Hairy Cell Leukemia Cells Research Articles

Real-world data on diagnostics, treatment and outcomes of patients with hairy cell leukemia: The HCL-CLLEAR study.

Hairy cell leukemia (HCL) and HCL-like disorders have to be distinguished because of their different biology and treatment response. Thus, we conducted a retrospective study on patients with HCL and hairy cell leukemia variant (HCLv) to assess diagnostic algorithms and treatment outcomes in a real-world setting. We analyzed 225 HCL and 26 HCLv patients with median follow-up of 67.9months (HCL) and 20.1months (HCLv). Median age at diagnosis was 56.2 (HCL) and 69.5years (HCLv), male predominance was observed in both groups (76.0% vs. 73.1%). Diagnostics was mostly based on morphological evidence of hairy cells in the peripheral blood and bone marrow. At diagnosis, BRAF V600E mutation was detected in 94.7% of examined HCL patients and in no HCLv patient. Front-line treatment was indicated in 205 (91.1%) HCL and 18 (69.2%) HCLv patients. The majority of HCL patients were administered a cladribine-based regimen (91.2%). Overall response rate (ORR) was higher in cladribine-treated patients compared to those given other treatments (97.7% vs. 81.3%), the same applied with achieving Complete remission (CR) (91.2% vs. 62.5%). HCLv treatment was heterogeneous, but cladribine remained the most frequent option (44.4%) with ORR 81.3% and CR rates 43.8%. Second-line treatment was indicated in 52 HCL and 8 HCLv patients, 25.4% and 44.4% of those treated in first-line. In the whole HCL group, median time to next treatment (TTNT) was not reached and 10-year TTNT was estimated at 74.1%. HCLv patients who underwent first-line treatment had a median TTNT of 56months. The median overall survival (OS) in HCL patients was not reached compared to HCLv with a median OS of 9.5years. These data confirm an excellent prognosis for HCL patients treated with cladribine-based therapy. On the contrary, HCLv with its aggressive behavior represents a group of patients in whom novel treatment approaches are needed.

SINGLE‐CELL RNA‐SEQ OF CLASSIC HAIRY CELL LEUKEMIA REVEALS DISEASE DRIVERS LINKED TO INTRINSIC TREATMENT RESISTANCE AND IDENTIFIES DUSP1 AS POTENTIAL NEW THERAPEUTIC TARGET

Introduction: Standard treatment with purine analogs such as cladribine (2-CdA) achieves excellent remissions in classic hairy cell leukemia (HCL), but up to 25% of patients relapse early and there is a non-diminishing risk of late relapse in complete responders. Even when targeting the recurrent genetic driver mutation BRAF V600E, HCL cells frequently persist in the bone marrow suggesting cooperating biologic alterations facilitating disease survival and persistence. Methods: Single-cell RNA sequencing (scRNA-Seq) was performed in primary HCL cells derived from three long-term (>10 years progression-free survival, PFS) and three short-term responders after 2-CdA treatment (SR, <3 years PFS) and from up to three time points (HCL diagnosis, first, and second relapse). Functional studies were performed using the BRAF D594E-mutated HAIR-M cell line after validating BRAF D594E as an activating mutation with similar downstream signaling and inhibitory potential with BRAF inhibitors as compared to BRAF V600E in primary HCL cells. Results: scRNA-Seq revealed distinct HCL cell clusters characterized by marked overexpression of DUSP1, FOS and JUND in all six patients that were outgrowing at first and second relapse in all three SR. In these DUSP1hi/FOShi/JUNDhi HCL cells, PROGENy cancer pathway analysis demonstrated induced activation of MAPK pathways and gene ontology analysis found negative regulation of the pro-apoptotic p38-MAPK-cascade as potential underlying biologic alteration. BRAF D594E-mutated HAIR-M cells were selected as substitute for primary HCL cells in cell culture due to comparable changes upon exposure to BRAF inhibitors in BRAF kinase activity conformation as assessed by the modular kinase conformation biosensor platform (KinCon) and similar reduction in ERK phosphorylation levels in HEK293T cells transiently overexpressing either flag-tagged BRAF D594E or BRAF V600E. In HAIR-M, we confirm stable expression levels of DUSP1, FOS and JUND, which were highly increased when co-cultured with HS5 stromal cells. Whereas increased expression of FOS and JUND was reversible upon exposure to BRAF inhibitors, expression of DUSP1, a direct target of BRAF-MEK-ERK signaling, and phosphorylation of p38 remained unaffected. As DUSP1 preferentially dephosphorylates/inactivates p38, we studied exposure of the DUSP1-inhibitor BCI in co-culture and found re-phosphorylated p38. Conclusion: DUSP1hi/FOShi/JUNDhi HCL cells may represent a distinct subset of HCL cells highly dependent on environmental cues with an inherently more resistant phenotype to treatment with 2-CdA or BRAF-inhibitors. Inhibition of DUSP1 may represent a novel therapeutic approach by effectively truncating HCL cells from extracellular pro-survival stimuli. Finally, our results suggest that BRAF D594E-mutated HAIR-M cells may serve as a representative disease model for functional studies. Keywords: bioinformatics, computational and systems biology, indolent non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

The protective role of the microenvironment in hairy cell leukemia treatment: Facts and perspectives.

Hairy cell leukemia (HCL) is an incurable, rare lymphoproliferative hematological malignancy of mature B cAlthough first line therapy with purine analogues leads to positive results, almost half of HCL patients relapse after 5-10 years, and standard treatment may not be an option due to intolerance or refractoriness. Proliferation and survival of HCL cells is regulated by surrounding accessory cells and soluble signals present in the tumor microenvironment, which actively contributes to disease progression. In vitro studies show that different therapeutic approaches tested in HCL impact the tumor microenvironment, and that this milieu offers a protection affecting treatment efficacy. Herein we explore the effects of the tumor microenvironment to different approved and experimental therapeutic options for HCL. Dissecting the complex interactions between leukemia cells and their milieu will be essential to develop new targeted therapies for HCL patients.

