Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cell therapies for B cell leukemias and lymphomas target CD19, which is widely expressed across the B cell lineage, often leading to on-target, off-tumor B cell depletion, prolonged immune suppression, and antigen-negative escape in a subset of patients. In contrast, B cell receptor (BcR) signaling is essential for the survival of most mature B cell neoplasms, and BcRs carrying the immunoglobulin heavy variable gene IGHV4-34 are highly enriched in B cell malignancies compared with normal B cells. Further, self-reactive IGHV4-34+ serum autoantibodies are enriched in aggressive systemic lupus erythematosus (SLE) and other autoimmune diseases. Here, we developed CAR T cells targeting the BcR carrying IGHV4-34 (CART4-34). We found that CART4-34 showed specific cytotoxicity and cytokine secretion toward IGHV4-34+ malignant B cells. In addition, although CD19 was down-regulated upon relapse after treatment with CART19, IGHV4-34+ BcR levels remained intact upon relapse after treatment with CART4-34, suggesting reduced risk of antigen-negative escape. In IGHV4-34+ HBL1 cell line-derived xenograft mouse models, CART4-34 showed robust expansion and antitumor activity comparable to those of CART19. Optimized CAR:BcR binding using shorter CAR hinge domains improved immune synapse morphology and in vivo activity. In addition, we showed that CART4-34 could target human IGHV4-34+ SLE B cells and deplete IGHV4-34+ autoantibodies ex vivo, without targeting healthy B cells or affecting total IgG titers. In conclusion, we developed a CAR T cell product that specifically targets pathogenic B cells in lymphoid malignancies and SLE, offering potential for precision cell therapy for these indications.

ABSTRACT:The primary objective in multiply relapsed hairy cell leukemia and variant (HCL/HCLv) was to determine whether pentostatin-rituximab (DCFR) and bendamustine-rituximab (BR) each achieve an overall response rate (ORR) exceeding that historically achieved by rituximab alone (∼40%) in favor of 65%. Prospective data were unreported for either regimen. Fifty-six patients received 6 28-day cycles of rituximab (375 mg/m2, days 1 and 15) with either bendamustine (90 mg/m2, days 1 and 2) or pentostatin (4 mg/m2, days 1 and 15). Eligibility required ≥2 purine analogs, or 1 purine analog plus rituximab for response of <1 year to the initial purine analog. Although patients were assigned to either regimen through randomization to increase homogeneity of the 2 treatment groups, the DCFR arm had fewer previous purine analogs (P = .021) and lower baseline marrow HCL/HCLv infiltration (P = .013). ORRs for DCFR and BR were 93% (95% confidence intervals [CI], 83-102) and 86%, (95% CI, 73-99), respectively, exceeding 40% (P< .0001) for each group. Rates for complete remission (CR) and minimal residual disease-free CR and median progression-free survival (141 vs 50 months; HR, 0.63; 95% CI, 0.32-1.25) numerically favored DCFR, but that arm was significantly enriched with less previous purine analogs and marrow infiltration, each of which was associated post hoc with better response. Post hoc subgroup analysis, particularly for 41 patients with classic HCL, suggested any superiority of DCFR vs BR might apply to patients with more favorable disease. DCFR and BR were highly effective in multiply relapsed HCL/HCLv. Possible DCFR superiority was hypothesis-generating, given uneven baseline risks and trial design. This trial was registered at www.clinicaltrials.gov as #NCT01059786.

ABSTRACT:Hairy cell leukemia (HCL) and HCL variant (HCLv) are distinct, rare, and chronic splenic B-cell lymphomas/leukemias that partially overlap in clinicopathologic presentation but differ in genetic basis, prognosis, and management. HCL is caused by the BRAF-V600E kinase-activating mutation in >95% of the patients, usually has excellent responses to chemotherapy with purine analogues, and is also amenable to BRAF inhibitor-based targeted treatments. In contrast, HCLv lacks BRAFV600E mutation, requires combined therapy with purine analogues in addition to rituximab, and generally shows less durable responses. Here, an international team of hematologists, experts on these rare diseases, was convened by the Hairy Cell Leukemia Foundation to update the previous guidelines (published in 2017) by providing a summary of current methods to diagnose and manage patients with HCL and HCLv as well as a prospective on newer targeted therapies to further improve outcomes.

