Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

Computational quantification of global effects induced by mutations and drugs in signaling networks of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most deadly and commonly diagnosed tumors worldwide. Several genes are involved in its development and progression. The most frequent mutations concern APC, KRAS, SMAD4, and TP53 genes, suggesting that CRC relies on the concomitant alteration of the related pathways. However, with classic molecular approaches, it is not easy to simultaneously analyze the interconnections between these pathways. To overcome this limitation, recently these pathways have been included in a huge chemical reaction network (CRN) describing how information sensed from the environment by growth factors is processed by healthy colorectal cells. Starting from this CRN, we propose a computational model which simulates the effects induced by single or multiple concurrent mutations on the global signaling network. The model has been tested in three scenarios. First, we have quantified the changes induced on the concentration of the proteins of the network by a mutation in APC, KRAS, SMAD4, or TP53. Second, we have computed the changes in the concentration of p53 induced by up to two concurrent mutations affecting proteins upstreams in the network. Third, we have considered a mutated cell affected by a gain of function of KRAS, and we have simulated the action of Dabrafenib, showing that the proposed model can be used to determine the most effective amount of drug to be delivered to the cell. In general, the proposed approach displays several advantages, in that it allows to quantify the alteration in the concentration of the proteins resulting from a single or multiple given mutations. Moreover, simulations of the global signaling network of CRC may be used to identify new therapeutic targets, or to disclose unexpected interactions between the involved pathways.

Radiomics and Artificial Intelligence for Outcome Prediction in Multiple Myeloma Patients Undergoing Autologous Transplantation: A Feasibility Study with CT Data.

Multiple myeloma is a plasma cell dyscrasia characterized by focal and non-focal bone lesions. Radiomic techniques extract morphological information from computerized tomography images and exploit them for stratification and risk prediction purposes. However, few papers so far have applied radiomics to multiple myeloma. A retrospective study approved by the institutional review board: n = 51 transplanted patients and n = 33 (64%) with focal lesion analyzed via an open-source toolbox that extracted 109 radiomics features. We also applied a dedicated tool for computing 24 features describing the whole skeleton asset. The redundancy reduction was realized via correlation and principal component analysis. Fuzzy clustering (FC) and Hough transform filtering (HTF) allowed for patient stratification, with effectiveness assessed by four skill scores. The highest sensitivity and critical success index (CSI) were obtained representing each patient, with 17 focal features selected via correlation with the 24 features describing the overall skeletal asset. These scores were higher than the ones associated with a standard cytogenetic classification. The Mann-Whitney U-test showed that three among the 17 imaging descriptors passed the null hypothesis. This AI-based interpretation of radiomics features stratified relapsed and non-relapsed MM patients, showing some potentiality for the determination of the prognostic image-based biomarkers in disease follow-up.

Identification of Biochemical and Molecular Markers of Early Aging in Childhood Cancer Survivors.

Survival rates of childhood cancer patients have improved over the past four decades, although cancer treatments increase the risk of developing chronic diseases typical of aging. Thus, we aimed to identify molecular/metabolic cellular alterations responsible for early aging in childhood cancer survivors (CCS). Biochemical, proteomic, and molecular biology analyses were conducted on mononuclear cells (MNCs) isolated from peripheral blood of 196 CCS, the results being compared with those obtained on MNCs of 154 healthy subjects. CCS-MNCs showed inefficient oxidative phosphorylation associated with low energy status, and increased lipid peroxidation and lactate fermentation compared with age-matched normal controls. According to a mathematical model based on biochemical parameters, CCS-MNCs showed significantly higher metabolic ages than their real ages. The dysfunctional metabolism of CCS-MNCs is associated with lower expression levels of genes and proteins involved in mitochondrial biogenesis and metabolism regulation, such as CLUH, PGC1-alpha, and SIRT6 in CCS, not observed in the age-matched healthy or elderly subjects. In conclusion, our study identified some biochemical and molecular alterations possibly contributing to the pathophysiology of aging and metabolic deficiencies in CCS. These results identify new targets for pharmacological interventions to restore mitochondrial function, slowing down the aging-associated pathologies in CCS.

