Chezi Ganzel

Recurrent RAS and PIK3CA mutations in Erdheim-Chester disease

Erdheim-Chester disease (ECD) is a rare histiocytic disorder that is challenging to diagnose and treat. We performed molecular analysis of BRAF in the largest cohort of ECD patients studied to date followed by N/KRAS, PIK3CA, and AKT1 mutational analysis in BRAF wild-type patients. Forty-six of 80 (57.5%) of patients were BRAFV600E-mutant. NRAS mutations were detected in 3 of 17 ECD BRAFV600E wild-type patients. PIK3CA mutations (p.E542K, p.E545K, p.A1046T, and p.H1047R) were detected in 7 of 55 patients, 4 of whom also had BRAF mutations. Mutant NRAS was present in peripheral blood CD14(+) cells, but not lymphoid cells, from an NRASQ61R mutant patient. Our results underscore the central role of RAS-RAF-MEK-ERK activation in ECD and identify an important role of activation of RAS-PI3K-AKT signaling in ECD. These results provide a rationale for targeting mutant RAS or PI3K/AKT/mTOR signaling in the subset of ECD patients with NRAS or PIK3CA mutations.

Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

UNLABELLED Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (non-LCH), respectively. The discovery of BRAF(V600E) mutations in approximately 50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of patients with BRAF(V600E)-wild-type non-LCH are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole-exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in patients with BRAF(V600E)-wild-type non-LCH. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of patients with MAP2K1- and ARAF-mutated non-LCH using MEK and RAF inhibitors, respectively, resulted in clinical efficacy, demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders. SIGNIFICANCE We provide the first description of kinase fusions in systemic histiocytic neoplasms and activating ARAF and MAP2K1 mutations in non-Langerhans histiocytic neoplasms. Refractory patients with MAP2K1- and ARAF-mutant histiocytoses had clinical responses to MEK inhibition and sorafenib, respectively, highlighting the importance of comprehensive genomic analysis of these disorders.

Hyperleukocytosis, leukostasis and leukapheresis: practice management.

Hyperleukocytosis, arbitrarily defined in acute leukemia as a white blood cell count greater than 100,000/mL, often is associated with increased morbidity and mortality in patients with leukemic processes. It can induce leukostasis, tumor lysis syndrome and disseminated intravascular coagulopathy and has significant prognostic implications with or without one of these clinical complications. The main sites that tend to be injured from the obstructions are the central nerve system and lungs. Despite characteristic clinical presentations, the diagnosis of leukostasis is rarely made with high confidence. The main goal of the management of hyperleukocytosis and/or leukostasis is to reduce the white blood cell count before starting induction chemotherapy. The cytoreduction can be achieved by either leukapheresis and/or hyroxyurea. The technical aspects, complications and efficacy of leukapheresis are discussed in the current article.

DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3AR882), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3AR882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3AR882 cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3ITD) and the nucleophosmin gene (Npm1c) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3AR882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3AR882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3AR882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.

How I treat hematologic emergencies in adults with acute leukemia.

Acute myeloid leukemia and acute lymphoblastic leukemia remain devastating diseases. Only approximately 40% of younger and 10% of older adults are long-term survivors. Although curing the leukemia is always the most formidable challenge, complications from the disease itself and its treatment are associated with significant morbidity and mortality. Such complications, discussed herein, include tumor lysis, hyperleukocytosis, cytarabine-induced cellebellar toxicity, acute promyelocytic leukemia differentiation syndrome, thrombohemorrhagic syndrome in acute promyelocytic leukemia, L-asparaginase-associated thrombosis, leukemic meningitis, neutropenic fever, neutropenic enterocolitis, and transfussion-associated GVHD. Whereas clinical trials form the backbone for the management of acute leukemia, emergent clinical situations, predictable or not, are common and do not readily lend themselves to clinical trial evaluation. Furthermore, practice guidelines are often lacking. Not only are prospective trials impractical because of the emergent nature of the issue at hand, but clinicians are often reluctant to randomize such patients. Extensive practical experience is crucial and, even if there is no consensus, management of such emergencies should be guided by an understanding of the underlying pathophysiologic mechanisms.

Severe and persistent heparin-induced thrombocytopenia despite fondaparinux treatment.

