DETECTION OF CALR MUTATIONS USING HIGH RESOLUTION MELTING CURVE ANALYSIS (HRM-A); APPLICATION ON A LARGE COHORT OF GREEK ET AND MF PATIENTS

Background and Objectives Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibosis (MF), lacking the JAK2and MPLmutations. To determine the prevalence of CALRframeshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALRmutants.   Methods MPN patient samples were collected from different clinical units and screened for JAK2and MPLmutations after informed consent was obtained. Negative samples were analyzed for the presence of CALRmutations. To this end, we developed a modified post Real Time PCR High Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing.   Results Using this protocol we screened 173 MPN, JAK2and MPLmutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALRexon 9 mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study reavealed 6 rare novel mutations which are to be added in the COSMIC database.    Conclusions Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups

Antiretroviral activity of 5-azacytidine during treatment of a HTLV-1 positive myelodysplastic syndrome with autoimmune manifestations

Myelodysplastic syndromes (MDS) are often accompanied by autoimmune phenomena. The underlying mechanisms for these associations remain uncertain, although T cell activation seems to be important. Human T-lymphotropic virus (HTLV-1) has been detected in patients with myelodysplastic syndromes, mostly in regions of the world which are endemic for the virus, and where association of HTLV-1 with rheumatological manifestation is not rare. We present here the case of a 58 year old man who presented with cytopenias, leukocytoclastic vasculitis of the skin and glomerulopathy, and was diagnosed as MDS (refractory anemia with excess blasts - RAEB 1). The patient also tested positive for HTLV-1 by PCR. After 8 monthly cycles of 5-azacytidine he achieved a complete hematologic remission. Following treatment, a second PCR for HTLV-1 was carried out and found to be negative. This is the first report in the literature of a HTLV-1-positive MDS with severe autoimmune manifestations, which was treated with the hypomethylating factor 5-azacitidine, achieving cytogenetic remission with concomitant resolution of the autoimmune manifestations, as well as HTLV-1-PCR negativity. HTLV-1-PCR negativity may be due to either immune mediated clearance of the virus, or a potential antiretroviral effect of 5-azacytidine. 5-azacytidine is known for its antiretroviral effects, although there is no proof of its activity against HTLV-1 infection in vivo

Atypical Chronic Myelogenous Leukemia, BCR-ABL1 Negative: Diagnostic Criteria and Treatment Approaches

Atypical chronic myelogenous leukemia (aCML), BCR/ABL1 negative is a rare myelodysplastic/myeloproliferative neoplasm, usually manifested with hyperleukocytosis without monocytosis or basophilia, organomegaly, and marked dysgranulopoiesis. In this review, we will discuss the classification and diagnostic criteria of aCML, as these have been formulated during the past 30 years, with a focus on the recent advances in the molecular characterization of the disease. Although this entity does not have a definitive molecular profile, its molecular characterization has contributed to a better understanding and more accurate classification and diagnosis of aCML. At the same time, it has facilitated the identification of adverse prognostic factors and the stratification of patients according to their risk for leukemic transformation. What is more, the molecular characterization of the disease has expanded our therapeutic choices, thoroughly presented and analyzed in this review article

Progressive multifocal leukoencephalopathy in the context of newer therapies in hematology and review of new treatment strategies

Progressive multifocal leukoencephalopathy (PML) is a rare, often fatal demyelinating disease of the central nervous system (CNS) caused by the reactivation of JC polyomavirus in the CNS. We present a case of a 54-year-old man with follicular lymphoma diagnosed with PML after being treated with anti-CD20 monoclonal antibody-based regimens for several years. Due to the lack of effective treatment choices for PML, the patient was treated with nivolumab, based on recent reports, but succumbed to his disease a few months after diagnosis. In this paper, we focus on reviewing the literature of PML cases correlated with newer agents used in hematology, possible factors affecting disease prognosis, as well as the available data on upcoming therapeutic options for patients with PML. Though newer promising treatments such as anti-PD1 monoclonal antibodies arise, a definitive treatment option is yet to be found. Vigilance, early detection, and prompt intervention play a crucial role in the prognosis of PML in patients with hematological malignancies