Genetic Screening for Potential New Targets in Chronic Myeloid Leukemia Based on Drosophila Transgenic for Human BCR-ABL1.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9;22)(q34;q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the gmrGal4,UAS-BCR-ABL1 4M/TM3 transgenic Drosophila as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.

Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.

Iron overload alters the energy metabolism in patients with myelodysplastic syndromes: results from the multicenter FISM BIOFER study.

Myelodysplastic syndromes (MDS) are hematological malignancies characterized by ineffective hematopoiesis and increased apoptosis in the bone marrow, which cause peripheral cytopenia. Mitochondria are key regulators of apoptosis and a site of iron accumulation that favors reactive oxygen species (ROS) production with detrimental effects on cell survival. Although the energy metabolism could represent an attractive therapeutic target, it was poorly investigated in MDS. The purpose of the study was to analyze how the presence of myelodysplastic hematopoiesis, iron overload and chelation impact on mitochondrial metabolism. We compared energy balance, OxPhos activity and efficiency, lactic dehydrogenase activity and lipid peroxidation in mononuclear cells (MNCs), isolated from 38 MDS patients and 79 healthy controls. Our data show that ATP/AMP ratio is reduced during aging and even more in MDS due to a decreased OxPhos activity associated with an increment of lipid peroxidation. Moreover, the lactate fermentation enhancement was observed in MDS and elderly subjects, probably as an attempt to restore the energy balance. The biochemical alterations of MNCs from MDS patients have been partially restored by the in vitro iron chelation, while only slight effects were observed in the age-matched control samples. By contrast, the addition of iron chelators on MNCs from young healthy subjects determined a decrement in the OxPhos efficiency and an increment of lactate fermentation and lipid peroxidation. In summary, MDS-MNCs display an altered energy metabolism associated with increased oxidative stress, due to iron accumulation. This condition could be partially restored by iron chelation.

Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques.

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.

Discrete Changes in Glucose Metabolism Define Aging.

Aging is a physiological process in which multifactorial processes determine a progressive decline. Several alterations contribute to the aging process, including telomere shortening, oxidative stress, deregulated autophagy and epigenetic modifications. In some cases, these alterations are so linked with the aging process that it is possible predict the age of a person on the basis of the modification of one specific pathway, as proposed by Horwath and his aging clock based on DNA methylation. Because the energy metabolism changes are involved in the aging process, in this work, we propose a new aging clock based on the modifications of glucose catabolism. The biochemical analyses were performed on mononuclear cells isolated from peripheral blood, obtained from a healthy population with an age between 5 and 106 years. In particular, we have evaluated the oxidative phosphorylation function and efficiency, the ATP/AMP ratio, the lactate dehydrogenase activity and the malondialdehyde content. Further, based on these biochemical markers, we developed a machine learning-based mathematical model able to predict the age of an individual with a mean absolute error of approximately 9.7 years. This mathematical model represents a new non-invasive tool to evaluate and define the age of individuals and could be used to evaluate the effects of drugs or other treatments on the early aging or the rejuvenation.

Loss of whole chromosome X predicts prognosis of neuroblastoma patients with numerical genomic profile.

BACKGROUND: Neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is characterized by very frequent chromosomal aberrations at the onset of the disease. Identification of further risk factors for relapse, which could lead to increased survival and potentially reduced late effects among survivors, is still urgently needed. Segmental chromosome aberrations (SCA) are associated with poor prognosis, whereas numerical whole-chromosome aberrations (NCA) are found in patients with a good prognosis; however, a small percentage of the latter patients (10%-15%) subsequently relapse and/or die of disease. PROCEDURE: DNA copy-number data from 174 NB patients with an NCA genomic profile were analyzed. Association between NCA and event-free survival (EFS) was investigated by the Kaplan-Meier estimator and prognostic decision tree (DT). RESULTS: DT identified 65 patients with normal chromosome X and an excellent five-year EFS (100%) independently from the stage at diagnosis. The association between poor EFS and whole chromosome X alterations was confirmed after stratification into two groups of different expected prognosis and by internal validation via bootstrap analysis. Furthermore, the association was also observed in an independent cohort of NB patients extracted from the data set of the National Cancer Institute TARGET Project for Neuroblastoma, but sample size was small (n = 75) and statistical significance was not achieved. CONCLUSIONS: Loss of whole chromosome X may represent a new prognostic marker for NB patients with an NCA genomic profile. If confirmed by further studies, this finding could indicate that such patients should be reclassified as intermediate risk and treated accordingly.