An 85-year-old woman was admitted to the orthopedic department of Shaare Zedek, Medical Center in Jerusalem, Israel, due to hip fracture. Prior to undergoing surgery she was started on dalteparin treatment (5,000 U day) as prophylaxis for thrombosis. On the eighth day of treatment, while suffering from a urinary tract infection, her platelet count fell from 163,000/ml to 3,000/ml. No bleeding was observed. Thrombocytopenia occurring 8 days after the initiation of heparin raised the suspicion of heparin-induced thrombocytopenia (HIT). HIT is an immune-mediated disorder characterized by the formation of antibodies against platelet factor 4 (PF4)/heparin complexes. It usually begins 5–10 days after the initiation of heparin or low molecular weight heparin (LMWH). The platelet count usually falls by at least 50% compared to the highest count after start of heparin; in 5–15% of patients, the platelet count falls only by 30–50% and in rare cases it falls by <30%. In 10–15%, the platelet count falls below 20,000/ml, but is typically not <10,000/ml [1]. A commonly used score to predict the likelihood of HIT is the “4 Ts” score. It predicts the likelihood of HIT by grading the Timing, severity of Thrombocytopenia, occurrence of new Thrombosis and the presence of alternative explanations. The score ranges from 0 to 8 points. A higher score predicts higher probability of HIT. A very low platelet count is very unusual in HIT and accordingly, in the 4Ts pretest probability, a platelet count below 10,000/ml gets zero points [2]. In contrast, immune thrombocytopenia (ITP) and especially druginduced immune thrombocytopenia often present with platelet counts <10,000/ml. Because such a low platelet count is unusual for HIT, acute ITP was suspected. Dalteparin was stopped and prednisone was started. The next day her platelets rose to 14,000/ml. The PF4/heparin-particle gel immunoassay (PaGIA) HIT test was strongly positive. The ELISA was positive for anti-PF4/heparin antibodies of the immuneglobulin class G (OD 2.610). The heparin-induced platelet activation (HIPA) test showed positive reaction. Platelet activation was inhibited by high concentration of heparin (100 IU/ml) and by the monoclonal antibody IV.3. There are several diagnostic tests for heparin-induced thrombocytopenia; solid phase immunoassays (ELISAs and particle gel immunoassay), functional assays using whole blood or platelet rich plasma, and functional assays using washed platelets, serotonin release assay (SRA) and the heparin-induced platelet aggregation assay (HIPA) [3]. The SRA is considered the “gold standard” assay for the detection of heparin-dependent antibodies in HIT. It is based on HIT antibodies causing platelet activation and subsequent serotonin release. Platelet-rich plasma of healthy individuals is incubated with C-serotonin and then mixed with patient serum, along with low (therapeutic) and high heparin concentrations. The test is positive for HIT if there is strong platelet activation (>20% serotonin release) at a therapeutic dosage of heparin and inhibition of platelet activation with high heparin concentration. HIPA is a washed platelet-activation test with similar characteristics as the SRA, in which the patient’s serum is mixed with donor platelets in the presence of heparin. Aggregation of the donor platelets indicates the presence of antibodies to the heparin-PF4-complex. The assay is evaluated visually and does not require radioactivity. She was started on fondaparinux 2.5 mg daily. The dose of fondaparinux was lowered due to her very low platelet count. Four days after the initiation of fondaparinux she developed bilateral deep vein thrombosis (DVT). Even though her platelet count was still only 10,000/ml, the dose of fondaparinux was raised to 7.5 mg day. Heparin-induced thrombocytopenia is a pro-thrombotic adverse effect of heparin. However, only a subgroup of patients with HIT develops thromboembolic complications. In a retrospective analysis, one of the most important risk factors for thrombosis was the magnitude of platelet count decrease [4]. There is a natural tendency of physicians to use lower doses of anticoagulation when the platelet counts are very low. However, this is one of the paradoxes in HIT, a very low platelet count indicates very severe HIT and needs aggressive anticoagulation. A bone marrow aspirate showed normal megakaryocytes without overt pathology. Prednisone was reduced gradually. Despite 13 days of fondaparinux treatment, her platelet count did not recover. This condition might represent “autoimmune HIT.” Indeed, the HIPA test showed positive reaction with 4 out of 4 test cells (time to activation 5 min), even in the absence of heparin. In regular cases of HIT, thrombocytopenia begins 5to 10-days post heparin or LMWH administration. The rare cases where the thrombocytopenia begins late in the course of heparin treatment or even after cessation of heparin are usually called “delayed-onset HIT.” In these patients, the antibodies no longer require heparin to activate platelets. Perhaps, they recognize PF4 bound to endogenous polyanions such as chondroitinsulfate or heparansulfate. Thrombocytopenia might last weeks or even months in such patients [5]. Therefore, long-lasting HIT without dependent on heparin is often called “autoimmune HIT.” The term “autoimmune HIT” is also used for the very rare cases where HIT appears without any exposure to heparin [6]. In these patients the

Efficacy and safety of salvage therapy using Carfilzomib for relapsed or refractory multiple myeloma patients: a multicentre retrospective observational study