PARP1-Driven Apoptosis in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is considered a malignancy resulting from defects in apoptosis. For this reason, targeting apoptotic pathways in CLL may be valuable for its management. Poly [ADP-ribose] polymerase 1 (PARP1) is the main member of a family of nuclear enzymes that act as DNA damage sensors. Through binding on DNA damaged structures, PARP1 recruits repair enzymes and serves as a survival factor, but if the damage is severe enough, its action may lead the cell to apoptosis through caspase activation, or necrosis. We measured the PARP1 mRNA and protein pretreatment levels in 26 patients with CLL and the corresponding posttreatment levels in 15 patients after 3 cycles of immunochemotherapy, as well as in 15 healthy blood donors. No difference was found between the pre- and posttreatment levels of PARP1, but we found a statistically significant relative increase of the 89 kDa fragment of PARP1 that is cleaved by caspases in the posttreatment samples, indicating PARP1-related apoptosis in CLL patients after treatment. Our findings constitute an important step in the field, especially in the era of PARP1 inhibitors, and may serve as a base for future clinical trials with these agents in CLL

PARP1 as a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome

Poly(ADP-ribose) polymerase 1 (PARP1) is a key mediator of various forms of DNA damage repair and plays an important role in the progression of several cancer types. The enzyme is activated by binding to DNA single-strand and double-strand breaks. Its contribution to chromatin remodeling makes PARP1 crucial for gene expression regulation. Inhibition of its activity with small molecules leads to the synthetic lethal effect by impeding DNA repair in the treatment of cancer cells. At first, PARP1 inhibitors (PARPis) were developed to target breast cancer mutated cancer cells. Currently, PARPis are being studied to be used in a broader variety of patients either as single agents or in combination with chemotherapy, antiangiogenic agents, ionizing radiation, and immune checkpoint inhibitors. Ongoing clinical trials on olaparib, rucaparib, niraparib, veliparib, and the recent talazoparib show the advantage of these agents in overcoming PARPi resistance and underline their efficacy in targeted treatment of several hematologic malignancies. In this review, focusing on the crucial role of PARP1 in physiological and pathological effects in myelodysplastic syndrome and acute myeloid leukemia, we give an outline of the enzyme's mechanisms of action and its role in the pathophysiology and prognosis of myelodysplastic syndrome/acute myeloid leukemia and we analyze the available data on the use of PARPis, highlighting their promising advances in clinical application

A Patient with Synchronous Gallbladder and Bone Plasmacytoma

Multiple myeloma (MM) is the most common primary bone-originating tumor, whereas extramedullary plasmacytoma (EMP) is a plasma cell tumor that arises outside the bone and is most commonly found in the head and neck area. Gastrointestinal and particularly gallbladder involvement is exceedingly rare, and symptoms, if any are present, are usually similar to those seen with cholelithiasis. Treatment options usually include surgical resection and/or chemotherapy. In this report, we present a rare case of a clinically unexpected plasmablastic extramedullary plasmacytoma that was found on abdominal ultrasound (US) and magnetic resonance imaging (MRI) in a 61-year-old asymptomatic patient and led him to undergo cholecystectomy. A fluorodeoxyglucose positron emission computed tomography (FDG PET-CT) that was performed due to the onset of left thigh pain also demonstrated concurrent bone plasmacytoma. The patient is currently receiving chemotherapy and is also being prepared for autologous stem cell transplantation. In this context, we further present the diagnostic, therapeutic and prognostic challenges of EMPs. Lastly, we point out the distinct features of the plasmablastic subtype and analyze its differences compared to other histologic subtypes in achieving a successful diagnosis and management

Are We Identifying Malnutrition in Hospitalized Patients with Hematologic Malignancies? Results from a Quality Clinical Audit

Disease-related malnutrition (DRM) is highly prevalent among patients with hematologic malignancies. The aim of the present study was to evaluate the prevalence of DRM in hospitalized patients with hematologic malignancies and investigate the level of awareness of DRM among the medical team treating this group of patients. A cross sectional quality clinical audit took place in two hematology units of a tertiary university hospital. Inpatients were screened within 48 h of their admission using the Malnutrition Universal Screening Tool (MUST) to identify their nutritional risk, and they were reassessed to identify the implemented interventions during their hospitalization. One hundred eighty-five patients were included in the audit analysis. On admission, 37.3% of the audited population was identified as being at high risk of malnutrition according to the MUST score. Forty-nine (26.5%) patients reported reduced food intake during the past 5 days, while four (2.2%) reported no food intake. During the hospitalization, only five patients (2.7%) received nutritional support, as indicated. Low levels of awareness of the early detection and treatment of DMS were found. Moreover, the prevalence of DRM and low nutritional intake was reported to be low. Measures to increase awareness of DMR in the medical team and better coordination of the nutrition support teams is vital to ensure better management and early nutrition intervention in hematological patients