Measurable residual disease in hairy cell leukemia: Technical considerations and clinical significance.

Hairy cell leukemia (HCL) is a rare type of chronic lymphoid leukemia originating from a mature B lymphocyte. A diagnosis of HCL is based on cytology, confirmed by multiparametric flow cytometry (MFC) studies using anti-B-cell monoclonal antibodies, together with a panel of antibodies more specific to HCL, such as CD11c, CD25, CD103 and CD123. Recently, the BRAF V600E mutation has been described as a disease-defining genetic event. Measurable residual disease (MRD) is defined as the lowest level of HCL cells that can be detected accurately and reproducibly using validated methods; as MRD negativity is associated with high rates of durable complete response, by clearing MRD, the long-term outcome may be improved in patients with advanced HCL. MRD is typically detected using bone marrow, and in some cases, peripheral blood; however, in HCL, discrepancies frequently exist between MRD results obtained from blood, bone marrow aspirate and core biopsy. Among the methods used for MRD detection, MFC appears to be a more sensitive technique than immunohistochemistry. Molecular tests are also used, such as real-time quantitative PCR for unique immunoglobulin heavy chain (IgH) gene rearrangements and PCR techniques with clone specificity for BRAF V600E. Clone-specific PCR (spPCR) is able to detect one HCL cell in 106 normal cells, and is particularly suitable for patients found to be negative for MRD by MFC. Recently, the Hairy Cell Leukemia Consortium created a platform to work on a definition for MRD, and establish the optimal time point, tissue type and method for measuring MRD. This.

Updates in hairy cell leukemia (HCL) and variant-type HCL (HCL-V): rationale for targeted treatments with a focus on ibrutinib.

Hairy cell leukemia (HCL) and HCL-like disorders such as hairy cell leukemia variant (HCL-V) and splenic diffuse red pulp lymphoma (SDRPL) are rare indolent B-cell malignancies. Purine analogs (PNAs), alone or in association with rituximab (R), are the standard of care for HCL in the first-line setting. However, PNAs are toxic and patients may become resistant to these drugs. Therefore, new therapeutic strategies are needed. Several recent in vitro studies highlighted the importance of the interactions between HCL cells and their microenvironment, in particular with bone marrow stromal cells, endothelial cells, and the extracellular matrix. In these interactions, chemokine receptors and adhesion molecules play a major role. Moreover, the importance of signaling pathways, like BRAF, BCR, and CXCR4 has been underlined. Bruton’s tyrosine kinase (BTK) is a fundamental signal transmitter of BCR and CXCR4 in HCL. Preclinical and recent clinical data showed an efficacy of ibrutinib, a BTK inhibitor (BTKi), in HCL and HCL-V. These promising results joined those of other emerging drugs like BRAF or MEK inhibitors and anti-CD22 immunotoxins.Plain Language Summary Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) in hairy cell leukemia (HCL) and variant-type HCL The treatment of hairy cell leukemia (HCL) has changed significantly in recent years. In the first-line settings, treatment with purine analogs (PNAs) with or without anti-CD20 monoclonal antibodies remains the gold standard in 2022. In relapsed/refractory HCL, other drugs are needed: BRAF inhibitors: vemurafenib monotherapy with or without rituximab or dabrafenib in combination with trametinib, an MEK inhibitor (MEKi), as well as the anti-CD22 antibody drug conjugate moxetumomab pasudotox.There are arguments for the use of Bruton’s tyrosine kinase inhibitors (BTKi). Ibrutinib was recently tested in a multisite phase 2 study in 37 patients with either HCL (28 patients: 76%) or HCL-V (nine patients: 24%) including two who were previously untreated. Patients received single-agent ibrutinib at 420 mg daily (24 patients) or 840 mg daily (13 patients) until disease progression or unacceptable toxicity. The overall response rate (ORR) at 32 weeks was 24%, increasing to 36% at 48 weeks and reaching 54% at any time since starting ibrutinib. Seven patients achieved a complete response (CR) as the best response at any time on study, while 13 patients had a partial response (PR) and 10 patients had stable disease (SD). Interestingly, the response rate was not statistically different between HCL and HCL-V patients, suggesting that ibrutinib could be an option in both entities. The estimated 36-month progression-free survival (PFS) was 73% and the estimated 36-month overall survival (OS) was 85%, with no differences between HCL and HCL-V. The frequency of cardiovascular grade 1–2 adverse events (AEs) was 16% for atrial fibrillation; 3% for atrial flutter; 32% for hypertension; and 0%, 3%, and 11%, respectively, for grade ⩾ 3 AEs. Unlike in chronic lymphocytic leukemia (CLL), where the mechanism of action of ibrutinib is well known, the mechanism of action of ibrutinib in HCL appears to be unclear. No mutations were identified in patients with progressive disease, suggesting that the mechanisms of resistance could be different between HCL and CLL. The BTKi that are not yet approved are challenged by the new other targeted treatments.

Successful treatment of synchronous hairy cell leukaemia and diffuse large B cell lymphoma in a patient with severe hypercalcemia and extensive osteolytic lesions

Introduction. Although secondary malignancies usually occur at different times after hairy cell leukemia (HCL) treatment, the occurrence of HCL and other malignancies at the same time is very rare. Synchronous HCL and diffuse large B-cell lymphoma (DLBCL) have not been described so far. Case report. The report presents a 62-year-old female patient with intense constitutional symptoms, hypercalcemia, pancytopenia, and osteolytic destruction of the left shoulder joint. Immunohistochemical analysis of the bone marrow revealed the presence of two cell populations: a population of HCL cells and a population of DLBCL cells with the expression of CMYC and BCL-2 proteins ("double expressor" DLBCL) and high proliferative activity (Ki-67+cells &gt; 90%). Fluorescence in situ hybridization (FISH) analysis showed amplification of the BCL-2 gene. In addition, BRAF gene V600E mutation was detected. After intensive treatment with immunochemotherapy, radiotherapy, and bisphosphonates, the patient achieved complete remission, lasting for more than two years. Conclusion. As the association of HCL and lymphoma is very rare, diagnosis of synchronous occurrence of two lymphoproliferative diseases is a diagnostic and therapeutic challenge. It remains unclear whether DLBC and HCL originated from two different malignant clones or DLBCL developed by the transformation of HCL as the result of clonal evolution of the B-cell clone.

