively). There was less TED (thyroid eye disease) in TgAb-positive patients than TgAb-negative patients at diagnosis (11 out of 73 (15.1%) versus 21 out of 63 (33.3%) P = 0.012). In contrast, the presence of TPOAb at diagnosis was not associated with TED (P = 0.354). The absence of TgAb at diagnosis (P = 0.05) and time to euthyroidism (P = 0.009), but not smoking or TRAb levels, were associated with TED in multivariate logistic regression. TPOAb and TgAb levels during treatment and after its discontinuation were not predictive of relapse, except for lower titers of TgAb at 18 months in patients who relapsed (P = 0.034). Conclusion: In GD patients treated with a first course of ATD in a B+R regimen we observed lower titers of TgAb at the end of treatment in patients who relapsed and a significant protection against TED in patients with positive TgAb at diagnosis, irrespectively of TPOAb.

Natural history and surgical outcome of incidentally discovered clinically nonfunctioning pituitary macroadenomas.

Objectives: The incidental diagnosis of nonfunctioning pituitary macroadenomas (NFPMAs) is becoming more prevalent with the spread of modern brain imaging techniques. We sought to uncover new data about their natural history and surgical outcome. Design: This is a retrospective single-center observational study. Methods: Among 210 patients seen for a NFPMA between 2010 and 2019, 70 (33%) were discovered incidentally (i-NFPMA). We analyzed outcomes in a total of 65 patients with available follow-up data. Results: Mean age at diagnosis (± s.d.) was 60 ± 14 years and mean maximal diameter was 20.0 ± 7.3 mm. At diagnosis, 29 patients (45%) had pituitary hormone deficits (LH/FSH 41%, TSH 29%, ACTH 15%) and 12% had visual field deficits. 26 patients underwent initial surgery, while 12 had delayed surgery after initial surveillance. In the surveillance group, the risk of tumor growth was estimated at 10%/year. Patients with hormonal deficits at diagnosis experienced earlier growth at 24 months (P < 0.02). Overall, surgical resection of the i-NFPMA led to stable or improved endocrine function in 91% of patients, with only 6% postoperative permanent diabetes insipidus. Moreover, surgery was more effective in preserving intact endocrine function (10/12) than restoring altered endocrine function to normal (6/22, P = 0.03). Conclusion: About one-third of NFPMAs are now discovered incidentally and a significant subset may be responsible for unrecognized endocrine and visual deficits. Under surveillance the risk of further tumor growth is significant (10%/year) and seems to occur faster in patients already harboring an endocrine deficit. Early surgical removal before onset of endocrine deficits appears to lead to better endocrine outcome.

Leukemic conversion involving RAS mutations of type 1 CALR-mutated primary myelofibrosis in a patient treated for HCV cirrhosis: a case report.

Somatic frameshift mutations in exon 9 of calreticulin (CALR) gene are recognized as disease drivers in primary myelofibrosis (PMF), one of the three classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Type 1/type 1-like CALR mutations particularly confer a favorable prognostic and survival advantage in PMF patients. We report an unusual case of PMF incidentally diagnosed in a 68-year-old woman known with hepatitis C virus (HCV) cirrhosis who developed a progressive painful splenomegaly, without anomalies in blood cell counts. While harboring a type 1 CALR mutation, the patient underwent a leukemic transformation in less than 1 year from diagnosis, with a lethal outcome. Analysis of paired DNA samples from chronic and leukemic phases by a targeted next-generation sequencing (NGS) panel and single-nucleotide polymorphism (SNP) microarray revealed that the leukemic clone developed from the CALR-mutated clone through the acquisition of genetic events in the RAS signaling pathway: an increased variant allele frequency of the germline NRAS Y64D mutation present in the chronic phase (via an acquired uniparental disomy of chromosome 1) and gaining NRAS G12D in the blast phase. SNP microarray analysis showed five clinically significant copy number losses at regions 7q22.1, 8q11.1-q11.21, 10p12.1-p11.22, 11p14.1-p11.2, and Xp11.4, revealing a complex karyotype already in the chronic phase. We discuss how additional mutations, detected by NGS, as well as HCV infection and antiviral therapy, might have negatively impacted this type 1 CALR-mutated PMF. We suggest that larger studies are required to determine if more careful monitoring would be needed in MPN patients also carrying HCV and receiving anti-HCV treatment.

Recent advances in therapies for primary myelofibrosis.

Primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET) form the classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs) that are driven by a constitutive activation of JAK2 signaling. PMF as well as secondary MF (post-ET and post-PV MF) are the most aggressive MPNs. Presently, there is no curative treatment, except allogenic hematopoietic stem cell transplantation. JAK inhibitors, essentially ruxolitinib, are the therapy of reference for intermediate and high-risk MF. However, presently the current JAK inhibitors behave mainly as anti-inflammatory drugs, improving general symptoms and spleen size without major impact on disease progression. A better understanding of the genetics of MF, the biology of its leukemic stem cells (LSCs), the mechanisms of fibrosis and of cytopenia and the role of inflammatory cytokines has led to new approaches with the development of numerous therapeutic agents that target epigenetic regulation, telomerase, apoptosis, cell cycle, cytokines and signaling. Furthermore, the use of a new less toxic form of interferon-α has been revived, as it is presently one of the only molecules that targets the mutated clone. These new approaches have different aims: (a) to provide alternative therapy to JAK inhibition; (b) to correct cytopenia; and (c) to inhibit fibrosis development. However, the main important goal is to find new disease modifier treatments, which will profoundly modify the progression of the disease without major toxicity. Presently the most promising approaches consist of the inhibition of telomerase and the combination of JAK2 inhibitors (ruxolitinib) with either a BCL2/BCL-xL or BET inhibitor. Yet, the most straightforward future approaches can be considered to be the development of and/or selective inhibition of JAK2V617F and the targeting MPL and calreticulin mutants by immunotherapy. It can be expected that the therapy of MF will be significantly improved in the coming years.