Hairy cell leukemia is a B-cell malignancy presenting with cytopenias and splenomegaly. It responds well to purine analogs cladribine (CDA) and pentostatin, but minimal residual disease (MRD) and relapses are common. Randomized 1st line data showed higher MRD-free CR rate using cladribine with (CDAR) vs without (CDA) concurrent rituximab (97% vs 24% at 6 months, 94% vs 32% long term, both p<0.0001). Published results from rituximab started 4 weeks after cladribine (CDA+R) include MRD-free CR rate 83/107 (78%) at 3-4 months. To validate the 94% MRD-free CR rate after CDAR, 25 additional patients received CDAR, and follow-up from the 34 randomized patients after CDAR updated. Reportedly, rituximab increases cell sensitivity to cladribine, resulting in ex vivo synergy; this would be possible after CDAR but not after CDA+R due to cladribine's short half-life. Patients (N=25) with untreated HCL received CDAR, cladribine 0.15 mg/kg IV days 1-5 with 8 weekly doses of rituximab 375 mg/m2 begun day 1. Rituximab was given before cladribine on day 1 to compare to the 34 randomized CDAR patients who received cladribine before rituximab on day 1. MRD was assessed by blood and bone marrow aspirate (BMA) flow cytometry (FC, sensitivity 0.002% of cells) and bone marrow biopsy immunohistochemistry (IHC). As in the published CDAR trial, patients received one 8-week course of delayed rituximab if blood MRD was detected by FC >6 months after day 1. After beginning CDAR, blood was evaluated every 3 months for 1 year, semiannually until 2.5 years, then annually. Bone marrow was evaluated at 1, 6, 18 and 30 months, then every 2 years indefinitely. Patients treated with CDAR on the earlier randomized trial (N=34) were followed similarly. Of 25 non-randomized patients after CDAR, 25/25 (100%) achieved CR, and 21/25 (86%) achieved MRD-free CR by 6 months. One patient achieved MRD-free CR by 1.5-years and another by 4.5-years, both repeatedly redemonstrating MRD-free CR thereafter. At 7.3-11.1 (median 9.0) years of follow-up, 23/25 (92%) patients achieved and remained in MRD-free CR, with median MRD-free survival of 9.0 years. One of 2 patients with MRD received delayed rituximab for blood MRD, which resolved blood but not BMA MRD for 3.5 years. No new safety signals were seen; 4/25 (16%) patients received prophylactic platelet transfusion on day 1-2 due to grade 4 thrombocytopenia. This was due to rituximab unrelated to cladribine, and no significant bleeding occurred. The order of rituximab and cladribine on day 1 had no impact on efficacy or toxicity. Regarding the 34-patient CDAR randomized cohort, 5.0-15.9 (median 13.4) year follow-up is now available. Three of 34 patients died of causes unrelated to HCL or CDAR, and 3 patients were lost to follow-up. The remaining 28 patients continue to be followed, including bone marrow studies for MRD. As previously reported, the 6-month MRD-free CR rate was 97%, and the 1 patient who relapsed at 1.5 years had >20 years of interferon treatment but was eligible since purine analog naïve. However, no other patients have relapsed from MRD-free CR, and all remaining 32 (94%) patients had MRD-free CR reconfirmed by the last bone marrow assessment. Interestingly, 3 patients had transient MRD; 1 patient became MRD+ at 10.5 years but negative at 12.5 and 14.5 years; 1 became MRD+ at 4.5 years but negative at 6.5, 8.5, 10.5, and 12.5 years; 1 became MRD+ at 2.5 years but negative at 4.5, 6.5, 8.5, and 12.5 years. Of the 59 total patients after CDAR, with 5.0-15.9 (median 11.5) years of follow-up, 55/59 (93%) patients remain MRD-free at the most recent assessment. First-line CDAR is highly effective for HCL, achieving MRD-free CR in 54/59 (92%) total patients at 6 months and 55/59 (93%) long term. The high MRD-free CR rate may be due to synergy of cladribine and rituximab starting the same day. Transient appearance of MRD which later resolved may be related to immune destruction. Thrombocytopenia on day 1-2 was a minor issue, and the randomized trial reported higher platelet (and neutrophil) counts after CDAR vs CDA probably due to more rapid bone marrow clearing. To evaluate the clinical importance of remaining MRD-free, patients after CDAR are being compared to patients in MRD+ CR after 1st line CDA or CDA+R to determine if there is a difference in progression free survival or time to next treatment.