CLL-393: Knockdown of WEE1 Gene Induces Cytotoxic Effects in Hairy Cell Leukemia

Background: Hairy cell leukemia (HCL) is a rare B-cell lymphoid neoplasm with morphological variations that can be difficult to distinguish. A BRAFV600E mutation is found in 80% of patients. Despite the fact that purine analogs (PNA) with or without anti-CD20 antibodies remain the first-line treatment, resistance is still a major issue that has a severe impact on patient outcomes. The successful therapeutic application of short interfering RNA (siRNA) in tumor suppression is being paved by gene silencing. In the cell cycle, the WEE1 gene is the master inhibitor of cyclin-dependent kinase 1. Cumulative data suggests that the WEE1 gene plays a definite function in cell biological processes and that silencing the WEE1 gene could have a variety of implications in cancer cells. Objectives: The purpose of this study was to examine how ex vivo knockdown of the WEE1 gene affects the viability of hairy cell leukemia (HCL) cells in order to see if it has therapeutic potential. Methods: The HCL cell line [Mo T] was used in an experimental in vi’ro investigation. To silence the WEE1 gene ex vivo, cells were transfected using siRNA. Furthermore, the effect of gene knockdown on cell viability was assessed using the MTT test, as well as cell cycle and apoptosis assays performed by flow cytometry. Furthermore, the WEE1 gene expression level in silenced cells was measured and correlated with cellular protein expression. Results: Silencing the WEE1 gene resulted in a significant decrease in cell viability, increased cell apoptosis, and arrested the cell cycle transition from the G1 to the S phase of the cell cycle. In addition, transfected cells showed a significant reduction in WEE1 gene expression, demonstrating the efficiency of gene silencing. Conclusion: In primary hairy cells, silencing the WEE1 gene resulted in significant cytotoxic effects, suggesting that knockdown of the WEE1 gene could be a promising therapeutic target in HCL. Hairy cell leukemia (HCL) is a rare B-cell lymphoid neoplasm with morphological variations that can be difficult to distinguish. A BRAFV600E mutation is found in 80% of patients. Despite the fact that purine analogs (PNA) with or without anti-CD20 antibodies remain the first-line treatment, resistance is still a major issue that has a severe impact on patient outcomes. The successful therapeutic application of short interfering RNA (siRNA) in tumor suppression is being paved by gene silencing. In the cell cycle, the WEE1 gene is the master inhibitor of cyclin-dependent kinase 1. Cumulative data suggests that the WEE1 gene plays a definite function in cell biological processes and that silencing the WEE1 gene could have a variety of implications in cancer cells. The purpose of this study was to examine how ex vivo knockdown of the WEE1 gene affects the viability of hairy cell leukemia (HCL) cells in order to see if it has therapeutic potential. The HCL cell line [Mo T] was used in an experimental in vi’ro investigation. To silence the WEE1 gene ex vivo, cells were transfected using siRNA. Furthermore, the effect of gene knockdown on cell viability was assessed using the MTT test, as well as cell cycle and apoptosis assays performed by flow cytometry. Furthermore, the WEE1 gene expression level in silenced cells was measured and correlated with cellular protein expression. Silencing the WEE1 gene resulted in a significant decrease in cell viability, increased cell apoptosis, and arrested the cell cycle transition from the G1 to the S phase of the cell cycle. In addition, transfected cells showed a significant reduction in WEE1 gene expression, demonstrating the efficiency of gene silencing. In primary hairy cells, silencing the WEE1 gene resulted in significant cytotoxic effects, suggesting that knockdown of the WEE1 gene could be a promising therapeutic target in HCL.

In Vitro Sensitivity to Venetoclax and Microenvironment Protection in Hairy Cell Leukemia.

Current standard treatment of patients with hairy cell leukemia (HCL), a chronic B-cell neoplasia of low incidence that affects the elderly, is based on the administration of purine analogs such as cladribine. This chemotherapy approach shows satisfactory responses, but the disease relapses, often repeatedly. Venetoclax (ABT-199) is a Bcl-2 inhibitor currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) in adult patients ineligible for intensive chemotherapy. Given that HCL cells express Bcl-2, our aim was to evaluate venetoclax as a potential therapy for HCL. We found that clinically relevant concentrations of venetoclax (0.1 and 1 µM) induced primary HCL cell apoptosis in vitro as measured by flow cytometry using Annexin V staining. As microenvironment induces resistance to venetoclax in CLL, we also evaluated its effect in HCL by testing the following stimuli: activated T lymphocytes, stromal cells, TLR-9 agonist CpG, and TLR-2 agonist PAM3. We found decreased levels of venetoclax-induced cytotoxicity in HCL cells exposed for 48 h to any of these stimuli, suggesting that leukemic B cells from HCL patients are sensitive to venetoclax, but this sensitivity can be overcome by signals from the microenvironment. We propose that the combination of venetoclax with drugs that target the microenvironment might improve its efficacy in HCL.