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation.

The thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F-mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F-driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F-positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F-specific activation.

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Senescence-related impairment of autophagy induces toxic intraneuronal amyloid-ß accumulation in a mouse model of amyloid pathology.

Aging is the main risk factor for Alzheimer's disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes underlying pathological aging of the nervous system are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. Senescence has also been shown to reduce the autophagic flux, a mechanism involved in clearing damaged proteins from the cell, and such impairment has been linked to AD pathogenesis. In this study, we investigated the role of cellular senescence on AD pathology by crossing a mouse model of AD-like amyloid-ß (Aß) pathology (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc-/-). We studied changes in amyloid pathology, neurodegeneration, and the autophagy process in brain tissue samples and primary cultures derived from these mice by complementary biochemical and immunostaining approaches. Postmortem human brain samples were also processed to evaluate autophagy defects in AD patients. Our results show that accelerated senescence produces an early accumulation of intraneuronal Aß in the subiculum and cortical layer V of 5xFAD mice. This correlates with a reduction in amyloid plaques and Aß levels in connecting brain regions at a later disease stage. Neuronal loss was specifically observed in brain regions presenting intraneuronal Aß and was linked to telomere attrition. Our results indicate that senescence affects intraneuronal Aß accumulation by impairing autophagy function and that early autophagy defects can be found in the brains of AD patients. Together, these findings demonstrate the instrumental role of senescence in intraneuronal Aß accumulation, which represents a key event in AD pathophysiology, and emphasize the correlation between the initial stages of amyloid pathology and defects in the autophagy flux.

Oncogenic CALR mutant C-terminus mediates dual binding to the thrombopoietin receptor triggering complex dimerization and activation.

Calreticulin (CALR) frameshift mutations represent the second cause of myeloproliferative neoplasms (MPN). In healthy cells, CALR transiently and non-specifically interacts with immature N-glycosylated proteins through its N-terminal domain. Conversely, CALR frameshift mutants turn into rogue cytokines by stably and specifically interacting with the Thrombopoietin Receptor (TpoR), inducing its constitutive activation. Here, we identify the basis of the acquired specificity of CALR mutants for TpoR and define the mechanisms by which complex formation triggers TpoR dimerization and activation. Our work reveals that CALR mutant C-terminus unmasks CALR N-terminal domain, rendering it more accessible to bind immature N-glycans on TpoR. We further find that the basic mutant C-terminus is partially α-helical and define how its α-helical segment concomitantly binds acidic patches of TpoR extracellular domain and induces dimerization of both CALR mutant and TpoR. Finally, we propose a model of the tetrameric TpoR-CALR mutant complex and identify potentially targetable sites.

SRSF2-P95H decreases JAK/STAT signaling in hematopoietic cells and delays myelofibrosis development in mice.

Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2P95H with Jak2V617F, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFß1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H, an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Therapeutic activity of GARP:TGF-ß1 blockade in murine primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-ß1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-ß1, only a few activate the cytokine via cell type-specific mechanisms. The cellular source of the active TGF-ß1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-ß1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-ß1 activation by GARP-expressing cells. GARP:TGF-ß1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-ß1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-ß1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

Structural Determinant of ß-Amyloid Formation: From Transmembrane Protein Dimerization to ß-Amyloid Aggregates.

Most neurodegenerative diseases have the characteristics of protein folding disorders, i.e., they cause lesions to appear in vulnerable regions of the nervous system, corresponding to protein aggregates that progressively spread through the neuronal network as the symptoms progress. Alzheimer's disease is one of these diseases. It is characterized by two types of lesions: neurofibrillary tangles (NFTs) composed of tau proteins and senile plaques, formed essentially of amyloid peptides (Aß). A combination of factors ranging from genetic mutations to age-related changes in the cellular context converge in this disease to accelerate Aß deposition. Over the last two decades, numerous studies have attempted to elucidate how structural determinants of its precursor (APP) modify Aß production, and to understand the processes leading to the formation of different Aß aggregates, e.g., fibrils and oligomers. The synthesis proposed in this review indicates that the same motifs can control APP function and Aß production essentially by regulating membrane protein dimerization, and subsequently Aß aggregation processes. The distinct properties of these motifs and the cellular context regulate the APP conformation to trigger the transition to the amyloid pathology. This concept is critical to better decipher the patterns switching APP protein conformation from physiological to pathological and improve our understanding of the mechanisms underpinning the formation of amyloid fibrils that devastate neuronal functions.