Background: Hairy cell leukemia (HCL) is an indolent B-cell leukemia characterized by durable complete remissions to purine analogs cladribine or pentostatin, but repeated relapses and cumulative toxicity to repeated purine analog courses. Vemurafenib, targeting the BRAF V600E mutation expressed by >90% of classic HCL, achieved complete remission (CR) rates of 35-42% after a 16-18-week course. All evaluated CRs were positive for HCL minimal residual disease (MRD) by bone marrow immunohistochemistry (IHC) and median relapse free survival (RFS) was 19 months. Vemurafenib was combined with rituximab, achieving 57% MRD-free CRs. Combined BRAF-MEK inhibition using dabrafenib-trametinib respectively was superior to vemurafenib for BRAF V600E+ melanoma, and in 55 HCL patients achieved CR rate 65.5%, with 12.7% MRD-free by bone marrow aspirate (BMA) flow cytometry (FC), 2-year response duration 97.7% and progression-free survival 94.4%. The most common toxicity was pyrexia (fever, 58.2%) often associated with severe rigors and/or need for discontinuation or steroids. To avoid pyrexia, we began a phase 2 trial of combined BRAF-MEK inhibition using encorafenib-binimetinib, which in melanoma was reported to effectively target BRAF-MEK without fever. Patients and Methods: Twenty-eight patients with BRAF V600-mutated HCL were treated with encorafenib 450 mg/day and binimetinib 45 mg twice daily, both orally. The primary endpoint was to rule out a CR rate of 35% in favor of 55%. The most important secondary endpoint was to reduce the rate of pyrexia compared to dabrafenib-trametinib. Eligibility required prior purine analog and need for treatment including neutrophils <1/nL, hemoglobin <10 g/dL, platelets <100/nL, symptomatic splenomegaly, or HCL mases >2cm in short axis. CR criteria were consistent with HCL consensus guidelines. Response was assessed after three 4-week cycles (pre-cycle 4), pre-cycle 7, yearly until 2.5 years after starting, then biannually. MRD was assessed by marrow IHC and FC of blood and BMA. Encorafenib and/or binimetinib could be interrupted or dose-reduced for toxicity, to a minimum of 75 and 15 mg/day, respectively. Results: Of 28 patients enrolled, 26 (93%) achieved CR. Time to CR was 2.5-40.5 (median 3.0) months, with 19 (68%) of 28 patients achieving CR by the earliest (12-week) restaging time point. At 3.3-56.5 (median 26.1) months of follow-up, only 1 of the 26 CRs relapsed. CRs achieved by encorafenib-binimetinib were MRD-free in 25/26 (96%) by IHC but only 3/26 (12%) by BMA FC. One patient achieved MRD-free CR at 12 weeks, was still MRD-free prior to cycle 20, stopped encorafenib-binimetinib pre-cycle 25, and was still MRD-free 1 year later. One patient achieved MRD-free CR pre-cycle 34 but was MRD+ at the next assessment pre-cycle 55. The 3rd patient first achieved MRD-free CR pre-cycle 19 after over a year in CR. For patients with MRD+ CR lasting at least a year, the protocol allowed patients to continue encorafenib-binimetinib while receiving rituximab 375 mg/m2 every 2 weeks for 8 doses. Of 6 patients so far who received rituximab during MRD+ CR, 3 (50%) achieved MRD-free CR by all tests and stopped therapy without MRD-relapse after up to 2 years of follow-up. Most patients underwent one or several dose reductions from the initial dose level to better tolerate chronic dosing. Toxicity was consistent with events reported in melanoma and other malignancies, and nearly always resolved rapidly with dose-reduction. Common (>20%) adverse events related to encorafenib included elevated lipase (57%) and amylase (36%) only rarely associated with symptomatic pancreatitis, reversible thinning (43%) and abnormal texture (21%) of hair, abdominal pain (36%), nausea (32%), loose stools (29%), skin lesions (36%), myalgias (29%) and fatigue (25%). Those related to binimetinib included serous retinopathy (43%), usually asymptomatic, blurred vision (25%), elevated creatine kinase (32%), and dry skin (25%). Conclusion: Encorafenib-binimetinib is highly effective in relapsed/refractory HCL and was well tolerated when dose reductions occurred as needed. Compared to dabrafenib-trametinib, the lower incidence of fever (11% vs 58%, p<0.0001) is a major advantage. The CR rate of 93% without rituximab is unprecedented for BRAF inhibition in HCL. With rituximab, MRD-free CR is achievable. Encorafenib-binimetinib adds to the options for BRAF treatment of this disease.

ABSTRACT:We found 20 patients with an immunophenotype consistent with classic hairy cell leukemia and BRAF mutations other than just V600E. Fourteen had 1 non-V600E BRAF mutation and 6 had V600E with 1 (n = 5) or 2 (n = 1) non-V600E BRAF comutations. This study was registered at https://clinicaltrials.gov as #NCT01087333.