Genomic Analysis of Hairy Cell Leukemia Identifies Novel Recurrent Genetic Alterations

Hairy cell leukemia (HCL) comprises the clonal malignancies classical and variant hairy cell leukemia (vHCL). Classical HCL (cHCL) is characterized by a near 100% frequency of the BRAFV600E mutations while ~50% of vHCL have MAP2K1 mutations. However, recurrent genetic alterations cooperating with BRAFV600E or MAP2K1 mutations in HCL, as well as those in MAP2K1 - wild type vHCL are not well defined. Therefore, we performed deep targeted mutational and copy number (CN) analysis of cHCL (n=53) and vHCL (n=8) to gain additional insights into the pathogenesis and mechanisms of therapeutic resistance in HCL.Diagnostic bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs) were obtained from 53 cHCL and 8 vHCL patients from multiple centers. Mutational and CN analysis of MNCs (and FACS-purified HCL cells for some cases) was performed using a targeted next-generation sequencing assay, which sequences all coding regions of 585 genes recurrently mutated in leukemias, lymphomas, and solid tumors.Of the 53 cHCL patients, 100% were marked by BRAFV600E mutations, and the next most commonly mutated genes were the histone methyltransferase KMT2C (MLL3) and CDKN1B occurring in 15% (8/53) and 11% (6/53) of patients, respectively. Other recurrent mutations in cHCL affected genes involved in transcriptional regulation (BRD4, CEBPA, CREBBP, RUNX1, EP300, and MED12), Notch signaling (NOTCH1 and NOTCH2), and DNA repair (RAD50) (Fig. A). The most recurrent copy alterations in cHCL were deletions of chromosome 7q and 13q and gains of chromosome 5. Chromosome 7q and 13q deletions were confirmed by FISH. While recurrent 7q deletions have previously been reported in cHCL, genes in the minimally deleted region of 7q were not known and identified here to include SMO (7q32) and BRAF (7q34). Recurrent 13q deletions in cHCL include the tumor suppressor RB1 and the miR-15a and miR-16-1 microRNA cluster at 13q14.3 (Fig. B-D).Sequencing across 8 additional vHCL patients identified change-of-function mutations in both CCND3 and U2AF1, each in 13% (1/8) of vHCL patients and hotspot mutations in TP53 (38%; 3/8) (Fig. E). The CCND3 and U2AF1 mutations were absent in cHCL suggesting additional genetic differences between cHCL and vHCL. These findings may have therapeutic relevance as CCND3 mutations are thought to confer sensitivity to CDK4/CDK6 inhibitors while those in U2AF1 confer sensitivity to spliceosome inhibitors. Other mutations affecting genes involved in transcriptional regulation (CEBPA, CREBBP, DDX3X, and PBRM1) and chromatin remodeling (KMT2C, KDM6A, and KDM5C) were also identified (Fig. E). As with cHCL, chromosome 7q deletions were also present in vHCL and always included BRAF (7q34). Additionally, we identified recurrent 3p deletions in vHCL, which include a critical tumor suppressor locus encoding VHL, SETD2, BAP1, and PBRM1 (Fig. F).Prior studies have identified acquired KRAS mutations as driving vemurafenib resistance in HCL. Here we identified an activating mutation in NRAS in a treatment naïve cHCL patient. In addition, genomic analysis of the pre-treatment sample from a patient that developed de novo vemurafenib resistance uncovered a clonal hemizygous BRAFV600E mutation, as well as heterozygous deletions of BRAF, NF1, NF2, and TP53 (Fig. G). Consistent with the heterozygous deletion of NF1 and NF2, the de novo vemurafenib-resistant patient showed decreased expression of both NF1 and NF2 while vemurafenib-sensitive patients without NF1 or NF2 copy loss did not demonstrate decreased expression of NF1/NF2 by qRT-PCR. To understand the functional role of Nf1 and Nf2 loss and the potential contribution to RAF inhibitor resistance in the hematological system, we performed shRNA-mediated downregulation of Nf1 or Nf2 in Ba/F3 cells stably expressing BRAFV600E. Silencing of either Nf1 or Nf2 alone or concomitant downregulation of Nf1 and Nf2 simultaneously conferred vemurafenib resistance in vitro (Fig. H).Combined, these data identify several novel drivers of HCL. While activating MAPK mutations are critical for both cHCL and vHCL, our data suggest additional shared cooperating alterations, as well as disease-specific alterations targeting BRAF, KMT2C, and CDKN1B in cHCL and MAP2K1, CCND3, U2AF1, TP53, and KMT2C in vHCL. Finally, these data nominate several novel potential therapeutic approaches for vHCL and identify a novel mechanism of vemurafenib resistance seen clinically. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Phase 1 trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox combined with rituximab for relapsed/refractory hairy cell leukemia.

7036 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) is FDA-approved for hairy cell leukemia (HCL) patients who have received at least two prior systemic therapies including a purine nucleoside analog. In phase 3 testing the complete remission (CR) rate was 41%, and response was higher in patients with lower tumor burden and lower titers of antidrug antibodies (ADA). Phase 1 testing indicated that most CRs were without minimal residual disease (MRD) and eradication of MRD was associated with prolonged CR duration. Monoclonal antibody (Mab) rituximab binds to CD20 on HCL cells and induces apoptosis or immune-mediated killing, but as a single-agent achieved only 13% CRs in relapsed HCL requiring therapy. In a phase 1 trial to determine safety, rituximab was combined with Moxe, with the goal to help reduce tumor burden and to prevent or delay ADA by killing normal B-cells. Methods: To allow rituximab sufficient time to accomplish both goals, it was infused 3 days before day 1 of cycle 1 at 375 mg/m2, and Moxe was given by 30-minute infusion on days 1, 3 and 5. On repeat cycles of Moxe days 1, 3 and 5, rituximab was given on day 1. Cycles were generally spaced 4 weeks apart. Moxe was begun at a lower dose, 30 rather than the 40 mcg/kg dose used in phase 3 in case the rituximab would increase its toxicity. Bone marrow aspirate flow cytometry, which can detect 0.002% HCL cells, was the most sensitive test used for MRD detection, much more sensitive than BRAF V600E digital droplet PCR (ddPCR) or bone marrow biopsy immunohistochemistry (IHC). Patients could receive 4 cycles past MRD-free CR, but not more than 8 cycles. Results: Three patients received Moxe at 30 mcg/Kg/dose and 6 received 40 mcg/Kg/dose, all without dose limiting toxicity (DLT). There was no evidence of hemolytic uremic syndrome or capillary leak syndrome. To prevent intravascular hypovolemia due to expected third spacing, patients were encouraged to drink one cup per hour of water or other fluid from days 1 to 8 and take dexamethasone 4 mg orally if headache or nausea prevented good oral hydration. Of the 9 patients, 7 (78%) achieved CR after 2 (n = 6) or 3 (n = 1) cycles, and achieved MRD-free CR after 2 (n = 3), 4 (n = 3) or 6 (n = 1) cycles. No patients became infected with COVID-19. Conclusions: This phase 1 trial met its primary endpoint of determining whether rituximab could be safely combined with Moxe and will enroll 4 additional patients to further access clinical activity. Further testing will determine whether addition of a CD20 Mab to Moxe significantly improves clinical outcome compared to Moxe alone, particularly long-term MRD-free CR rate. Clinical trial information: NCT03805932.