Phase I/II Study of Stem-Cell Transplantation Using a Single Cord Blood Unit Expanded Ex Vivo With Nicotinamide.

PURPOSE: Increasing the number of hematopoietic stem and progenitor cells within an umbilical cord blood (UCB) graft shortens the time to hematopoietic recovery after UCB transplantation. In this study, we assessed the safety and efficacy of a UCB graft that was expanded ex vivo in the presence of nicotinamide and transplanted after myeloablative conditioning as a stand-alone hematopoietic stem-cell graft. METHODS: Thirty-six patients with hematologic malignancies underwent transplantation at 11 sites. RESULTS: The cumulative incidence of neutrophil engraftment at day 42 was 94%. Two patients experienced secondary graft failure attributable to viral infections. Hematopoietic recovery was compared with that observed in recipients of standard UCB transplantation as reported to the Center for International Blood and Marrow Transplant Research (n = 146). The median time to neutrophil recovery was 11.5 days (95% CI, 9 to 14 days) for recipients of nicotinamide-expanded UCB and 21 days (95% CI, 20 to 23 days) for the comparator ( P < .001). The median time to platelet recovery was 34 days (95% CI, 32 to 42 days) and 46 days (95% CI, 42 to 50 days) for the expanded and the comparator cohorts, respectively ( P < .001). The cumulative incidence of grade 2 to 4 acute graft-versus-host disease (GVHD) at day 100 was 44%, and grade 3 and 4 acute GVHD at day 100 was 11%. The cumulative incidence at 2 years of all chronic GVHD was 40%, and moderate/severe chronic GVHD was 10%. The 2-year cumulative incidences of nonrelapse mortality and relapse were 24% and 33%, respectively. The 2-year probabilities of overall and disease-free survival were 51% and 43%, respectively. CONCLUSION: UCB expanded ex vivo with nicotinamide shortens median neutrophil recovery by 9.5 days (95% CI, 7 to 12 days) and median platelet recovery by 12 days (95% CI, 3 to 16.5 days). This trial establishes feasibility, safety, and efficacy of an ex vivo expanded UCB unit as a stand-alone graft.

Novel Immunotherapeutic Approaches for Neuroblastoma and Malignant Melanoma.

Neuroblastoma (NB) and malignant melanoma (MM), tumors of pediatric age and adulthood, respectively, share a common origin, both of them deriving from the neural crest cells. Although NB and MM have a different behavior, in respect to age of onset, primary tissue involvement and metastatic spread, the prognosis for high stage-affected patients is still poor, in spite of aggressive treatment strategies and the huge amount of new discovered biological knowledge. For these reasons researchers are continuously attempting to find out new treatment options, which in a near future could be translated to the clinical practice. In the last two decades, a strong effort has been spent in the field of translational research of immunotherapy which led to satisfactory results. Indeed, several immunotherapeutic clinical trials have been performed and some of them also resulted beneficial. Here, we summarize preclinical studies based on immunotherapeutic approaches applied in models of both NB and MM.

Late Development of FcεRγneg Adaptive Natural Killer Cells Upon Human Cytomegalovirus Reactivation in Umbilical Cord Blood Transplantation Recipients.

In human natural killer (NK) cells, human cytomegalovirus (HCMV) has been shown to be a driving force capable of inducing the expansion of a highly differentiated NKG2C+CD57+ subset, persisting over time in both HCMV+ healthy subjects and umbilical cord blood transplantation (UCBT) recipients experiencing HCMV viral reactivation. In HCMV+ healthy subjects, such expanded NK-cells are characterized by epigenetic modifications that modulate their phenotypic and functional characteristics. In particular, an enhanced ADCC activity is detectable in NK cells lacking the signaling protein FcεRγ. Timing and mechanisms involved in the acquisition of HCMV-induced, adaptive-like features by NK cells are currently unknown. In this study, we investigated the de novo acquisition of several adaptive features in NK cells developing after UCBT by monitoring NK-cell differentiation for at least 2 years after transplant. In UCBT recipients experiencing HCMV reactivation, a rapid phenotypic reconfiguration occurred resulting in the expected expansion of CD56dim NKG2C+CD57+ NK cells. However, while certain HCMV-driven adaptive hallmarks, including high KIR, LILRB1, CD2 and low/negative NKG2A, Siglec-7, and CD161 expression, were acquired early after UCBT (namely by month 6), downregulation of the signaling protein FcεRγ was detected at a later time interval (i.e., by month 12). This feature characterized only a minor fraction of the HCMV-imprinted NKG2C+CD57+ CD56dim NK cell subset, while it was detectable in higher proportions of CD57+ NK cells lacking NKG2C. Interestingly, in patients developing a hyporesponsive CD56-CD16bright NK-cell subset, FcεRγ downregulation occurred in these cells earlier than in CD56dim NK cells. Our data suggest that the acquisition of a fully "adaptive" profile requires signals that may lack in UCBT recipients and/or longer time is needed to obtain a stable epigenetic reprogramming. On the other hand, we found that both HCMV-induced FcεRγneg and FcεRγ+ NK cells from these patients, display similar CD107a degranulation and IFN-γ production capabilities in response to different stimuli, thus indicating that the acquisition of specialized effector functions can be achieved before the "adaptation" to HCMV is completed. Our study provides new insights in the process leading to the generation of different adaptive NK-cell subsets and may contribute to develop new approaches for their employment as novel immunotherapeutic tools.

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation.

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.

EphA3 targeting reduces in vitro adhesion and invasion and in vivo growth and angiogenesis of multiple myeloma cells.

PURPOSE: Multiple myeloma (MM) is a hematologic malignancy characterized by a clonal expansion of plasma cells (PCs) in the bone marrow (BM). Since MM has so far remained incurable, further insights into its pathogenesis and the concomitant identification of new therapeutic targets are urgently needed. The tyrosine kinase receptor EphA3 is known to be involved in various cellular processes including cell viability, cell movement and cell-cell interactions. Recently, EphA3 has emerged as a potential therapeutic target in several hematologic and solid tumors. Here, we aimed to uncover the role of EphA3 in MM. METHODS: EphA3 mRNA and protein expression in primary MM bone marrow plasma cells (BMPCs), in MM-derived cell lines and in healthy controls (HCs) was assessed using qRT-PCR, Western blotting and flow cytometry. The effects of siRNA-mediated EphA3 silencing and anti EphA3 antibody (EphA3mAb) treatment on MM PC trafficking and viability were evaluated using in vitro assays. The effects of EphA3mAb treatment were also assessed in two MM-derived mouse xenograft models. RESULTS: We found that EphA3 was overexpressed in primary MM BMPCs and MM-derived cell lines compared to HCs. We also found that siRNA-mediated EphA3 silencing and EphA3mAb treatment significantly inhibited the ability of MM PCs to adhere to fibronectin and stromal cells and to invade in vitro, without affecting cell proliferation and viability. Gene expression profiling showed that EphA3 silencing resulted in expression modulation of several molecules that regulate adhesion, migration and invasion processes. Importantly, we found that EphA3mAb treatment significantly inhibited in vivo tumor growth and angiogenesis in two MM-derived mouse xenograft models. CONCLUSIONS: Our findings suggest that EphA3 plays an important role in the pathogenesis of MM and provide support for the notion that its targeting may represent a novel therapeutic opportunity for MM.

A novel assay to detect calreticulin mutations in myeloproliferative neoplasms.

The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) were detected in 56-88% of JAK2/MPL-negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs.We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.