Hairy cell leukemia (HCL) is an indolent B-cell neoplasm characterized by the expression of CD20, CD22, CD25, CD11c, CD103, CD123, and annexin A1. Treatment with purine analog monotherapy is highly effective, resulting in durable complete remissions (CR). In contrast, the HCL variant (HCLv), which typically lacks CD25, CD123, and annexin A1, responds poorly to purine analogs with poorer overall response rate (ORR) <50% and CR <10%, and median 6-9 year overall survival (OS). Classic HCL is driven by the BRAF V600E mutation or non-V600E BRAF mutations in >95% of cases, constituting both a diagnostic biomarker and a therapeutic target for BRAF inhibitors. Notably, both HCLv and some cases immunophenotypically consistent with classic HCL lack BRAF mutation. BRAF wild-type cases often harbor unmutated IGHV rearrangements with significant representation of IGHV4-34 gene, associated with activating mutations in MAP2K1 encoding MEK1, downstream of BRAF. Since 2014, our report and 5 others described a total of 28 patients with 10 different MAP2K1 mutations. These cases were too few to report clinical implications, and MAP2K2 mutations in HCL/HCLv have not been described. We performed whole exome sequencing (WES) and/or TrueSight Oncology (TSO500) and/or other sequencing including next generation sequencing (NGS) in 225 HCL/HCLv patients, including 91 with HCLv and 24 with unmutated IGHV4-34+ HCL. To be evaluable for WES/NGS, HCL/HCLv cells comprised >80% of B-cells and were partially purified using CD19 microbeads followed by positive fraction isolation. We identified 26 different MAP2K1 genetic alterations in 52 BRAF V600E-negative HCL/HCLv patients. 21 patients had HCL and 31 patients HCLv immunophenotype. Unmutated IGHV rearrangements were observed in 19/21 HCL and 15/31 HCLv patients. One HCLv patient harbored two MAP2K1 mutations, and another carried three. MAP2K1 mutations included both missense mutations and in-frame deletions. The most common mutations in HCL/HCLv were K57N and C121S each found in 9 patients, but K57N was observed in 7 HCL and C121S in 7 HCLv patients (p=0.057). Besides K57N and C121S, the most frequent MAP2K1 mutations in HCL were Q56P (n=4) and F53L (n=3); the most frequent in HCLv were I103N and K55E (n=3 each). MAP2K2 mutations were identified in 7 HCLv and 2 HCL patients, most commonly R231C in 4 HCLv patients. The other 3 HCLv patients had R231L, F57C and F57L, while the 2 HCL patients had A100T and P166T. One HCLv patient harbored concurrent mutations in both MAP2K1 and MAP2K2. BRAF V600E was found in none of 21 HCL patients with MAP2K1 mutations but was found in both HCL patients with MAP2K2 mutations. In comparing evaluable patients with C121S (n=9) vs K57N (n=8) MAP2K1 mutations after 1st-line treatment, there was a trend for shorter median relapse free survival for C121S (RFS 4.0 vs 6.3 months, p=0.091) but no difference in OS (p=0.48). Five HCL patients with MAP2K1 mutations did not have high-risk features such as IGHV4-34 usage or an HCLv phenotype. Compared to 39 classic HCL patients with only BRAF V600E mutation after 1st line purine analog treatment, they had significantly shorter RFS (p<0.0001) and OS (p=0.0034). However, 3 of these 5 patients also had unmutated non-IGHV4-34 rearrangements. To investigate the impact of MAP2K1 mutations on leukemic cell behavior and to explore potential therapeutic strategies, we developed an ex vivo model using the Raji cell line. The four most common MAP2K1 mutations observed in HCL and HCLv, C121S, K57N, K57E, and K57T, were introduced into Raji cells using the TALEN gene editing method. Comparative analysis of the resulting stable mutant clones revealed differences in cell proliferation, gene expression profiles, and activation of kinases. These included MEK and ERK phosphorylation, which are directly involved in the MAPK signaling pathway, as well as other kinases such as CREB and AKT, suggesting crosstalk between MAPK and additional signaling pathways. We report the largest series to date of MAP2K1 mutations in HCL/HCLv and the first report of MAP2K2 mutations. HCL/HCLv patients with MAP2K1 mutations constitute a high-risk group associated with other known high-risk features including unmutated and/or IGHV4-34+ rearrangements and/or HCLv phenotype. B-cell lines edited to carry MAP2K1 mutations are being studied to test existing and new therapies for these high-risk mutations.

Chronic lymphocytic leukemia (CLL) cells may bear mutations in IGHV genes, the 2%-cutoff allowing to discriminate two subsets, unmutated (U)- or mutated (M)-CLL, with different clinical course. IGHV genes may also incorporate additional ongoing mutations, a phenomenon known as intraclonal diversification (ID). Here, through an original bioinformatic workflow for NGS data, we used the inverse Simpson Index (iSI) as diversity measure among IGHV sequences to dichotomize cases with different ID levels into IDhigh (iSI ≥ 1.2) vs. IDlow (iSI < 1.2) both in CLL (n = 983) and in other lymphoproliferative disorders (LPD; n = 127). In CLL, IDhigh cases accounted for 14.6%, overrepresented in M-CLL (P = 0.0028), while higher percentages were documented in GC-derived LPD. In M-CLL (n = 396), IDhigh patients (n = 69) experienced longer time-to-first treatment than IDlow patients (P = 0.015), and multivariate analyses (n = 299) confirmed ID as independent variable. IGHV gene mutations of IDhigh cases had molecular signatures indicating ongoing activity of the AID)/Polη-dependent machinery; consistently, IDhigh M-CLL expressed higher levels of AID transcripts than IDlow M-CLL (P = 0.012). In conclusion, we propose a robust NGS protocol to quantitatively evaluate ID in CLL, demonstrating that: i) all CLL patients presented ID although at various degree; ii) high degree of ID has clinical relevance identifying a M-CLL subset with better outcome.

Patients with the B-cell malignancy hairy cell leukemia (HCL) and the poorer-prognosis variant HCLv often receive anti-CD20 monoclonal antibodies (mAbs), which kill normal B cells, impairing humoral immunity. We measured COVID-19 antibodies after doses of COVID-19 vaccine in patients with HCL (n = 415) and HCLv (n = 32). After the second COVID-19 vaccine dose, spike antibody level most strongly correlated with normal B-cell levels (r = 0.365, P < .0001), followed by CD4+ T-cell count (r = 0.244, P = .0002), and was less related to immunoglobulin G level (r = 0.101, P = .14). Spike antibody also correlated with normal B cells after the third to fifth vaccine doses and with CD4+ count after the third dose. Normal B-cells were undetectable in 87% of patients within 6 months after the last dose of anti-CD20 mAb and were lower than in patients at 6 to 12 months (P = .0003), which, in turn, were lower than at 12 to 18 months (P = .0002). Infection with COVID-19 became more common after use of the third vaccine dose; spike antibody levels were higher in patients with prior infection (positive vs negative nucleocapsid antibodies; P < .0001). Spike antibodies decreased faster after ibrutinib or anti-CD20 mAb. We conclude that decreased levels of normal B cells in patients with HCL/HCLv, due to disease and/or anti-CD20 therapy, are associated with lower COVID-19 vaccination efficiency and such patients may not respond well to vaccines. The associated studies were registered at www.ClinicalTrials.gov as #NCT01087333 (HCL/HCLv) and #NCT04362865 (COVID-19).

e19015 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) was FDA-approved for relapsed/refractory hairy cell leukemia (HCL) but is now unavailable due to vial expiration until a company resumes development. The phase 3 complete remission (CR) rate was 41%, higher in patients with lower anti-drug antibodies (ADA). Minimal residual disease (MRD) eradication led to longer CR duration. HCL and the poorer prognosis variant HCLv strongly express CD22 and CD20. A clinical trial was done to determine if Rituximab could decrease immunogenicity by killing normal B-cells in HCL patients receiving Moxe and hasten MRD-free CR by reducing HCL tumor burden. Methods: To permit Rituximab enough time to reduce ADA and tumor burden, it was administered 3 days before cycle 1 day 1 at 375 mg/m2, and Moxe was given by 30-minute infusion days 1, 3 and 5. On subsequent 28-day cycles, patients received Moxe days 1, 3 and 5, and Rituximab prior to Moxe on day 1. Patients received up to 4 cycles past MRD-free CR, or up to 8 total cycles if MRD-free CR was achieved after cycle 4. The ADA assay determined percent neutralization by serum of the cytotoxicity of Moxe on CD22+ Raji cells. Results: After 13 patients received Moxe-Rituximab (MoxeR) without dose-limiting toxicity (DLT), meeting the phase 1 endpoint, 5 additional patients received Moxe with the biosimilar Ruxience (MoxeR). The first 3 patients received Moxe at 30 mcg/Kg/dose and subsequent patients 40 mcg/Kg/dose. All 18 were evaluable for toxicity and response. Although no DLT, one patient had transient grade 3 hemolytic uremic syndrome during cycle 3 without significant symptoms or need for therapy. Of the 18 patients, 15 (83%) responded, 14 (78%) achieved CR and 13 (72%) MRD-free CR by blood and bone marrow (BM) aspirate flow cytometry and BM biopsy immunohistochemistry. MRD-free CRs included one with HCLv. Even though Moxe vial expiration prevented enrollment of the 26 planned patients needed for a 1-sided p-value <0.025 compared to Moxe alone where 30 (47%) of 64 phase 1-3 patients achieved MRD-free CR, the 53% relative improvement with MoxeR achieved a 1-sided p-value of 0.05. Compared to 29 (48%) of 61 evaluable phase 1-3 patients who received Moxe alone with high ADA levels, 4 (29%) of 18 patients had high ADA levels to MoxeR (p=0.048). At 14-51 (median 35.3) months of follow-up, all but 2 of 13 MRD-free CRs are continuing, with relapse-free survival 14-45 (median 34.5) months. Conclusions: Despite enrolling slightly fewer patients than planned, MoxeR was safe and more effective than Moxe alone at achieving MRD-free CR, probably due to lower immunogenicity and faster reduction of HCL/HCLv tumor burden. Since non-Hodgkin’s lymphoma (NHL) cells from patients are sensitive to Moxe like HCL, MoxeR could be tested after NHL treatment to convert MRD+ to MRD-free CRs. Clinical trial information: NCT03805932 .