Importance of Flow Cytometry in the Differential Diagnosis of Hairy Cell Leukemia in the State of Rio Grande Do Norte, Brazil

Introduction:Hairy Cell Leukemia (HCL) is a B-cell non-Hodgkin's Lymphoma (B-NHL) representing about 2% of chronic leukemias, is manifested in adults with an average age of 55 years old or more and the ratio of male: female is 5:1, being more common among white people. It is characterized by the presence of neoplastic lymphocytes with cytoplasmic projections (villous cells), a characteristic commonly observed in other DLPCs such as variant HCL (HCL-v) and splenic villous cell lymphoma (SVCL), being the immunophenotyping by flow cytometry determinant in the differential diagnosis of these neoplasms. HCL is characterized by splenomegaly, hepatomegaly, pancytopenia in peripheral blood (PB) with leukopenia, anemia, neutropenia, monocytopenia, and thrombocytopenia. It has a low number of circulating tumor cells, spleen, liver, and bone marrow (BM) infiltration.Objective:To investigate, by flow cytometry, patients with lymphocytosis and presence of villous lymphocytes in the characterization of HCL and HCV-v and SMZL.Methodology:Were investigated samples of peripheral blood (SP) and bone marrow (MO) from 27 patients previously diagnosed with DLPC and presence of villous lymphocytes which were by flow cytometry with a panel of monoclonal antibodies (MoAb) conjugated to fluorochromes and targeted to T-lymphocytes: CD1a, CD2, CD3, CD5, CD7, subpopulation T-helper (CD3/CD4) and T-cytotoxic (CD3/CD8), in addition to TCR a/b and TCR g/d; Natural Killer cells: CD16-56; B-lymphocytes: CD19, CD20, CD21, CD22, CD23, CD79b, CD200, IgM, IgG, IgD, anti-kappa and anti-lambda, in addition to CD10, TdT (Terminal deoxynucleotidyl Transferase), CD103, CD123, CD11c, CD25, CD38, CD138, CD45 and CD14. At the same time, a complete blood count with differential white blood cell count and investigation of clinical and demographic data such as age, sex and ethnicity / race were also performed.Results:The distribution of patients according to ethnicity and gender, there was a predominance of white individuals and males. The age group most affected was in patients older than 60 years. All patients expressed pan-B antigens on leukemic cells with expression of CD19, CD22 / CD20 (Forte), sIgH, associated with clonal restriction for immunoglobulin light chain (kappa= 20 and lambda= 7), associated with FMC7 expression, HLADR, CD38 and CD45 strong and negativity to CD10, CD138, CD200, CD23, CD5, TdT and related T antigens. Sixteen cases were categorized as HCL, six HCLv and five SVCL. The immunophenotyping of HCL cases was positive for CD103, CD25, CD123 and CD11c. HCLv was negative for CD103 in three cases and CD25 and SVCL negative for CD103, CD123 and CD11c and CD25 in all cases.Conclusions:The precise diagnosis of HCL has fundamental importance because each NHL-B has a specific treatment, besides emphasizing the sensitivity and speed of the IFC regarding diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

A rare case of hairy cell leukemia with co-expression of CD5 and cyclin D1: A diagnostic pitfall.

Hairy cell leukemia (HCL) is an uncommon chronic B-cell lymphoproliferative disease with an indolent course. It mainly occurs in elderly men, although abdominal lymphadenopathy is rare. HCL cells are mostly found in the bone marrow, peripheral blood, and spleen and typically express CD11c, CD20, CD25 and CD103. We present a case of HCL with a novel immunophenotype. A 48-year-old woman presented with pancytopenia and splenomegaly. The diagnosis was HCL with lymph node infiltration. Unlike previously described HCL cases, the current case showed strong expression of CD5 and cyclin D1 in the lymph nodes. The patient underwent cladribine chemotherapy, and the leukocyte count increased during and after treatment. The 8-month follow-up revealed that she had recovered well. This case highlights the distinctive immunophenotype of HCL infiltrating the lymph nodes and the potential misdiagnosis of HCL as mantle cell lymphoma. It also adds to our limited understanding of HCL.

Development of Recombinant Immunotoxins for Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is an indolent B-cell malignancy with excellent initial response to purine analogs pentostatin or cladribine, but patients are rarely, if ever, cured. Younger patients will usually need repeat chemotherapy which has declining benefits and increasing toxicities with each course. Targeted therapies directed to the BRAF V600E mutation and Bruton’s tyrosine kinase may be helpful, but rarely eradicate the minimal residual disease (MRD) which will eventually lead to relapse. Moxetumomab pasudotox (Moxe) is an anti-CD22 recombinant immunotoxin, which binds to CD22 on HCL cells and leads to apoptotic cell death after internalization and trafficking of the toxin to the cytosol. Phase I testing achieved a complete remission (CR) rate of 57% in relapsed/refractory HCL. Most CRs were without MRD and eradication of MRD correlated with prolonged CR duration. Patients were often MRD-free after five years. Important mild-moderate toxicities included capillary leak and hemolytic uremic syndromes which could be prevented and managed conservatively. A phase 3 trial met its endpoint of durable CR with acceptable toxicity, leading to FDA approval of Moxe for relapsed/refractory HCL, under the name Lumoxiti. Moxe combined with rituximab is currently being evaluated in relapsed/refractory HCL to improve the rate of MRD-free CR.