Carfilzomib has been established in previous years as a treatment for patients with relapsed and/or refractory multiple myeloma (RR‐MM). A retrospective multicentre study to evaluate the clinical use of carfilzomib for RR‐MM outside of a clinical trial setting was conducted by our group. One hundred and thirty‐five patients were included. All patients had been previously exposed to bortezomib and 93% had also been treated with lenalidomide. The vast majority of patients received carfilzomib as part of a two‐ or three‐drug combination. The overall response rate was 47·2%. Multivariate analysis revealed bortezomib resistance, lenalidomide resistance and albumin <35 g/l to negatively impact the likelihood of achieving response. The median duration of response was 8·4 months, and was significantly higher in patients receiving three‐drug combination and patients presenting without extramedullary disease. The median progression‐free survival and overall survival for the entire cohort was 4·9 months (95% confidence interval [CI] 3·8–6·4) and 12·2 months (95% CI 9‐not reached), respectively. Toxicity was manageable, although treatment‐related death was seen in 5% of patients. In the setting of progressive multiple myeloma, carfilzomib in a combination regimens yields effective results with a manageable toxicity.

Autologous transplant remains the preferred therapy for relapsed APL in CR2

Despite their favorable prognosis, 10–20% of acute promyelocytic leukemia (APL) patients relapse. Reinduction therapy is often followed by autologous hematopoietic cell transplantation (auto-HCT). Arsenic trioxide (ATO) has become part of standard reinduction and is often followed by auto-HCT. Data on patients in CR2 were collected from two large transplant registries (Center for International Blood and Marrow Transplant Research (CIBMTR) and European Group for Blood and Marrow Transplant (EBMT)) and two specialty referral centers. The outcome of patients in CR2 who received only ATO-based therapy as reinduction was retrospectively compared with those who got an auto-HCT, with or without ATO. Prognostic factors included age, disease risk, extramedullary disease and duration of CR1. Of 207 evaluable patients, the median age was 31.5 years, 15.3% had extramedullary disease and median WBC at diagnosis was 4.8 × 109/L. Sixty-seven patients received ATO alone and 140 underwent auto-HCT. The groups were comparable for age, gender, extramedullary disease, risk group and duration of CR1. At 5 years, overall survival (OS) was 42% and 78% for the ATO-only and auto-HCT groups, respectively (P<0.001). In addition, OS was associated with longer duration of CR1 (P=0.002), but not with disease risk at diagnosis. These data suggest that auto-HCT for APL patients in CR2 results in better OS than ATO-based therapy alone.

Clinical considerations and key issues in the management of patients with Erdheim-Chester Disease: A seven case series

BackgroundErdheim-Chester Disease (ECD), a non Langerhans’ cell histiocytosis of orphan nature and propensity for multi-systemic presentations, comprises an intricate medical challenge in terms of diagnosis, treatment and complication management.ObjectivesThe objectives are to report the clinical, radiological and pathological characteristics, as well as cardinal therapeutic approaches to ECD patients and to provide clinical analyses of the medical chronicles of these complex patients.MethodsPatients with biopsy proven ECD were audited by a multi-disciplinary team of specialists who formed a coherent timeline of all the substantial clinical events in the evolution of their patients’ illness.ResultsSeven patients (five men, two women) were recruited to the study. The median age at presentation was 53 years (range: 39 to 62 years). The median follow-up time was 36 months (range: 1 to 72 months). Notable ECD involvement sites included the skeleton (seven), pituitary gland (seven), retroperitoneum (five), central nervous system (four), skin (four), lungs and pleura (four), orbits (three), heart and great vessels (three) and retinae (one). Prominent signs and symptoms were fever (seven), polyuria and polydipsia (six), ataxia and dysarthria (four), bone pain (four), exophthalmos (three), renovascular hypertension (one) and dyspnea (one). The V600E BRAF mutation was verified in three of six patients tested. Interferon-α treatment was beneficial in three of six patients treated. Vemurafenib yielded dramatic neurological improvement in a BRAF mutated patient. Infliximab facilitated pericardial effusion volume reduction. Cladribine improved cerebral blood flow originally compromised by perivenous lesions.ConclusionsECD is a complex, multi-systemic, clonal entity coalescing both neoplastic and inflammatory elements and strongly dependent on impaired RAS/RAF/MEK/ERK signaling.

Prognostic Factors in Adult Acute Leukemia

The prognostic factors in acute leukemia have undergone a major change over the past decade and are likely to be further refined in the coming years. While age is the single most important prognostic factor in both AML and in ALL, recurring cytogenetic abnormalities and molecular markers have become crucial for the prognosis of patients and for new directions in the development of targeted therapies. No less important is the development of a personalized approach for therapy as determined by the response to therapy using increasingly sensitive technologies. The assessment of MRD is rapidly superseding other prognostic factors in ALL and, somewhat lacking behind, coming into its own in AML. The next decade should see further refinement of response-driven prognostication, to include epigenetics as well as pharmacogenetics and pharmacodynamics of individual drugs used and the responses to them. It is hoped that these refinements and better predictors of response will also lead to a significantly improved overall outcome of patients with both AML and ALL.