Background: Hairy cell leukemia (HCL) is an indolent B-cell malignancy typically expressing CD20, CD22, CD25, CD11c, CD103, CD123, annexin A1, and tartrate-resistant acid phosphatase (TRAP). Treatment with purine analog monotherapy is highly effective, leading to durable complete remission (CR). In contrast, HCL variant (HCLv), lacking CD25, CD123, annexin A1, and TRAP, achieves poor response to purine analogs, with partial responses in <50%, CRs in <10%, and poorer overall survival from diagnosis. Classic HCL is associated with the BRAF V600E mutation in >90% of cases, serving as both a diagnostic marker and target for class I BRAF inhibitors. In contrast, not only HCLv but also a fraction of immunophenotypically classic HCL cases lack BRAF V600E. Some of these cases exhibited unmutated IGHV4-34 immunoglobulin rearrangement, which is associated with mutations in MAP2K1 (MEK1) downstream of BRAF. Reports of non-V600E BRAF mutations have been rare, including 3 patients with D449E, F468C, or F595L, and 2 patients with both V600E and either S602T or W607L. Methods: Looking for unusual mutations, we performed whole exome sequencing (WES) and/or TrueSight Oncology (TSO500) in 213 HCL/HCLv patients, including 88 with HCLv and 21 with IGHV4-34+ HCL. The HCL/HCLv cells constituted >80% of B-cells and were partially purified using CD19 microbeads followed by positive fraction isolation. BRAF V600E was also tested by droplet digital PCR. Results: We found non-V600E BRAF genetic alterations in 18 patients. Surprisingly, all 18 patients had the classic rather than variant phenotype. Of the 18 patients, 12 had a single non-V600E BRAF mutation, and 6 had V600E along with 1 (n=5) or 2 (n=1) non-V600E BRAF co-mutations. These non-V600E mutations included missense mutations, deletions, and insertions located in exons 8, 12, and 15. None of the 19 non-BRAF mutations observed in the 18 patients had been previously reported in HCL/HCLv, some had been reported in other malignancies, and others were novel alterations found in the protein kinase domain. The most common non-BRAF mutation was p.N486_P490del (n=6), always in the absence of BRAF V600E. Those observed in 2 patients each included p.V487_T491del, p.V504_L505insGKT, p.S607P, and p.D587N. One patient had a V600D mutation, which has been sensitive to BRAF inhibition in melanoma. Initial treatment and relapse data was known for 15 patients. Unless patients received rituximab as part of initial purine analog (n=7, none relapsed), patients after purine analog monotherapy (n=8) had all relapsed (p=0.0002) and had inferior relapse-free survival (RFS, 19.9 months vs not reached, p=0.0004). RFS of these 8 patients after purine analog monotherapy was also inferior compared to a control group of 34 patients who received cladribine monotherapy and began prospective follow-up before any relapses (19.9 vs 271.8 months, p<0.0001). Compared to the 8 patients with non-V600E BRAF mutations treated with purine analog monotherapy, all of whom relapsed at 2.5-73 (median 19.8 months), in the control group 5 of 34 patients (p<0.0001) relapsed at 57-272 (median 98.6) months and 29 remain in remission at 25.0-225.8 (median 47.8) months of follow-up. Five of the 18 patients with non-V600E BRAF mutations had IGHV4-34+ HCL which has been associated with poor prognosis if unmutated. Two of these 5 had 100% and 3 had 97.49%, 97.72% and 98.18% homology to germline. One of these patients who had 100% homology began treatment with cladribine monotherapy and relapsed at 20 months, while the other 4 had initial treatment containing rituximab and have not relapsed. Conclusion: Non-V600E BRAF mutations were observed in patients with classic HCL immunophenotype, some of which were co-mutations in BRAF with V600E. In view of the inferior RFS in these patients when purine analog was not combined with rituximab, these mutations may constitute a higher risk for relapse or chemoresistance. While those with BRAF V600 mutations might be candidates for BRAF inhibition, those with other BRAF mutations represent an opportunity for development of other specific inhibitors, not only for HCL/HCLv, but also for other malignancies. We believe more patients with HCL should be tested for non-V600E BRAF mutations.