Abstract CN07-03: Recombinant immunotoxins for hematologic malignancies

Abstract Recombinant immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p&amp;lt;0.0001) compared to those remaining blood RQ-PCR+. Both BMA flow cytometry and RQ-PCR were associated with prolonged CR duration if negative, but blood flow cytometry was not helpful since only one patient with CR remained flow-positive. Molecular remissions assessed by blood analysis may be useful to determine the number of cycles of Moxe to administer to achieve long-term CR or possibly cure. To achieve molecular remissions with fewer cycles, Moxe is being tested with rituximab to facilitate tumor reduction and decrease anti-drug antibody formation. Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03

PB2019 MOLECULAR ANALYSIS OF A COMPOSITE B‐CELL TUMOR – HAIRY CELL LEUKEMIA AND MANTLE CELL LYMPHOMA COMBINATION

Background:Composite lymphomas are unique models to study the clonal transformation process according stages of lymphoid cell differentiation. Clonal relationship is a key point in the research. At the moment, molecular analysis of V(D)J‐recombination is the most accurate method to evaluate related lymphoid populations.Aims:To study clonal rearrangements of IGH genes in isolated tumor populations in order to determine the mechanism of their occurrence during pathogenesis of composite lymphoma – hairy cell leukemia (HCL) and mantle cell lymphoma (MCL) combinationMethods:Patient was diagnosed and treated at the National Research Center for Hematology. Informed consent was obtained. Isolation of MCL and HCL cells was carried out by magnetic selection of bone marrow leukocyte fraction using anti‐CD103 and anti‐CD5 magnetic particles according to the standard Miltenyi Biotec protocol (www.miltenyibiotec.com). B‐cell clonality testing and IGHV sequencing was performed according to BIOMED‐2 protocol. Fragment analysis and Sanger sequencing was performed on ABI PRISM 3100 genetic analyzer, followed by comparison with the germinal sequence databases www.imgt.org.Results:Patient was performed with leukopenia and lymphocytosis, splenomegaly, lymphadenopathy in May, 2018 and was diagnosed with HCL. The diagnosis was confirmed by the BRAF mutation detection and peripheral blood lymphocytes immunophenotyping (table 1). Patient was consistently treated with interferon α2b and cladribine with good partial response. Within 4 months after therapy was completed spleen repeatedly enlarged. Diagnostic splenectomy was performed and MCL was revealed. The diagnosis was confirmed by the t(11;14) detection. Immunophenotyping of spleen suspension allowed to identify not only MCL cells but minimal residual HCL population (table 1). Analysis of clonally rearranged IGH genes is isolated CD103+ and CD5+ population revealed two clonal products corresponding to components of composite lymphoma (figure 1). The sequence analysis identified the dissimilar repertoire of gene segments involved in V(D)J‐recombination in MCL (IGHV3‐9/IGHD2/IGHJ4) and HCL (IGHV1‐69/IGHD3/IGHJ4). This distinction indicated the independence of two clones’ origin, at least at the stage of B‐lymphocyte precursors as we do not have other molecular markers for progenitor lymphoid cells. Nucleotide sequence was 100% germinal in MCL and only 90% matches with the germinal sequence were detected in HCL reflecting the postgerminal phenotype. Thus not only the existence of separate tumor precursors was strictly supposed but also the different way of parallel lymphoid cell differentiation was proven. Patient was treated with R‐BAC cycles for MCL. Complete remission was achieved and has been maintained for 6 months.Summary/Conclusion:At the moment, there is no known description of the synchronous development of MCL and HCL. The presented clinical case is the first observation of the clinical course of such the composite lymphoma. We demonstrated independent origin of two tumor components and proved the different way of their differentiation. The proposed algorithm for the analysis of isolated cell populations and the obtained results make an additional contribution to the lymphomagenesis understanding, which is necessary for the development of effective methods of diagnosis and therapy.image

S104 THE BRAF INHIBITOR VEMURAFENIB COMBINED WITH RITUXIMAB PRODUCES A HIGH RATE OF DEEP AND DURABLE REMISSIONS IN RELAPSED OR REFRACTORY HAIRY CELL LEUKEMIA: UPDATED RESULTS OF A PHASE‐2 TRIAL

