Hairy Cell Research Articles

Efficacy and Safety of a Third Generation CD20 CAR-T (MB-106) for Treatment of Relapsed/Refractory Indolent B-Cell Non-Hodgkin Lymphoma: Phase-1 Results from a Multicenter Trial

Background: Indolent B-cell non-Hodgkin lymphomas (NHL) remain incurable despite multiple therapeutic options, including chemoimmunotherapy, targeted therapies, bispecific antibodies, and CD19-targeted chimeric antigen receptor T (CAR-T) cells. MB-106, a fully human 3 rd-generation CD20-targeted CAR-T product with both 4-1BB and CD28 costimulatory domains, had high overall response rate (100%) and complete response (CR) rate (91%) in follicular lymphoma (FL) patients at doses ≥ 3.3×10 6 cells/kg in a single institution trial (Shadman et al., 17-ICML, 2023). We seek to replicate the favorable efficacy and safety profile of MB-106 in a multicenter trial with centralized manufacturing. Methods: MB-106 is being evaluated in a Phase 1/2, open label, multicenter study utilizing a 3+3 dose-escalation design in R/R B-cell NHL patients with confirmed CD20 expression. Prior treatment with CD19-targeted CAR-T therapy is permitted. Following lymphodepletion (cyclophosphamide 300 mg/m 2/day and fludarabine 30 mg/m 2/day for 3 days), MB-106 is administered to patients with indolent B-cell NHL at one of two dose levels (DL): DL1, 3.3×10 6; and DL2, 1.0×10 7 cells/kg. MB-106 is infused in the outpatient setting if allowed by the institution, except for the first patient in each dose level cohort (overnight observation). Dose-limiting toxicities (DLT) are monitored through day 28. The treatment response is assessed on day 28 and on subsequent visits, and best overall response is reported here. CAR-T persistence is assessed by both flow cytometry and qPCR in peripheral blood mononuclear cells. Results: Three patients with indolent NHL (2 FL, 1 Waldenström macroglobulinemia [WM]) received MB-106 at DL1. In addition, one patient with hairy cell leukemia variant (HCL-v) received non-conforming material (drug product that failed to meet release criteria) at DL1, following FDA authorization. These four patients were heavily pre-treated with a median of 5.5 prior lines of treatment. High-risk features included progression of disease within 24 months of first-line treatment (POD24, n=1), prior autologous stem cell transplant (n=1) and prior CD19-targeted CAR-T therapy (n=1). No DLTs were observed at DL1. Three patients experienced Grade 1 cytokine release syndrome. There have been no occurrences of immune effector cell-associated neurotoxicity syndrome (ICANS). The 2 FL patients achieved CR by both PET-CT and bone marrow biopsy, one of whom had been previously treated with CD19-targeted CAR-T. The WM patient, who had nine prior treatments, including autologous stem cell transplant, and high disease burden, achieved very good partial response, with complete metabolic response by PET-CT, morphologic clearance of lymphoma in bone marrow, and resolution of the IgM paraprotein. The patient with HCL-v, who had been transfusion dependent, continues to have stable disease with decreased disease in the bone marrow, and achieved complete transfusion independence which is ongoing at 6 months. All patients displayed MB-106 expansion with peak levels between 7-14 days post-infusion. Persistence of CAR T-cells is ongoing at 6 months. Conclusion: Treatment with MB-106, a third generation CD20 targeting CAR-T, resulted in responses, including CRs, and CAR-T persistence in patients with R/R indolent NHL and was associated with favorable safety profile with no occurrence of Grade 3 or Grade 4 cytokine release syndrome and no ICANS of any grade. CRs have been observed in patients previously treated with CD19-targeted CAR-T in both this multicenter trial and the original single institution trial. Dosing is ongoing in the final dosing level (1.0×10 7 cells/kg) to establish the recommended Phase 2 dose for a planned pivotal trial in WM. It is anticipated that the complete data set from the Phase 1 indolent NHL arm will be presented.

Defining Signaling Interactomes Involved in Non-Canonical Wnt Signaling By ROR1 or ROR2 in Hairy Cell Leukemia That Can Influence Cell Morphology and Migration

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) and 2 (ROR2) are highly conserved oncoembryonic surface proteins expressed on neoplastic cells of patients with a variety of human cancers, but not on most normal postpartum tissues. ROR1 and ROR2 are each a receptor for non-canonical Wnt factors that direct migration and planar cell polarity, which is essential for organ development. We developed humanized monoclonal antibodies (mAbs) each specific for ROR1 (UC-961) or ROR2 (6E6-70) and found the leukemia cells of patients with classic hairy cell leukemia (cHCL) or variant HCL (vHCL) express both ROR1 and ROR2 and that these receptors function in HCL non-canonical Wnt signaling (Widhopf, G. et al). We hypothesized that ROR1 and ROR2 each may associate with overlapping and distinctive sets of proteins, which may contribute to ROR1/2, ROR1, or ROR2 signaling in HCL. To test this hypothesis, we optimized conditions for generating immune precipitates (i.p.) with either UC-961 or 6E6-70, and examined the proteins of each i.p. via data dependent acquisition mass spectrometry. Consistent with the assumed capacity of each of these mAb to disrupt ROR1/2 heteroligomerization, we identified ROR1, but not ROR2, in i.p. generated using UC-961 and ROR2, but not ROR1, in i.p. generated using 6E6-70 on cell lysates of leukemia cells isolated from a patient with cHCL. In the anti-ROR1 i.p. we identified 1,414 proteins, 849 (60%) of which also were found in the anti-ROR2 i.p.. In the anti-ROR2 i.p. we identified 2,523 proteins, a third of which also were found in the ROR1 i.p.. Among proteins common to both anti-ROR1 and anti-ROR2 i.p. we noted hematopoietic cell-specific protein-1 (HS1 or HCLS1), which we found associated a SH3-binding motif that was dependent on a proline residue at position 841 of activated ROR1 in chronic lymphocytic leukemia (CLL); we found this SH3-binding motif was critical for ROR1-dependent Wnt5a-induced chemokine-directed migration ( Leukemia 12:2615, 2017). Also found common to both ROR1 and ROR2 in HCL were proteins that drive actin polymerization and cytoskeletal dynamics that influence cell morphology and migration. To validate that ROR2 itself was involved in binding HS1 and in enhancing migration, we generated mutant forms of ROR2, each harboring a proline-to-alanine mutation in one of several proline residues found in the proline-rich domain of ROR2. We transduced MEC1 cells, which lack ROR1 or ROR2, with lentivirus vectors encoding wild-type ROR2 or each one of several P-A mutants of ROR2. Flow cytometry confirmed that each of the transduced cells had comparable expression of surface ROR2. We generated anti-ROR2 i.p. using lysates of each and found that all but one of the mutant forms of ROR2 could bind to HS1 by immunoblot analysis. In contrast to wild-type ROR2, or other mutant forms of ROR2, expression of this mutant form of ROR2 could not enhance chemokine-directed migration relative to that of wild-type MEC1 cells. Collectively, our studies define signaling interactomes involved in non-canonical Wnt signaling by ROR1 and/or ROR2 in hairy cell leukemia and define a residue within ROR2 that is necessary for binding HS1 and facilitating cellular migration.