Background: Hairy cell leukemia is a B-cell malignancy characterized by pancytopenia, splenomegaly, and long-term remissions to purine analogs cladribine (CDA) and pentostatin, but late relapses occur, presumably from minimal residual disease (MRD). For nearly 35 years, CDA monotherapy has been standard 1st and 2nd line therapy. Other agents are increasingly being used in 2nd line, including concurrent or delayed rituximab. Randomized 1st line data indicate a trend for higher CR rate (100% vs 88%) and much higher MRD-free CR rate (97% vs 24%, p<0.0001) using cladribine with (CDAR) vs without (CDA) concurrent rituximab. Randomized 2nd line data are not reported, but prospective 2nd-line data showed high CR and MRD-free CR rates (100% and 64%, n=14) when rituximab was begun 4 weeks after 2nd-line cladribine. We completed a randomized trial of 2nd line CDAR vs CDA in 62 patients with once-relapsed HCL. Similar to the 1st line randomized trial, all patients could receive up to 2 courses of rituximab to eliminate MRD detected in blood, with delayed rituximab begun at least 6 months after beginning the previous course of CDA or rituximab. Patients and Methods: Patients with relapsed HCL after initial purine analog were randomized to CDAR (2nd line cladribine 0.15 mg/kg intravenously days 1-5 with 8 weekly doses of rituximab 375 mg/m2 begun on day 1) or CDA alone. MRD was assessed by blood and bone marrow aspirate (BMA) flow cytometry (FC, sensitivity 0.002% of cells) and bone marrow biopsy immunohistochemistry (IHC). If blood MRD was detected by FC after 6 months, patients could receive delayed rituximab, one 8-dose course for those after CDAR, and up to 2 courses for those after CDA. After beginning each course of CDAR, CDA or delayed rituximab, blood was evaluated every 3 months for a year, then every 6 months until 2.5 years, then annually. Bone marrow was evaluated at 1, 6, 18 and 30 months, and then every 2 years. CR required resolution of enlarged spleen and lymph nodes, morphologic resolution of blood and bone marrow disease, and resolution of cytopenias to neutrophils >1.5/nL, hemoglobin >11 g/dL, and platelets >100/nL, unless patients were MRD-free by all tests. Results: Sixty-two patients, each with 1 prior course of CDA (n=60) or pentostatin (n=2), were randomized to either CDAR (n=32) or CDA (n=30). At 4 weeks posttreatment, CR rates were 50% vs 20% (p=0.018), and MRD-free CR rates were 38% vs 0% (p=0.0001). By 6 months (prior to delayed rituximab), CR rates were 91% vs 73% (p=0.10) and MRD-free rates were 63% vs 3% (p<0.0001). With subsequent resolution of MRD without delayed rituximab, MRD-free CR rates after CDAR vs CDA improved to 72% vs 10% (p<0.0001). Before delayed rituximab, relapse-free survival (RSF) was not different for CDAR vs CDA, with relapses in 3 of 29 vs 5 of 22 from CR (median not reached vs 157 months, p=0.29), and 5 of 23 vs 1 of 3 from MRD-free CR (medians both not reached, p=0.86). A first course of delayed rituximab was given 6.5-104 (median 16.3) months after CDAR (n=9) and 6.2-44 (median 7.9 months) after CDA (n=23). After 1 course of delayed rituximab, CR was observed in 8 (89%) of 9 CDAR vs 21 (91%) of 23 CDA patients, and MRD-free CR in 2 (22%) of 9 vs 8 (35%) of 23. Ten patients received a 2nd course of delayed rituximab after CDA and 6-91 (median 56) months after the 1st delayed rituximab course, with CRs in 8 of 10 and MRD-free CR in 1 of 10. At 7.0-182 (median 113) months of follow-up after CDAR vs CDA including delayed rituximab, 100% vs 90% (p=0.11) achieved CR and 75% vs 60% (p=0.28) MRD-free CR, without significant differences in RFS for these CRs. Two after CDAR vs 4 after CDA (p=0.42) progressed and required alternative therapy, several of whom remain MRD-free after moxetumomab pasudotox with rituximab. Conclusion: In once-relapsed HCL, like untreated HCL, CDAR compared to CDA trended towards a higher CR rate and achieved a much higher MRD-free CR rate. However, delayed rituximab after CDAR and CDA improved rates and durations of response to similar levels. This differs from untreated HCL, where MRD-free CR remained higher after CDAR than after CDA even after delayed rituximab. It is possible that CDA has less synergy with rituximab once CDA is already used. Thus, CDA with delayed rituximab at >6 months if/when blood becomes MRD-positive is still reasonable 2nd line treatment of HCL. Delayed rituximab is now being tested to eliminate MRD in patients who achieve MRD-positive CR to MEK +/- BRAF inhibition.

BRAF V600E is the key oncogenic driver mutation in hairy cell leukemia (HCL). We report the efficacy and safety of dabrafenib plus trametinib in patients with relapsed/refractory BRAF V600E mutation-positive HCL. This open-label, phase 2 study enrolled patients with BRAF V600E mutation-positive HCL refractory to first-line treatment with a purine analog or relapsed after >= 2 prior lines of treatment. Patients received dabrafenib 150 mg twice daily plus trametinib 2 mg once daily until disease progression, unacceptable toxicity, or death. The primary endpoint was investigator-assessed objective response rate (ORR) per criteria adapted from National Comprehensive Cancer Network-Consensus Resolution guidelines. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Fifty-five patients with BRAF V600E mutation-positive HCL were enrolled. The investigator-assessed ORR was 89.0% (95% confidence interval, 77.8%-95.9%); 65.5% of patients had a complete response (without minimal residual disease [MRD]: 9.1% [negative immunohistochemistry of bone marrow {BM} biopsy], 12.7% [negative BM aspirate flow cytometry {FC}], 16.4% [negative immunohistochemistry and/or FC results]; with MRD, 49.1%), and 23.6% had a partial response. The 24-month DOR was 97.7% with 24-month PFS and OS rates of 94.4% and 94.5%, respectively. The most common treatment-related adverse events were pyrexia (58.2%), chills (47.3%), and hyperglycemia (40.0%). Dabrafenib plus trametinib demonstrated durable responses with a manageable safety profile consistent with previous observations in other indications and should be considered as a rituximab-free therapeutic option for patients with relapsed/refractory BRAF V600E mutation-positive HCL. This trial is registered at www.clinicaltrials.gov as #NCT02034110.