Background:Up to 50% of hairy cell leukemia (HCL) patients relapse after purine analogs. We identified the BRAF‐V600E kinase mutation as the genetic cause of HCL (Tiacci et al., NEJM 2011). We then documented, in 26 relapsed/refractory patients treated with the oral BRAF inhibitor vemurafenib for a median of 16 weeks, 96% of overall responses (including 35% complete remissions ‐ CR), obtained after a median of 8 weeks (Tiacci et al., NEJM 2015). However, residual bone marrow (BM) HCL cells persisted even in CR patients (5–10% cells) and the median relapse‐free survival among all responding patients was 9 months.Aims:Since HCL strongly expresses CD20, rituximab could improve the efficacy of BRAF inhibition by targeting leukemic cells resistant to vemurafenib.Methods:In this academic, phase‐2, single‐arm, single‐center trial (EudraCT 2014‐003046‐27), relapsed/refractory HCL patients received vemurafenib (960 mg b.i.d.) for 8 weeks and concomitant rituximab (375 mg/m2 i.v.) every 2 weeks. Rituximab was then given as consolidation 4 times every 2 weeks after the end of vemurafenib. CR required normal blood counts, no palpable splenomegaly, and no leukemic cells at morphologic analysis of BM biopsies and blood smears.Results:We enrolled 31 patients (median age: 59 years) with a median of 3 previous therapies, including 8 primary refractory cases (26%). Toxicity was mostly of grade 1‐2, without myelosuppression, and overlapped that of either drug when used alone.Strikingly, a CR was achieved by 26/27 (96%) patients evaluable for efficacy, including 2 patients with incomplete platelet recovery at the end of treatment who however normalized the platelet count soon after; and 2 patients with incomplete resolution of splenomegaly who nonetheless remain in otherwise continuous CR at 22.5 and 25 months from the end of treatment. Among the 26 CR cases, all previously treated with purine analogs, there were also some patients previously refractory to rituximab (n = 5) and/or who had relapsed after a prior BRAF inhibitor (n = 7; 5 obtained a short‐lived PR and 2 a CR after treatment with the BRAF inhibitor). Notably, a CR was obtained after just 4 weeks of vemurafenib and two concomitant doses of rituximab in 15/24 evaluable patients (63%). Minimal residual disease (MRD) by allele‐specific PCR (sensitivity: 0.05% BRAF‐V600E alleles) was absent in the BM of 17/26 (65%) patients; in 8/17 patients (47%), MRD clearing was obtained before rituximab consolidation.Progression‐free survival (PFS) in the 29 evaluable patients was 83% at a median follow‐up of 29.5 months (range: 2–45) (Fig. 1A). Progressions (n = 5) always occurred in cases who were MRD‐positive, including 3/5 patients whose immediate prior treatment was a BRAF inhibitor (leading to PR in all 3 cases). Indeed, PFS was significantly longer in MRD‐negative CR cases (100% in 17 patients at 30.5 months of median follow‐up) than in MRD‐positive CR cases (44% in 9 patients at a median follow‐up of 24.5 months) (p = 0.001; Fig. 1B). In subsequent BM evaluations, 16/17 (94%) CR patients maintained a MRD‐negative status after a median of 22.5 months (range 12.5–36.5); in the remaining patient, MRD reappeared 12.5 months later but he remains in continuous CR at 38 months after the end of treatment.Summary/Conclusion:Vemurafenib plus rituximab is a brief, safe and non‐myelotoxic regimen that induces MRD‐negative durable responses in the majority of relapsed/refractory HCL patients. Randomized testing of this regimen against the chemotherapy‐based standard of care in the frontline setting is warranted.image

Hairy cell leukemia: present and future directions

Hairy cell leukemia (HCL) is an indolent B-cell malignancy, with long-term responses to purine analogs, but with decreasing efficacy and increasing toxicity with repeated courses. Leukemic cells express CD22, CD20, CD25, tartrate-resistant acid phosphatase (TRAP), annexin 1A (Anxa1), and BRAF V600E mutation. HCLv, lacking CD25, Anxa1, TRAP, and BRAF V600E, is more aggressive and less purine analog-sensitive. A molecularly defined IGHV4-34+ variant is also resistant whether HCL or HCLv immunophenotypically. Traces of HCL cells, termed minimal residual disease (MRD), accompany most with complete remission (CR) and may cause relapse. Rituximab has limited single-agent activity, but frequent CR without MRD when combined with purine analog, albeit with chemotherapy toxicities. The anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox can achieve MRD-negative CR in multiply relapsed HCL without chemotherapy toxicities and was FDA approved in 2018 as Lumoxiti. Investigational oral non-chemotherapy options also include Vemurafenib or Dabrafenib/Trametinib targeting BRAF V600E ± MEK, and Ibrutinib targeting Bruton’s tyrosine kinase.

The Role of Novel Dual Color Immunohistochemistry in Detection of Minimal Hairy Cell Leukemia in Bone Marrow: A Study of 148 Cases

Background:Hairy-cell leukemia (HCL) is a B-cell lymphoproliferative disorder characterized by distinct immunophenotype (positive for CD19, CD20, PAX5, CD22, CD11c, CD25, CD103, CD123 and CD200). Both flow cytometry (FC) and immunohistochemistry (IHC) can be used to determine these markers, while the expression of another marker (TRAP) is IHC specific. Both trephine bone marrow biopsy and aspirate are vital for assessment of extent of bone marrow infiltration. However, in some cases a cellular aspirate cannot be obtained due to extensive fibrosis, ie “dry tap”. In such cases, IHC stains are crucial for assessment of bone marrow leukemic involvement. So far, IHC detection of HCL in the bone marrow sections has been limited to overt disease and could not be reliably used in cases with minimal HCL involvement. Novel automated dual-antibody immunohistochemistry techniques can identify aberrant antigen co-expression in neoplastic cells with high sensitivity. Consistent detection of minimal disease involvement is crucial given that treatment decisions are sometimes based on these data (Grever et. al. Blood 2017). In this study, we evaluated the efficacy of two novel dual IHC assays in assessing minimal HCL involvement in the core biopsies and compared the results with concurrently collected flow cytometric data.Design:We analyzed data on 148 cases of HCL (123 male, 25 female; mean age 59.8, range 25-81). All cases had multiparameter flow cytometry performed using CD19, CD20, CD22, CD11c, CD25, CD103, CD123, surface light chains, CD5 and CD23. In parallel, bone marrow IHC was done using PAX5/CD103 and PAX5/TRAP dual stains and the automated stainer (Ventana Ultra).Results:Cases were divided into three groups based on the combined results of PAX5/CD103 and PAX5/TRAP stains: negative (82; 55.4%); rare dual positive cells (less than 5% of total cells) (21; 14.1%) and positive (45; 30.4%). Flow cytometry data concurred with IHC results in all IHC positive cases (median 7.05% HCL cells out of all lymphoid cells by FC; range 0.05%-91.7%) and all rare dual-cell IHC positive cases (median 0.98% HCL cells; range 0.02%-19.04%). Overall sensitivity of dual IHC was 81.4%, positive predictive value 100% and negative predictive value 81.7%. In dual IHC negative group, 15/82 cases (18.3%) were low level positive by FC analysis (median 0.13% HCL cells; range 0.01%-9.21%). When dual IHC results were analyzed separately, PAX5/CD103 results were similar to the combined results. PAX5/TRAP staining alone was slightly less sensitive in IHC negative cases; 22/82 (26.8%) of PAX5/TRAP negative or non-evaluable cases were positive by FC analysis (median 0.27% HCL cells, range 0.01%-29.5%).Conclusion:Dual color IHC is a sensitive tool in detecting HCL, even in cases with minimal disease involvement. All IHC positive cases concurred with flow cytometry data, even when HCL burden was extremely low (as low as 0.02% of all lymphoid cells by flow cytometric analysis). Only 18.3% of dual IHC negative cases were positive for low level involvement by FC analysis. Therefore, dual IHC is a sensitive new tool for evaluation of minimal marrow involvement by HCL. DisclosuresKreitman:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox.