Immunophenotypic Profile of Chronic Lymphoproliferative Diseases in the State of Rio Grande Do Norte, Northeast, Brazil

INTRODUCTION: Chronic lymphoproliferative diseases (CLPD) are considered as a heterogeneous group of diseases characterized by monoclonal expansion and accumulation of apparently mature lymphocytes, which have a proliferative and/or survival advantage over normal lymphocytes in different organs such as bone marrow, peripheral blood and lymph nodes. This translates into the progressive accumulation of clonal cells and their products, causing peripheral blood and bone marrow lymphocytosis, in addition to lymphadenopathy, splenomegaly or other organomegaly. According to the World Health Organization, which reflects the international consensus and is based on pathological, genetic and clinical factors, lymphoproliferative neoplasms are classified according to the stage of maturation and the lineage where the neoplastic transformation occurs through flow cytometry (FC). OBJECTIVES: The aim of this study was to demonstrate the immunophenotypic diagnostic profiles of patients with CLPD in Rio Grande do Norte, Brazil by FC. METHODOLOGY: Were investigated 884 patients with CLPD of both sexes through CF with a panel of monoclonal antibodies directed to CLPD. In addition, other patient information, such as age, gender, and clinical and hematological data were also collected. RESULTS: The results showed that 89.7% of the cases were B Cell CLPD (B-CLPD) and 10.3% T and natural killer cells (T/NK-CLPD). The distribution of B-CLPD cases were as follows: 487 Chronic lymphocytic leukemia; 18 B-Cell Pro-lymphocytic leukemia; 25 Hairy cell leukemia; 6 variant Hairy cell leukemia; 6 Follicular lymphoma leukemized; 4 Splenic lymphoma with villous lymphocytes; 33 Mantle cell lymphoma; 4 Waldenstrom's macroglobulinemia; 69 Multiple myeloma; 14 Plasma cell leukemia; 11 Burkitt's lymphoma and 117 Leukemic Non-Hodgkin lymphoma (Table 1). The distribution of CLPD-T/NK was as follows: 12 Large granular lymphocytic leukemia; 14 T-Cell prolymphocytic leukemia; 18 Adult T-cell leukemia; 20 Sézary syndrome; and 24 Peripheral T-Cell lymphoma and 2 DLPC-NK (Table 1). DISCUSSION: The B-CLPD are the most commonly observed corresponding to more than 85% of all cases and are characterized by the presence of clonal B lymphocytes with expression of a single type of immunoglobulin light chain (kappa or lambda) in a proportion greater than 1/10, associated the strong positivity of Pam-B antigens such as CD19, CD22 and CD20 and the absence or low expression of Pam-T antigens. However, other antigens such as CD200, CD5, FMC7, CD79b and CD103, among others, may be present or absent, specifically characterizing these entities as Chronic lymphocytic leukemia (weak CD22+; CD200+, CD5+, FMC7-, CD79b-), Hairy cells leukemia (strong CD22+; FMC7+; CD79b+; CD5-; CD200-; CD103+; CD123+; CD11c+ and Follicular lymphoma (strongCD22+/CD20+, CD10+, CD200-, CD79B+, FMC7+). T/NK-CLPD are less frequent and are characterized by the expression of Pan-T antigens CD2, CD3, CD5, CD7, CD28 and TCL-1, with clonality evidenced by strong positivity of TCRα/β in most cases and increase of TCD4+ or TCD8+ lymphocytes in a ratio above 1:10 in most cases. In this group, large granular lymphocyte leukemia (LGLG) is a rare type of T/NK cell CLPD characterized by peripheral and medullary infiltration of large hypergranular lymphocytic cells, comprising about 2-5% of all cases, the most commonly seen being CD8+ T cells. CONCLUSION: The results presented demonstrate that immunophenotyping is a defining technique for clarifying the diagnosis of patients with CPLD, clonality was also successfully characterized in the investigated cases of CPLP of B ,T and NK lineages.

A Single Arm Phase II Pilot Study of Low Dose Vemurafenib Plus Rituximab in the Front-Line and Relapsed/Refractory Treatment of Hairy Cell Leukemia

Introduction: Hairy Cell Leukemia (HCL) is a rare, low-grade B-cell malignancy that is characterized by cytopenias, infections and splenomegaly. Given the chronic and incurable nature of this disorder, limiting therapy-related toxicity is of paramount importance. Though purine nucleoside analogs have an established track record of durable efficacy, they are associated with significant myelosuppression and immunosuppression as well as late secondary malignancies. Mechanistic exploitation of aberrant BRAF V600E activation with vemurafenib in combination with CD20 targeting with rituximab has provided an effective means of eradicating leukemic cells without significant myelosuppression or immunosuppression. However, vemurafenib at the standard dose of 960 mg twice daily is often poorly tolerated and frequently requires dose reduction. We conducted a pilot study of rituximab and low-dose vemurafenib (240 mg twice daily) in patients with untreated or relapsed HCL, with a focus on providing a non-cytotoxic regimen that minimizes toxicity with potentially comparable treatment outcomes. Methods: This phase 2, open-label, single-arm, investigator-initiated pilot trial was designed, conducted, and managed at Scripps Clinic in La Jolla, California. We enrolled adult patients with HCL and mutated BRAF V600E who met standard treatment initiation criteria defined by an ANC ≤1.0, Hgb ≤ 10.0 or PLT ≤100K. The treatment plan consisted of eight weeks of oral vemurafenib 240 mg twice daily and eight intravenous rituximab infusions at 375 mg per square meter of body-surface area administered every two weeks. The primary outcome was complete hematologic response. Secondary endpoints included time to complete hematologic response, clearance of minimal residual disease determined by BRAF V600E allele frequency in the bone marrow, safety and progression-free, relapse-free and MRD-free survival. Results: From March 2022 to July 2023, a total of six patients were enrolled in the study. Three patients had previously received a purine analog and three patients were previously untreated. The median age of patients was 61 years, and all were male. At the interim efficacy analysis, all five (5/5) evaluable patients had demonstrated a complete hematologic response with resolution of cytopenias and splenomegaly. 6-month bone marrow biopsy data were available for 3 patients. In 2 of these patients, MRD assessment by allele-specific PCR for BRAF V600E was absent in the bone marrow. In the third patient, though residual disease was present by MRD assessment, he had a hematologic response and requires no subsequent treatment to date. At a median follow-up time of 303 days, the relapse-free survival rate was 100%. There were no adverse grade 3 or 4 adverse events and no vemurafenib dose limiting toxicity was observed. Conclusions: Interim results to-date suggest that rituximab and low-dose vemurafenib (240 mg twice daily) have efficacy and good tolerability in patients with untreated or relapsed HCL. With accruing patient data and further enrollment on study, more information will guide the durability, efficacy and tolerability of this treatment approach.

A RESPON KEMOTERAPI FLUDARABINE PADA HAIRY CELL LEUKEMIA

HCL is part of a rare B-cell lymphoproliferative disorder and is a subtype of Chronic Lymphocytic Leukemia. It is characterized by progressive pancytopenia, splenomegaly, and bone marrow hypercellularity. Patients often come because of abnormal complete blood count results or complications such as infection. This case report explains a case of a 41-year-old woman who had recurrent shortness of breath and cough as symptoms, where the patient had a history of acute myeloblastic leukemia (AML) with non-routine fludarabine chemotherapy. After receiving treatment and underwent further investigations in the form of peripheral blood smear and bone marrow aspiration examination, it was found that the patient had hairy cell leukemia. Fludarabine was given to the patient and it is reported to give effect partial remission (based on last bone marrow aspiration) along with pancytopenia as a clinical manifestation from myelosuppression as a side effect for long-term used fludarabine.
 HCL is part of a rare B-cell lymphoproliferative disorder and is a subtype of Chronic Lymphocytic Leukemia. It is characterized by progressive pancytopenia, splenomegaly, and bone marrow hypercellularity. Patients often come because of abnormal complete blood count results or complications such as infection. This case report explains a case of a 41-year-old woman who had recurrent shortness of breath and cough as symptoms, where the patient had a history of acute myeloblastic leukemia (AML) with non-routine fludarabine chemotherapy. After receiving treatment and underwent further investigations in the form of peripheral blood smear and bone marrow aspiration examination, it was found that the patient had hairy cell leukemia. Fludarabine was given to the patient and it is reported to give effect partial remission (based on last bone marrow aspiration) along with pancytopenia as a clinical manifestation from myelosuppression as a side effect for long-term used fludarabine.

Broadening the horizon: potential applications of CAR-T cells beyond current indications.

Engineering immune cells to treat hematological malignancies has been a major focus of research since the first resounding successes of CAR-T-cell therapies in B-ALL. Several diseases can now be treated in highly therapy-refractory or relapsed conditions. Currently, a number of CD19- or BCMA-specific CAR-T-cell therapies are approved for acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), and follicular lymphoma (FL). The implementation of these therapies has significantly improved patient outcome and survival even in cases with previously very poor prognosis. In this comprehensive review, we present the current state of research, recent innovations, and the applications of CAR-T-cell therapy in a selected group of hematologic malignancies. We focus on B- and T-cell malignancies, including the entities of cutaneous and peripheral T-cell lymphoma (T-ALL, PTCL, CTCL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), classical Hodgkin-Lymphoma (HL), Burkitt-Lymphoma (BL), hairy cell leukemia (HCL), and Waldenström's macroglobulinemia (WM). While these diseases are highly heterogenous, we highlight several similarly used approaches (combination with established therapeutics, target depletion on healthy cells), targets used in multiple diseases (CD30, CD38, TRBC1/2), and unique features that require individualized approaches. Furthermore, we focus on current limitations of CAR-T-cell therapy in individual diseases and entities such as immunocompromising tumor microenvironment (TME), risk of on-target-off-tumor effects, and differences in the occurrence of adverse events. Finally, we present an outlook into novel innovations in CAR-T-cell engineering like the use of artificial intelligence and the future role of CAR-T cells in therapy regimens in everyday clinical practice.

Long term follow-up of refractory/relapsed hairy cell leukaemia patients treated with low-dose vemurafenib between 2013 and 2022 at the Department of Internal Medicine and Oncology, Semmelweis University.

Introduction: Hairy cell leukemia (HCL) is an indolent B-cell lymphoproliferative disease. BRAF V600E mutation is detected in nearly all classical HCL cases which offers the possibility of targeted therapy. Objective: The aim of our study was to assess the efficacy of low-dose vemurafenib as well as to assess the long term outcome of HCL patients treated with this drug at the Department of Internal Medicine and Oncology at Semmelweis University. Methods: We report on 10 patients with classical HCL treated with low-dose vemurafenib at our Department between 2013 and 2022. Results: As a result of fixed time low-dose vemurafenib treatment, 5 of 10 patients (5/10) achieved partial remission, 4 (4/10) had stable disease, and 1 (1/10) had MRD positivity. No patients achieved complete remission. The median progression-free survival was 28.5months while the overall survival was 82months. Conclusion: We confirm that low dose of vemurafenib is effective and safe in the vast majority of patients with HCL. This small-molecule oral treatment allows to gain valuable time-months or even years-before further, usually parenteral treatment options have to be given or before previous treatment has to be repeated. There are also promising data supporting the combination of vemurafenib with other drugs for the treatment of HCL patients which could provide even further possibility to bridge treatment.

Hairy Cell Leukemia with Severe Leukocytosis

Introduction Hairy cell leukemia (HCL) is a rare indolent mature B-cells neoplasm that typically manifests with pancytopenia and splenomegaly. Nevertheless, significant splenomegaly, high hairy cells count in the blood, elevated β 2 microglobulin, and increased LDH can predict resistance to purine analogs. The absence of BRAF V600E mutation in HCL is rare and is associated with poorer outcomes. Case Presentation A 68-year-old female patient presented with symptomatic massive splenomegaly, leukocytosis (89,000 /microliter), monocytopenia, and thrombocytopenia. β 2microgobulin level was 4.4 mcg/L. Lactate dehydrogenase was 521 U/L. Peripheral blood and bone marrow examinations revealed typical hairy cells morphology, and the cells were positive for CD19, CD20, CD103, CD25, CD11c, CD200 (dim), and surface-Kappa. CD38 was negative. BRAF V600E result was wild-type. IGHV was mutated (IGHV3-49). She received first-line therapy with a 5-day course of Cladribine (0.15 mg/kg/day) with initial resolution of leukocytosis, normalization of platelets, and a 50% reduction in spleen size. This was followed by consolidation with an 8-weekly course of Rituximab (375 mg/m 2). Soon after the 2 nd dose of Rituximab, leukocytosis recurred, and she was initiated on second-line therapy with Bendamustine (90 mg/m 2 on days 1 and 2) along with Rituximab (BR). She has not achieved any durable response following two cycles of BR. Unfortunately, she suffered a septic shock requiring admission to our intensive care unit. Upon routine mental status evaluation, brain MRI revealed innumerable supratentorial and infratentorial bilateral acute/subacute and old microhemorrhages (Figure 1). This prompted further evaluation of unexplained recurrent episodes of pulmonary hemorrhages while on mechanical ventilation that were found to be secondary to pulmonary end-arterial cavitating lesions suggestive of hairy cells infiltration (Figure 2 arrowheads). As she was clinically unstable, we attempted to control leukocytosis with corticosteroids, leukapheresis aggressively and started the patient on Ibrutinib, but she succumbed to her disease. Discussion Recently, the 5 th edition of the World Health Organization classification of hematolymphoid tumors highlighted the splenic B-cell lymphoma and leukemia family that encompasses hairy cell leukemia (HCL), splenic B-cell lymphoma/leukemia with prominent nucleoli (SBLPN), formerly known as hairy-cell leukemia variant, splenic diffuse red pulp small B-cell lymphoma (SDRPL) and splenic marginal zone lymphoma (SMZL). HCL is typically an indolent mature B-cell neoplasm with characteristic pathologic features that preferentially insinuate the bone marrow and the spleen causing impaired hematopoiesis and splenomegaly. Unlike other disorders within this group, HCL commonly harbors BRAF V600E somatic mutation in almost all cases. Rarely, patients with HCL can have extramedullary involvement by the malignant cells. Leukocytosis in HCL appears to increase the risk of developing extramedullary lesions that can present unique clinical challenges necessitating early and effective therapy. According to the literature, brain lesions in HCL were described in three other cases, and leukocytosis was a common denominator. Our patient with significant leukocytosis had brainand pulmonary lesions, and the latter was the cause of her demise due tounusually aggressive and refractory disease. Therefore, we hypothesize that leukocytosis in HCL can be “the tip of the iceberg”. The higher the leukocytosis, the higher the burden of extramedullary disease, as in our case, who had simultaneous lesions in the brain and lungs. Patients with HCL and leukocytosis may warrant initial diagnostic evaluation to ensure the absence of potentially sinister lesions, which can inform subsequent monitoring and management plans. Additionally, more novel therapies could be considered sooner in the treatment course to control the disease and these lesions. Finally, our patient did not harbor BRAF V600E mutation, which likely resulted in further worse outcomes and limited the usage of targeted therapies like BRAF inhibitors.

DNA Methylation-Based Classification of Hairy Cell Leukemia and Splenic B Cell Lymphoma

Improved classification of rare lymphoid neoplasms would be aided by a deeper understanding of their underlying molecular features and is important for diagnosis, prognosis and therapy. Tumor entities classified within the WHO category of splenic B cell lymphomas and leukemias often exhibit heterogenous, transecting features, and include hairy cell leukemia (HCL), splenic diffuse red pulp lymphoma (SDRPL), splenic marginal zone lymphoma (SMZL), and the newly described WHO entity, splenic B cell lymphoma/leukemia with prominent nucleoli (SBLPN); the latter including patients formerly classified as HCL-variant (HCL-V). Genome-wide epigenetic information provides a tumor cell fingerprint combining cell-of-origin and tumor-specific events. Here we used DNA methylation to perform an unbiased molecular subclassification and to explore novel biological aspects of these patients. Samples from patients with a pathological diagnosis of HCL, HCL-V, SDRPL and SMZL (made prior to the 5 th WHO revision and ICC classifications) were obtained from 19 institutions across 9 countries, totaling 367 patients. Cells were FACS-purified where necessary and DNA was analyzed by 450/850K Illumina DNA methylation arrays. Genetic mutations were assessed by whole-exome or targeted sequencing, IGHV-D-J sequences by Sanger sequencing, and copy number alterations (CNAs) by Illumina arrays. The 1000 most variable CpG methylation sites were used for k-means clustering. Recursive feature elimination/random forest algorithms were used to develop a classifier for DNA methylation-based subgroups with 98% accuracy. Unsupervised clustering of 197 patients diagnosed with HCL, HCL-V or SDRPL revealed 5 distinct DNA methylation (M) subgroups ( Figure 1). Subgroup assignment was stable throughout longitudinal sampling (including pre/post-treatment) and consistent between splenic, bone marrow and PBMC derived cells. A subgroup with universally clonal BRAF-V600E mutations and majority diagnosed as HCL was termed the M-HCL subgroup ( Table 1). Four other groups termed M-SBLPN1-4 contained all HCL-V and SDRPL diagnosed samples and were devoid of BRAF-V600E mutations. M-SBLPN1 comprised MAP2K1 mutations (91%) and was enriched for CREBBP, ARIDIA and TERT-promoter mutations. These patients displayed an HCL-like immunophenotype (64.3% CD25+) with 1/3 diagnosed as HCL. M-SBLPN2 exhibited the highest prevalence of TP53 mutations and concomitant genomic instability. Patients in M-SBLPN1,2 were enriched in unmutated IGHV4-34 rearrangements. M-SBLPN3,4 subgroups displayed an immunophenotype more dissimilar to HCL, mutated IGHV genes, and enrichment of IGLL5, SYK and BIRC3 mutations. M-SBLPN4 contained the most SDRPL samples, suggesting it may represent the SDRPL entity retained by the WHO. We next uncovered that 29/170 SMZL patients displayed DNA methylation patterns mapping to M-SBLPN2-4. These patients were phenotypically and molecularly similar to SBLPN (70% displaying villous morphology and depleted in IGHV1-2*04, NOTCH2, KLF2 mutations), likely representing SMZL patients suggested for reassignment to SBLPN in the updated WHO classification. To elucidate molecular pathways governing the biology of M-SBLPN subgroups, transcription factor motif enrichment analysis in hypomethylated genomic regions revealed selective activation of AP-1 in M-HCL along with ETS in M-SBLPN1,2. Both transcription factors are downstream of MAPK signaling, consistent with activating BRAF and MAP2K1 mutations in these subgroups. However, we observed strong ETS enrichment in the absence of MAP2K1 in M-SBLPN along with mutual exclusivity of MAP2K1 and TP53 mutations, suggesting TP53 mutations are driving ETS activation. Although lymphoid neoplasms rarely exhibit TERT promoter mutations, 83% of M-SBLPN1 patients showed the c.-124C>T mutation commonly observed in other cancers producing an ETS binding site and ectopic TERT activation. ETS activation and gain of an ETS site by mutation implies oncogenesis involves aberrant TERT activation in this subgroup. In summary, we have developed a DNA methylation-based classifier that resolves 4 SBLPN subgroups with distinct molecular features, and reclassifies a subset of SMZL and HCL patients, adding further information to the updated WHO/ICC entities. We reveal distinct biological pathways operating in M-SBLPN subgroups that may aid targeted therapy approaches.

Efficacy of Chemo-Free Combination Therapy with BRAF Inhibitor and Rituximab in Classic Hairy Cell Leukemia: Insights from the French Experience

Background: Hairy cell leukemia (HCL) is a rare form of leukemia. The introduction of purine analogs has revolutionized its prognosis, leading to a life expectancy closer to that of the general population. However, patients experience relapses and require other treatment options. The BRAF V600E mutation is present in 80-100% of patients with HCL, which has prompted the use of BRAF inhibitors (vemurafenib or dabrafenib) in this condition. Recently, the combination of vemurafenib with rituximab has shown efficacy with durable responses in relapsed patients. Here, we present, for the first time, real-life data on the combination of a BRAF inhibitor with rituximab through a retrospective study involving 9 French centers. Data collection was completed by July 1, 2023. Results: We collected data from 17 patients with a male-to-female ratio of 13:4; the median age was 69 years (38-90), and 6 patients were aged over 75. The median number of previous treatments was 4 (0-7), and 3 patients were treatment-naive because they were deemed ineligible for purine analogs based on age or performance status. All 14 relapsed patients had previously received purine analogs once or multiple times (1-3). The median progression-free survival (PFS) after purine analogs was 48 months, and the median PFS after the last line of therapy was 24 months. Five patients were exposed to moxetumumab pasudotox, and 3 patients received single-agent vemurafenib previously. The BRAF V600E mutation was tested and positive for 16 patients. In our cohort, 13 patients received a combination of vemurafenib with rituximab (V+R), and 4 patients received a combination of dabrafenib with rituximab (D+R). All patients received the same regimen (Tiacci, NEMJ 2021), which included an induction phase (V+R or D+R for 8 weeks) followed by rituximab consolidation (4 injections every 15 days), except for one patient who received a rituximab sequence followed by vemurafenib. At the time of analysis, 15 out of 17 patients had completed the treatment, and the remaining 2 had completed the induction phase. Fourteen patients initiated treatment at the standard dose (960 mg bid for vemurafenib and 150 mg bid for dabrafenib), and 3 patients at a reduced dose (240-480 mg bid for vemurafenib). Toxicity of any grade was reported in 15 out of 17 patients, with 3 of them experiencing grade 3-4 toxicity. Skin toxicity was the most frequent, recorded in 12 patients (grade 1-2), leading to temporary treatment interruption in 2 patients and dose reduction in 6 patients. Other toxicities included musculoskeletal, hepatic, hematological, and cardiac, with only one patient experiencing a grade 2 infection during treatment. No secondary cancers were reported during or after treatment. Treatment was discontinued due to toxicity in 5 patients, with 4 of them resuming treatment later. Eight patients required a dosage reduction, with a median final vemurafenib dosage of 480 mg bid. At the end of treatment, 14 patients achieved a complete hematological response, and 1 patient achieved a partial response. Among the 9 patients with early response data, the median time for platelet recovery was 15 days, and for neutrophil recovery, it was 14 days. End-of-treatment bone marrow aspiration and/or biopsy was performed for 8 patients, showing a complete response in 7 patients. With a median follow-up of 12 months (4-56), only 2 patients had progressed, and as of the last update, no patients had died, and the 2 relapsed patients achieved a complete response after a new line of therapy. Conclusion: Despite a limited median follow-up of 12 months, the combination of a BRAF inhibitor with rituximab proves to be an effective regimen in heavily treated patients with HCL, achieving a 100% overall response rate. Our study confirms a rapid hematological response and an acceptable toxicity profile, even in elderly patients. The most frequent toxicity observed was cutaneous toxicity, mainly grade 1-2, and it never led to a definitive treatment discontinuation. A comparison of the two BRAF inhibitors in terms of efficacy or toxicity cannot be established due to the small sample size of patients treated with dabrafenib. Lastly, the combination of rituximab with vemurafenib or dabrafenib appears to be an interesting option in frontline treatment for patients ineligible for purine analogs.

Serum Soluble Interleukin-2 Receptor Levels in Hairy Cell Leukemia As a Marker of Tumor Burden with Prognostic Value and As a Tool for Disease Monitoring

Background Neoplastic cells in hairy cell leukemia (HCL) express the α chain of the interleukin-2 receptor on their surface, which is secreted in large amounts in the serum in a soluble form (sIL-2R). High serum levels of sIL-2R have previously been reported in HCL. Thus, we assessed whether serum sIL-2R levels correlated with tumor burden, represented a prognostic factor, and if they could be used as a tool for disease monitoring. Methods Serum levels of sIL-2R were measured in 59 patients affected by classical HCL followed at the Hematology Unit of Padova University Hospital. To assess whether sIL-2R levels were representative of tumor burden, we evaluated the relationship of pre-therapy sIL-2R levels with other hematologic features such as leukocyte count, the presence of splenomegaly, β-2 microglobulin levels, lactate dehydrogenase (LDH) levels, CD38 expression, the percentages of circulating hairy cells and bone marrow infiltration. In 50 patients we investigated whether post-therapy sIL-2R levels correlated with clinical response and measurable residual disease (MRD) status evaluated by immunohistochemistry according to consensus guidelines. To determine the usefulness of serum sIL-2R levels for disease monitoring, yearly serial sIL-2R levels were measured in 59 patients, including 5 (8.4%) cases in watch & wait phase and 54 (91.6%) with disease remission after therapy with purine analogues. sIL-2R kinetics were correlated with time to cytopenia (TTC) (hemoglobin <100g/L, neutrophils < 1000/µL or platelets <100.000/µL) and time to next treatment (TTNT). Serum sIL-2R levels were measured by enzyme-linked immunosorbent assay. The correlation of sIL-2R levels with other variables was evaluated by linear or logistic regression as appropriate. Medians were compared using the Mann-Whitney U test. Survival and univariate analyses for TTC and TTFT were conducted according to the Kaplan-Meier method and Cox proportional hazards model. Results The mean age of patients was 60.6 years; 10 were females (17%), and 49 were males (83%). Among treated patients, 47/54 (87%) received cladribine and 7/54 (13%) pentostatin. Pre-therapy sIL-2R levels significantly correlated with leukocyte count (r 2 = 0.13; p = 0.009), β-2 microglobulin levels (r 2 = 0.44; p < 0.001), CD38 expression (p = 0.018), the percentage of circulating hairy cells (r 2 = 0.17; p = 0.017) and the percentage of bone marrow infiltration (r 2 = 0.15; p = 0.009), but not splenomegaly (p = 0.167) or LDH levels (r 2 = 0.02; p = 0.369). We found a significant difference between serum sIL-2R levels measured before and after therapy (median 16.185 vs 599 kU/L respectively; p < 0.001), with a median reduction in sIL-2R levels of 15.628 kU/L after treatment with purine analogues. A similar decrease in sIL-2R levels after therapy was observed in 4 relapsed patients treated with rituximab-vemurafenib (median pre-therapy sIL-2R 11.460 vs. 467 kU/L after treatment, p = 0.03; median reduction 10.848 kU/L). Among responding patients, the absolute value of log10(sIL-2R) levels measured 6 months after therapy was a predictor of shorter TTNT in univariate analysis (HR 15.14; 95% CI 1.3 - 176; p = 0.03). Furthermore, sIL-2R correlated with the depth of response. Post-therapy median sIL-2R levels were significantly lower in patients achieving a complete remission (CR) versus those who did not (median 568 kU/L for CR vs. 3.272 kU/L for partial remission/stable disease; p = 0.002). A statistically significant difference was also observed in post-therapy sIL-2R levels between MRD- and MRD+ patients (median 502 vs 724 kU/L; p = 0.003) (Figure 1A). In the patients that underwent yearly serial sIL-2R measurements during follow-up, a rise from the nadir after therapy in serum sIL-2R levels of ≥ 25% between two samples was associated with both shorter TTC (HR 26.2; 95 % CI 3.44 - 200; p < 0.001) (Figure 1B) and TTNT (HR 8.83; 95 % CI 1.9 - 40.9; p < 0.001). Conclusion The consistent reduction in sIL-2R levels after therapy and their correlation with other standard disease parameters show how serum sIL-2R levels could be an effective marker of tumor burden in HCL. While more data is required to validate its use in clinical routine, sIL-2R could be used as an effective marker for disease monitoring. Moreover, given the prognostic significance of post-therapy levels, sIL-2R may represent a prognostic factor alongside MRD to identify those patients that are more likely to develop early relapse.

Precision Targeting of the Malignant Clone in Diffuse Large B Cell Lymphoma Using Chimeric Antigen Receptor T Cells Against the Clonotypic IGHV4-34 B Cell Receptor

Introduction: CD19-directed Chimeric Antigen Receptor T cell (CART19) immunotherapy has revolutionized the treatment of B cell lymphoma. However, most CART19-treated patients either fail to respond or show disease progression after an initial response. In fact, up to 30% of lymphoma relapses following CART19 show loss of CD19 expression. In addition, several patients who respond well to CART19 display complete loss or severe reduction of their normal B cell repertoire, leaving them exposed to recurrent infections, limited response to vaccines, and requiring prophylactic measures such as intravenous immunoglobulins. A significant portion of mature B cell malignancies express B cell receptors (BCR) that use the same immunoglobulin heavy variable gene: IGHV4-34. In particular, ~30% of activated B cell (ABC) diffuse large B cell lymphomas (DLBCL), ~35% of primary central nervous system lymphomas, ~65% of vitreoretinal lymphomas, and ~35% of hairy cell leukemia variant express IGHV4-34. This suggests that the IGHV4-34 heavy chain is critical for driving the disease by delivering cell survival and proliferation signals, and multiple studies have shown that BCR signaling is required for ABC-DLBCL survival. However, while highly enriched in several types of B cell lymphomas, IGHV4-34 expressing B cells compose only ~5% of the normal B cell repertoire of healthy individuals. Therefore, we hypothesized that anti-IGHV4-34 CAR T cells would be highly effective and safe against B cell malignancies, as they would: (i) efficiently recognize IGHV4-34+ lymphoma cells while sparing normal B cells; and (ii) target a tumor driver that is essential for lymphoma cell survival ( Fig 1A). Methods and Results:We developed a novel CAR construct (CD8-41BB-CD3z) targeting the IGHV4-34+ BCR (CART4-34) using a single-chain variable fragment (scFv) derived from the 9G4 rat monoclonal antibody. We used tumor B cells that endogenously (HBL1) or exogenously (Jeko1, Bonna12, Mec1) express the IGHV4-34 BCR. Using cytotoxicity, cytokine secretion and proliferation assays in vitro we showed that CART4-34 specifically target all IGHV4-34+ tumor B cells tested while sparing IGHV4-34- tumor B cells and, most importantly, healthy B cells. However, while the initial CART4-34 showed strong cytotoxicity towards IGHV4-34+ cells in short-term in vitro assays, they were significantly inferior to CART19 in in vivo xenograft models. We hypothesized that the poor performance of CART4-34 compared to CART19 was due to the unique challenge of targeting the membrane-distal portion of the BCR, 18 nm from the plasma membrane. In contrast, CART19 targets an epitope of CD19 located just 5 nm from the membrane ( Fig 1B). Therefore, to improve immune synapse (IS) formation, we designed new CAR constructs with smaller extracellular domains by replacing the original CD8 hinge (44 amino acids (aa)) with either: (i) a short version of the IgG4 hinge (12aa); or (ii) a G4S linker (5aa). We found that both CART4-34 (IgG4) and CART4-34 (G4S) exhibited significantly improved cytotoxicity towards IGHV4-34+ tumor cells in short-term and long-term in vitro assays in all IGHV4-34+ cell lines tested, while maintaining specificity. Importantly, in in vivo human xenograft models of IGHV4-34+ lymphoma (HBL1), CART4-34 with short hinges showed significantly improved tumor control comparable to, or better than, CART19 ( Fig 1B). We also found that while CART19-resistant lymphoma cells had complete loss of CD19 surface expression, CART4-34-resistant tumor cells retained high expression of IgM, suggesting a reduced likelihood of antigen-negative escape owing to the critical role of the BCR for lymphoma B cell survival. Mechanistically, we found that while the original CART4-34 (CD8 hinge) formed IS with striking morphological differences compared to IS formed by CART19, the IS formed by CART4-34 with short hinges highly resembled those formed by CART19, providing a rationale for the improved anti-tumor potency of short-hinge CART4-34. Conclusion:We implemented a novel paradigm for the treatment of B cell malignancies: the specific targeting of the malignant clone, while sparing the remainder of the healthy B cell repertoire. We designed and optimized a best-in-class anti-IGHV4-34 CAR T cell product with potent anti-tumor effects and minimal toxicity towards IGHV4-34-negative B cells. The activity of CART4-34 will be investigated in a Phase I clinical trial.

Efficacy and Safety of Obinutuzumab in Relapsed or Refractory Hairy Cell Leukemia (R/R HCL): An Italian Multicenter Phase-2 Academic Trial (HCL-PG04)

INTRODUCTION About 50% of HCL patients (pts) relapse after chemotherapy with purine analogs (PA). After identifying the BRAF-V600E mutation as the genetic cause of HCL (Tiacci et al. NEJM 2011), we documented a high efficacy of the BRAF inhibitor vemurafenib in R/R pts (Tiacci, Park et al. NEJM 2015), especially when added to rituximab (Tiacci et al. NEJM 2021). HCL expresses bright CD20 and obinutuzumab (OBI) is a more effective anti-CD20 agent than rituximab (RTX) in other indolent B-cell neoplasms such as chronic lymphocytic leukemia and follicular lymphoma. RTX produces ~20% complete remissions (CR) in R/R HCL (Kreitman, ASH Educ Program 2012), while OBI activity in this setting is unknown. METHODS In the ongoing academic, phase-2, multi-center trial HCL-PG04, R/R HCL pts needing treatment due to cytopenia(s) received OBI 1000 mg intravenously on days 1-8-15 of cycle 1 and on day 1 of cycles 2-6 (1 cycle = 28 days). CR required resolution of cytopenias (platelets ≥ 100,000/mmc; neutrophils ≥ 1500/mmc; hemoglobin ≥ 11 g/dl), no palpable splenomegaly and no leukemic cells on morphologic analysis of the bone marrow biopsy, as assessed one month after the last OBI dose. Minimal residual disease (MRD) was analyzed by BRAF-V600E specific PCR in the bone marrow aspirate (sensitivity: 0.05% mutant alleles). RESULTS We enrolled 26 pts (M/F 24/2; median age 62 years, range 39-80) with a median of 2 prior therapies (range 1-7), including 7 refractory to PA (26%) and 2 to RTX (8%). Median values of disease burden parameters at baseline were: 70% BM HCL infiltration, 830 neutrophils/mmc, 62000 platelets/mmc, 14 g/dl hemoglobin, and 16 cm of longest spleen diameter (in 16/26 splenomegalic patients; 1 pts. had been splenectomized and 9 were not splenomegalic at baseline). Toxicity was as expected, largely of grade (G) 1-2, always reversible and mainly consisting in: infusion reactions (G1-2 92%; G3 4%, in a single pt allergic to previous RTX who discontinued OBI); asymptomatic increase of liver enzymes (G1-2 19%; G3 GGT 15%; G3 ALT 4%); transient worsening of baseline G1-G3 thrombocytopenia to G3 (23%) or G4 (35%), with only 1 minor bleeding (4%, G1 epistaxis); transient neutropenia (G2 4%; G3 15%; G4 15%); and rare infections (G1-2 herpes labialis 8%; G3 pneumonia 4%). CR was reached in 12/25 evaluable pts (48%), including 2 pts with incomplete platelet recovery (~70000-90000/mmc) and 2 pts with delayed resolution of palpable splenomegaly. Notably, CR was achieved in 5/7 pts refractory to PA and 1/2 pts refractory to RTX. Furthermore, CR was negative for MRD in 9/12 pts (75%, including 3 pts with delayed MRD clearance), something never observed with vemurafenib. However, the kinetics of cytopenia resolution was slower compared to our previous vemurafenib trial, in that in pts obtaining CR with OBI neutrophil recovered to ≥1500/mmc after a median of 7 weeks (vs 4 weeks with vemurafenib) and platelets recovered to ≥100000/mmc after a median of 8 weeks (vs 2 weeks with vemurafenib). Prior exposure to RTX (n=12 pts) did not compromise OBI efficacy (5 CR, all MRD-negative, in 11 evaluable pts). In pts achieving CR with OBI, progression-free and MRD-free survival were both 100% at 56 months (range 20-66) and 48 months (range 6.5-60) of median follow-up, respectively (Fig.1). The 13 non-CR pts (minor response 7; no response 3; progression 3) were effectively salvaged with another course of OBI combined with vemurafenib, which will be presented at the meeting. CONCLUSIONS OBI frequently produced deep and durable responses in R/R HCL, to an extent apparently greater than RTX, thus qualifying as an active and safe chemotherapy-free strategy in this setting.

Refinement of MRD Assessment in Hairy Cell Leukemia by Integrated Molecular and Flow Cytometry Analysis

Refinement of MRD Assessment in Hairy Cell Leukemia by Integrated Molecular and Flow Cytometry Analysis

Hairy cell leukemia with an aggressive outcome: A case report with a review of the literature

Abstract Hairy cell leukemia (HCL), an uncommon cancer affecting B-lymphocytes primarily in the bone marrow and spleen, is identified by abnormal projections on malignant B cells, which give the illness its name. This condition is less frequently observed in Asian populations compared with individuals from American and European backgrounds and tends to be diagnosed around the age of 55 years. The leading genetic association of this ailment is linked to the BRAFV600 mutation. Diagnosing HCL poses challenges due to its similarity to other conditions involving excessive lymphocyte growth. Here, we present a distinctive case of an aggressive disease course in a 46-year-old male.

The Use of CD200 in the Differential Diagnosis of B-Cell Lymphoproliferative Disorders.

Background: B-Cell Lymphoproliferative Disorders (B-LPDs) are a group of heterogenous disorders characterised by the accumulation of B-cells in peripheral blood, bone marrow, lymph nodes and spleen. They have a variable disease course and outcome and many share similar features making differential diagnosis challenging. Therefore, accurate diagnosis is fundamental in particular for determining treatment options. Immunophenotyping by flow cytometry plays a crucial role in the diagnosis of B-LPDs. However, overlapping immunophenotyping patterns exist and the use of novel monoclonal antibodies has become increasingly important in immunophenotyping analysis. More recently differential expression of CD200 has been reported in various B-LPDs and that CD200 may improve the differentiation between chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL). In this study CD200 expression is evaluated in different B-LPDs. Methods: A total of 100 samples were collected and analysed by immunophenotyping flow cytometry over a period of 1year (2017-2018), by a panel of monoclonal antibodies including CD200. The percentage of CD200 and its expression intensity was evaluated and compared between different groups of B-LPDs. Results: All of the 50 cases of CLL expressed CD200 with moderate to bright intensity, 6 MCL cases lacked the expression of CD200. Furthermore, all 5 cases of hairy cell leukaemia (HCL) expressed CD200. Out of all B-LPDs evaluated, CD200 expression in HCL cases was noted to be the brightest. The other 39 cases were not found to be B-LPDs. Conclusion: CD200 has an important role in differentiating CLL from MCL, HCL has a consistent bright expression of CD200. By adding CD200 to the combinations of markers in routine testing panel, Immunophenotyping by flow cytometry can be an effective tool in the diagnosis of B-LPDs especially in cases with atypical immunophenotyping pattern. Our result support that CD200 can be added to routine testing panel as it is useful in differentiating them.

Single-agent rituximab is an effective salvage therapy in pretreated patients with hairy cell leukemia

Single-agent rituximab can be a suitable treatment in hairy cell leukemia (HCL) patients relapsing after repeated courses of purine analogs, if purine analogs are contraindicated (e.g. in case of poor marrow cellularity, high disease infiltration predicting long-lasting aplasia) and if newer agents (moxetumomab or vemurafenib) are not easily available. Our institutional series of patients receiving single-agent rituximab as salvage therapy was reviewed. Patients received rituximab at the standard dose of 375 mg/m2 weekly for 4 weeks. The main study objectives were overall response rate (ORR), time-to-next treatment (TTNT), progression-free survival (PFS) and overall survival (OS). Responses have been categorized according to the Consensus Resolution Criteria. Thirty-three patients received 39 courses of rituximab (4 patients received it twice, one patient three times), as a median third line of therapy (range 2-8). Out of 39 courses, a complete response was obtained in 28.2% of cases, a partial response in 23.1% and a minimal response in 20.5%. In 28.2% of patients, we observed no response. Median TTNT was 33 months (65% at 2 years), median PFS was 24 months (51% at 2 years) and median OS resulted of 154 months (22% at 20 years). To our knowledge, this is the widest series of HCL patients receiving single-agent rituximab for disease relapse. Rituximab is an effective salvage therapy in pretreated HCL patients after failure of purine analogs. It may be repeated if no alternatives are available.

COVID-19 in patients with classic and variant hairy cell leukemia

AbstractHairy cell leukemia (HCL), similar to its variant HCLv, is a B-cell malignancy associated with decreased humoral immunity. We prospectively monitored the largest cohort of patients with HCL/HCLv to date (n = 503) for COVID-19 by symptoms, antibody, and polymerase chain reaction (PCR) and/or antigen positivity. Fifty percent (253 of 503) of the patients with HCL/HCLv (238 HCL and 15 HCLv) had evidence of COVID-19, with 210 (83%) testing positive by PCR or rapid-antigen test. Of the 43 patients without positive tests, all had nucleocapsid antibodies indicating COVID-19 exposure, 7 recalled no symptoms, and 36 had mild symptoms. Of the 210 who tested positive, 23, 46, 129, and 12 cases occurred in 2020, 2021, 2022, and 2023, respectively. Among them, 175 began treatment for HCL/HCLv 0.4 to 429 (median, 66) months before, and 132 had their last dose of anti-CD20 monoclonal antibody 0.2 to 229 (median, 63) months before. Two patients died, including a young woman who began rituximab 2 months after first-line cladribine before vaccine availability. Nearly all patients with HCL/HCLv recovered uneventfully from COVID-19 including those without vaccination or those with significant immunosuppression and recent treatment. However, decreased normal B cells from HCL or treatment was associated with lower spike antibody levels as a response to COVID-19 (P = .0094) and longer recovery time (P = .0036). Thus, in a large cohort of patients with HCL/HCLv and in the first to determine relationships between COVID-19 outcome and immune markers, mortality was relatively low (∼1%), sequelae were uncommon, and recovery from COVID-19 was longer if normal B cells were low after recent treatment. The trials are registered at www.clinicaltrials.gov as #NCT01087333 and #NCT04362865.

Vemurafenib and Obinutuzumab as Frontline Therapy for Hairy Cell Leukemia

BackgroundHairy cell leukemia (HCL) is characterized by the underlying genetic lesion of BRAFV600E and responsiveness to BRAF inhibitors. We assessed the safety and activity of the BRAF inhibitor vemurafenib combined with obinutuzumab in patients with previously untreated HCL.MethodsWe conducted a single-arm, multicenter clinical study of vemurafenib plus obinutuzumab. Vemurafenib 960 mg twice daily was administered for four cycles, and obinutuzumab was administered in cycles 2 to 4. The primary end point was complete remission (CR). Secondary end points included assessment of safety, minimal residual disease (MRD), and BRAF allele burden according to digital droplet polymerase chain reaction (ddPCR).ResultsThirty patients were enrolled in the study, and 27 patients completed all four cycles of treatments and achieved CR (90%; 95% confidence interval [CI], 73 to 98). Three patients discontinued the study early because of adverse events and were not evaluable for response. Of the 27 patients who achieved CR, 26 patients (96%; 95% CI, 81 to 99) achieved MRD negativity. BRAFV600E allele was undetectable by ddPCR in all 21 evaluable patients. At a median follow-up of 34.9 months (95% CI, 29.6 to 36.9), no patient experienced disease relapse. The most common vemurafenib-related adverse events were rash and arthralgia. Febrile neutropenia occurred in two patients, and blood or platelet transfusions were required in two patients.ConclusionsCombined time-limited vemurafenib and obinutuzumab achieved CR in more than 90% of patients with previously untreated HCL. In this small study, acquired vemurafenib resistance or dose-limiting toxicity was not observed. Patients were not observed long enough to reveal secondary malignancies. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT03410875.)

Biotechnological Approaches for Enhancing Polyhydroxyalkanoates (PHAs) Production: Current and Future Perspectives.

The benefits of biotechnology are not limited to genetic engineering, but it also displays its great impact on industrial uses of crops (e.g., biodegradable plastics). Polyhydroxyalkanoates (PHAs) make a diverse class of bio-based and biodegradable polymers naturally synthesized by numerous microorganisms. However, several C3 and C4 plants have also been genetically engineered to produce PHAs. The highest production yield of PHAs was obtained with a well-known C3 plant, Arabidopsis thaliana, upto 40% of the dry weight of the leaf. This review summarizes all biotechnological mechanisms that have been adopted with the goal of increasing PHAs production in bacteria and plant species alike. Moreover, the possibility of using some methods that have not been applied in bioplastic science are discussed with special attention to plants. These include producing PHAs in transgenic hairy roots and cell suspension cultures, making transformed bacteria and plants via transposons, constructing an engineered metabolon, and overexpressing of phaP and the ABC operon concurrently. Taken together, that biotechnology will be highly beneficial for reducing plastic pollution through the implementation of biotechnological strategies is taken for granted.