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.

Background: Investigation of the mutational status of the heavy chain variable region of the immunoglobulin (IGHV) genes entered the clinical practice in chronic lymphocytic leukemia (CLL), due to its clinical relevance as prognostic/predictive marker. Despite the canonical categorization in mutated (M) and unmutated (UM) IGHV, some CLL may exhibit evidence of IGHV clonal evolution through the introduction of new mutations, in a process known as intraclonal diversification (ID). In NGS era, a rigorous approach for ID evaluation is still lacking. Aims: To develop a bioinformatic workflow for ID quantification in CLL and evaluate its clinical impact. Methods: The study included 983 CLL patients (759 with Time-to-first treatment, TTFT) analyzed at the time of diagnosis/first presentation. Lymphotrack assay was used to generate IGHV NGS libraries and sequencing data were analyzed with an original error-suppression pipeline to avoid ID overestimation. The inverse Simpson Index (iSI) was calculated to evaluate ID; a value set at 1.2 was chosen to discriminate between samples with ID (intraclonal) from samples without ID (clonal). IGHV analysis (n=52) utilizing Unique Molecular Identifier (UMI) as gold-standard revealed a very close correlation in iSI scores between samples processed with our pipeline vs. the UMI-based protocol (22 cases with ID and 30 cases without ID according to both the analyses, Fig.A). The pipeline was validated in lymphoproliferative disorders with different ID levels, including 14 hairy cell leukemia (HCL), 28 diffuse large B cell lymphoma (DLBCL), 40 follicular lymphoma (FL), and 43 mantle cell lymphoma (MCL). Results: By applying our UMI-independent pipeline, most of DLBCL (68%) and FL (72%) revealed ID (Fig.B), according to their germinal center origin. Moreover, about 50% of HCL had ID, in keeping with the heterogeneity of the disorder (Fig.B), and as expected from the naïve B cell origin, only a minority of MCL (22%) had ID (Fig.B). Among 983 CLL (iSI range 1.0-20.4), 144 CLL (14%) had ID while 839 were without ID (Fig.B). Based on IGHV mutations, 508 and 475 cases were either M- or UM-CLL, respectively. By combining ID and IGHV status, we observed 422 UM-CLL without ID, 417 M-CLL without ID, 53 UM-CLL with ID, and 92 M-CLL with ID, with a significant overrepresentation of cases with ID among M-CLL without correlation with specific IGHV families and genes. Moreover, CLL cells with ID overexpressed Activation-Induced cytidine Deaminase (AID; P=0.027), an enzyme responsible for the somatic hypermutation process, and revealed AID-specific mutational signatures (WRC/GYW). Strikingly, M-CLL patients with ID had significantly longer TTFT respect to M-CLL patients devoid of ID (P=0.015; Fig.C). A multivariate analysis, carried out on M-CLL, identified ID classification as independent variable along with Rai Stage, CD49d, and del 11p/del 17p (Fig.D). RNASeq performed on 14 M-CLL (8 with ID vs. 6 without ID) revealed that M-CLL with ID upregulated gene pathways related to BCR downstream signalling, T/NK cell activation and cellular apoptosis/programmed cell death, while downregulating genesets associated to increased protein synthesis and transcription, in keeping with their indolent clinical behavior (Fig.EF). Summary/Conclusion: Here we report a novel UMI-independent method to assess ID in CLL. By applying this approach, we provide evidence that: i) ID prevalently affects M-CLL; ii) patients affected by M-CLL with ID have better outcome than M-CLL cases without ID; iii) ID identifies a subset of M-CLL with specific molecular/biological features and clinical characteristics.Keywords: IGH, Prognostic factor, Chronic lymphocytic leukemia

Although monoclonal antibodies to the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are known, B-cell receptor repertoire and its change in patients during coronavirus disease-2019 (COVID-19) progression is underreported. We aimed to study this molecularly. We used immunoglobulin heavy chain (IGH) variable region (IGHV) spectratyping and next-generation sequencing of peripheral blood B-cell genomic DNA collected at multiple time points during disease evolution to study B-cell response to SARS-CoV-2 infection in 14 individuals with acute COVID-19. We found a broad distribution of responding B-cell clones. The IGH gene usage was not significantly skewed but frequencies of individual IGH genes changed repeatedly. We found predominant usage of unmutated and low mutation-loaded IGHV rearrangements characterizing naïve and extrafollicular B cells among the majority of expanded peripheral B-cell clonal lineages at most tested time points in most patients. IGH rearrangement usage showed no apparent relation to anti-SARS-CoV-2 antibody titers. Some patients demonstrated mono/oligoclonal populations carrying highly mutated IGHV rearrangements indicating antigen experience at some of the time points tested, including even before anti-SARS-CoV-2 antibodies were detected. We present evidence demonstrating that the B-cell response to SARS-CoV-2 is individual and includes different lineages of B cells at various time points during COVID-19 progression.