Immunohistochemical Detection of Hairy Cell Leukemia in Paraffin Sections: The Role of Pax5 and CD103 Double Staining to Improve Specificity and Sensitivity.

- In hematopathology practice, abnormal expression of CD103 on B cells is detected by flow cytometry in hairy cell leukemia (HCL) and, in combination with other phenotypic and morphologic findings, provides diagnostic specificity and sensitivity. Immunostaining on paraffin sections makes it possible to perform immunophenotyping in situ. However, normal bone marrow contains CD103-positive cells, which are not B cells, making it difficult to be certain about low-level involvement by HCL. - To develop dual immunostaining for confirmation that CD103 is expressed in B cells, which may be highly desirable in assessing low-level involvement by HCL. - We developed a dual immunostaining approach using a B-cell marker, Pax5, expressed in the nucleus, in combination with a membrane marker, CD103. - We analyzed 6 HCLs, 7 marginal zone lymphomas, 12 lymphoplasmacytic lymphomas, 7 follicular lymphomas, 5 chronic lymphocytic leukemias, 5 mantle cell lymphomas, 1 multiple myeloma (lymphocytic variant), and 3 bone marrows not involved by any B-cell neoplasm. Our dual staining approach confirmed that only the neoplastic cells of HCL were positive for both CD103 and Pax5. - This dual-staining method is particularly helpful in cases with low-level involvement by HCL and can be used for determining minimal residual disease after treatment.

Vemurafenib Plus Rituximab in Hairy Cell Leukemia: A Promising Chemotherapy-Free Regimen for Relapsed or Refractory Patients

▪BACKGROUND: Hairy cell leukemia (HCL) responds well to purine analogs, but up to 50% of patients relapse. We previously identified the BRAF-V600E mutation as the genetic lesion underlying HCL (NEJM 364:230-2315, 2011), and successfully targeted this mutation in the clinic with the oral BRAF inhibitor vemurafenib through an academic phase-2 multi-center Italian trial in HCL patients relapsed after or refractory to purine analogs (NEJM 373:1733-1747, 2015). In these heavily pre-treated patients, vemurafenib given for a median of 16 weeks produced 96% of responses, including 9/26 (35%) complete remissions (CR) and 16/26 (61%) partial remissions (PR), which were obtained after a median of 8 weeks of treatment. Even in complete responders, immunohistochemistry showed residual (~10%) bone marrow HCL cells at the end of treatment, and relapses were common, occurring at a median of 19 months and 6 months in CR and PR patients respectively. Residual HCL cells resisting vemurafenib treatment might be targeted by concomitant immunotherapy with an anti-CD20 monoclonal antibody, an attractive strategy to potentially achieve a more profound response and a better clinical outcome through a chemotherapy-free approach.METHODS: We started an academic, phase-2, single-center trial (EudraCT 2014-003046-27) in relapsed/refractory HCL, which tests vemurafenib in combination with rituximab, another targeted non-myelotoxic drug with known single-agent activity in HCL. Eligibility was extended to patients relapsed also after monotherapy with a BRAF inhibitor. Vemurafenib was given at its standard dose (960 mg twice daily orally) for 8 weeks. Rituximab infusions (375 mg/m2intravenously) were given concomitantly with vemurafenib every 2 weeks, as well as sequentially (after the end of vemurafenib dosing) four times every 2 weeks.RESULTS: We have so far enrolled 22 patients in 16 months. Adverse reactions were reversible, usually mild and consistent with the known toxicity profile of the two drugs when used alone. Notably, a CR was achieved by all 14 patients already evaluable for efficacy (100%), including 4 who had relapsed after a BRAF inhibitor and 1 previously refractory to rituximab. Furthermore, 12/14 patients (86%) obtained the CR as early as after 4 weeks of vemurafenib and 2 concomitant rituximab infusions. This CR rate appears higher than that observed by us and others using vemurafenib alone in BRAF inhibitor-naive patients relapsed after or refractory to purine analogs (CR rate 35-42%; NEJM 373:1733-1747, 2015). Moreover, minimal residual disease (MRD) was undetectable in the bone marrow biopsy and aspirate of 8/11 patients evaluated (73%), both by immunophenotyping and by allele-specific PCR (limit of detection: 0.05% BRAF-V600E copies). In 5 of these 8 patients, MRD clearing was reached even before sequential rituximab dosing post-vemurafenib. In the remaining 3/11 patients, MRD was at most 5% in 2 vemurafenib-naive patients, and 10% in 1 patient relapsed after prior BRAF-inhibitor treatment. In contrast, residual bone marrow disease was a constant feature of all 26 patients treated by us with vemurafenib alone for a longer time period (NEJM 373:1733-1747, 2015).CONCLUSIONS: This study - which is the first one combining vemurafenib and rituximab in relapsed/refractory HCL - suggests that this non-myelotoxic regimen produces more numerous, faster and deeper CRs than vemurafenib alone. Enrollment continues. DisclosuresGaidano:Karyopharm: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau.