MYD88 L265P Research Articles

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive but potentially curable lymphoma. Rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone (R-CHOP) is the standard first-line regimen with cure rates nearing 60%. Over the last years many efforts have been made aiming at improving outcomes of first line therapy but none has resulted in improved overall survival over R-CHOP. Recently, genetic subtypes of DLBCL that go beyond the cell-of-origin and have distinct biology, clinical characteristics and different likelihood of response to specific therapies have been identified. One of these entities is represented by the MCD subtype harboring either the MYD88 L265P mutation, the CD79A/B mutation, or both. MCD has a dismal outcome when treated with R-CHOP but is sensitive to BTK inhibition. In addition to the genetic definition of specific subtypes, the possibility to use circulating tumour DNA (ctDNA) response assessment together with PET-response may establishe a new strategy for treatment decisions in patients with DLBCL. Methods: SAKK 38/19 (NCT04604067) is an ongoing exploratory multicohort phase II trial in patients (pts) with previously untreated DLBCL not otherwise specified (NOS). Adult pts with CD20-positive DLBCL NOS, ECOG performance status 0–2, candidates for 6 cycles of R-CHOP are eligible. At baseline, a liquid biopsy is perfomed and pts with MCD subtype are treated with the combination of R-CHOP plus acalabrutinib for 6 cycles (cohort A). Non-MCD pts receive 2 cycles of R-CHOP and based on the combined PET and ctDNA response assessment after cycle 2 they are allocated to one of three different cohorts: R-CHOP for 4 additional cycles in combination with acalabrutinib followed by 2 months of acalabrutinib single-agent (cohort B); 2 additional cycles of R-CHOP and 2 administrations of single-agent rituximab (cohort C); 4 additional cycles of R-CHOP (cohort D). Co-primary endpoints of the trial are: Progression free survival (PFS) (in Cohorts A, C and D) and complete remission (CR) rate (in Cohort B) Secondary endpoints: Adverse events in the cohorts treated with acalabrutinib-R-CHOP; overall survival; PFS in cohort B; CR rate in cohorts A, C and D; overall response rate; duration of response. The research was funded by: -Astra Zeneca -Hubacher Fund Keywords: aggressive B-cell non-Hodgkin lymphoma, liquid biopsy, ongoing trials Conflicts of interests pertinent to the abstract A. Stathis Honoraria: Eli Lilly, Bayer, Roche, Novartis, Janssen Oncology, AstraZeneca Research funding: ADC Therapeutics, Pfizer, Roche, Novartis, Bayer, Eli Lilly, MEI Pharma, Cellestia, Debiopharm Group, Merck/MSD, Abbvie, Amgen, AstraZeneca, Incyte, Loxo, and Philogen F. Hitz Consultant or advisory role Abbvie, Takeda and Roche G. Gritti Consultant or advisory role Roche, Takeda, Kite-Gilead, Ideogen, Genmab Educational grants: Janssen, Beigene, Sandoz Other remuneration: Clinigen, Ideogen, Beigene, Incyte, Novartis T. Zenz Honoraria: Roche, Abbvie, AZD, Beigene, Janssen, Gilead, Novartis

Design, synthesis and pharmacological evaluation of 2,3-dihydrobenzofuran IRAK4 inhibitors for the treatment of diffuse large B-cell lymphoma

Interleukin-1 receptor associated kinase 4 (IRAK4) is a critical mediator of MYD88 L265P-induced NF-κB activation, indicating it is a promising therapeutic target for diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of 2,3-dihydrobenzofuran IRAK4 inhibitors through structure-based drug design. The representative compound 22 exhibited strong IRAK4 inhibitory potency (IRAK4 IC50 = 8.7 nM), favorable kinase selectivity and high antiproliferative activity against the MYD88 L265P DLBCL cell line (OCI-LY10 IC50 = 0.248 μM). Compound 22 also exhibited the ability to inhibit the activation of IRAK4 signaling pathway and induce apoptosis in MYD88 L265P DLBCL cell line. In combination with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, 22 showed enhanced apoptosis-inducing effect and antiproliferative potency. The most advanced compound 22 in this inhibitor series holds promise for further development into efficacious and selective IRAK4 inhibitors for the treatment of DLBCL.

IgM monoclonal gammopathy of undetermined significance: clinicopathologic features with and without IgM-related disorders.

A subset of patients with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) develop IgM-related disorders (IgM-RD) including peripheral neuropathy, cryoglobulinemia and/or cold agglutinin disease (CAD). We examined the clinical and bone marrow pathologic findings in 191 IgM MGUS patients (2016 World Health Oragnization criteria). Clonal plasma cells were identified in 41 of 171 (24%) cases by immunohistochemistry (IHC) and clonal B cells in 43 of 157 (27%). IgM-RD was identified in 82 (43%) cases, including peripheral neuropathy (n=67, 35%), cryoglobulinemia (n=21, 11%), and CAD (n=10, 5%). Cases of CAD showed distinctive features including lack of MYD88 mutations (P=0.048), supporting the concept of primary CAD as a distinct clinicopathologic disorder. Following exclusion of CAD, comparison of the remaining cases with (n=72) or without (n=109) IgM-RD showed IgM-RD to be more frequent in men than women (P=0.02) and to be more highly associated with MYD88 L265P (P=0.011). Cases with and without IgM-RD otherwise showed similar features including serum IgM concentrations, presence of lymphoid aggregates, clonal B cells by flow cytometry or clonal plasma cells by IHC. No differences were observed in overall survival between cases with and without IgM-RD. No cases in this series met criteria for plasma cell type IgM MGUS as defined in the 2022 International Consensus Classification of lymphoid neoplasms. These results show IgM-RD to be common in patients with IgM MGUS. While CAD shows distinctive features, the remaining cases of IgM-RD largely show pathologic findings similar to IgM MGUS without IgM-RD.

Ibrutinib response in a patient with refractory mixed essential cryoglobulinemia.

A 74-year-old male presented with fatigue, parasthesiae, Raynaud's phenomena, visual disturbances, and recurrent lower extremity rash (left). Investigations revealed an elevated serum cryoglobulin, rheumatoid factor >1320 IU/ml (ref. <15 IU/mL), undetectable C4, and C3 of 25 mg/dL (ref. 87–200 mg/dL). A small amount of IgM kappa monoclonal protein (M spike of 2.5%) and polyclonal IgG were identified by immunofixation. A diagnosis of mixed (Type II) cryoglobulinemia was made. Testing for Hepatitis B and C was negative. Positron emission/computed tomography (PET-CT) showed no evidence of lymphoma. Although the bone marrow appearance raised suspicion for Waldenstrom macroglobulinemia (WM), testing for MYD88 L265P mutation, which is commonly associated with this disorder, was negative. Of note, scattered CD20-positive B-cells comprised only ∼5% of bone marrow cellularity. The patient's rash gradually evolved into large ulcers over bilateral medial malleoli. Despite treatment with immunosuppression, rituximab, and plasmapheresis, they expanded to 9.3 × 8.9 × 0.3 cm on the left and 8.6 × 5.6 × 0.4 cm on the right (middle), requiring extensive use of opioids and significantly interfering with patient's mobility. A second bone marrow biopsy was again suggestive of WM. However, the frequency of abnormal B-cells was even lower (1.7%), likely reflecting prior therapy. Therefore, B-cells were purified by flow sorting using antibodies against CD19, CD20, and CD38 prior to molecular testing. Critically, this approach revealed the presence of MYD88 L265P mutation, supporting the diagnosis of WM and justifying therapy with Ibrutinib, a Bruton kinase inhibitor which is highly effective in this disease. Starting Ibrutinib 420 mg daily was a clear turning point in the patient's clinical course. After just a few days, he reported a drastic improvement in fatigue and leg ulcer pain. Within the following 2 months, he became pain-free and no longer required narcotics. By the end of the 9 months of therapy, the ulcers were completely healed (right) and quantitative IgM went down from 1058 to 325 mg/dL after the first 3 months. Nearly 2 years since starting the treatment, the patient is asymptomatic except for mild parasthesiae. He continues on Ibrutinib at a lower dose (280 mg daily). Cryoglobulinemia (CG) is a rare multisystem disorder which is caused by immunoglobulins with a capacity to aggregate at lower temperatures (cryoglobulins). Notably, even a small amount of cryoglobulin in the serum is sufficient to cause disease. Hence, a low-burden lymphoproliferative disorder that serves as the source of monoclonal cryoglobulin may be difficult to diagnose, precluding the use of targeted therapies. In agreement with a single prior report [1], our case shows that Ibrutinib can be remarkably effective in refractory CG due to WM. Unexpectedly, we found that despite a near-complete resolution of symptoms with Ibrutinib, the patient's cryoglobulins/IgM paraprotein persisted. In addition, the rheumatoid factor was still elevated, and C4 complement remained undetectable. This suggests that the therapeutic benefit of Ibrutinib may be explained not only by anti-tumor effect but also anti-inflammatory properties, as demonstrated by its efficacy in COVID [2], graft-versus-host disease [3], and autoimmune conditions [4]. Finally, our case shows that enriching for B-cells prior to molecular analysis may aid the diagnosis of occult lymphoproliferative disorder in other patients, for example those with IgM monoclonal gammopathy of clinical significance, who may also benefit from a trial of targeted therapy. H. Jamali wrote the manuscript. D. Wu performed cell sorting and molecular analysis. L. Soma performed morphological assessment of the bone marrow. M. Linenberger contributed to clinical management of the patient (plasma exchange). M. Wener provided rheumatology expertise and co-managed the patient with LS. L. Silberstein provided haematology expertise, co-managed the patient with MW, and wrote the manuscript. The authors have nothing to report. The authors received no specific funding for this work. None of the authors received financial compensation from an external source in return for writing or publishing this paper. The patient consented to the use of their clinical data and photographs for this correspondence.

380 A Rapid CSF Assay Accelerates Diagnosis and Treatment Initiation for CNS Neoplasms

INTRODUCTION: The low yields of CSF studies and serologies in current diagnostic workflows for suspected CNS neoplasms often lead to neurosurgical biopsies of high-risk lesions. To address this need, we developed an assay to rapidly identify hallmark mutations of CNS neoplasms in CSF. METHODS: Targeted Rapid Sequencing (TetRS) was developed as a qPCR-based method to detect IDH1/2, TERT promoter, BRAF, H3F3A, and MYD88 mutations in CSF-derived DNA within 80 minutes of collection. CSF was prospectively collected over 18 months from 98 patients with suspected new CNS neoplasms. Results were utilized in clinical decision-making. RESULTS: 40 of 98 patients were ultimately diagnosed with a neoplasm. TetRS detected a mutation in 15 out of 40 patients with a neoplasm (37.5%), including cases of CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma and H3K27M-mutant diffuse midline glioma. Detection of a mutation accelerated diagnosis in comparison to cases where a mutation was not detected (median 0.5 vs 10.5 days, p &lt; 0.00001). In multiple cases, a positive TetRS result obviated the need for biopsy and enabled initiation of disease-directed therapy. In one such case, detection of the MYD88 L265P variant was diagnostic of CNS lymphoma, enabling methotrexate and rituximab initiation within 3 days of admission. Results of repeated CSF samplings correlated with treatment response and recurrence. In another case, detection of the IDH1 R132G variant was diagnostic of IDH-mutant brainstem glioma, enabling IDH inhibitor and radiation therapy, with clinical and radiographic response. CONCLUSIONS: The targeted cell-free DNA panel for CSF presented here is a novel tool to rapidly, cost-effectively and broadly diagnose CNS neoplasia, initiate therapies and follow disease progression in the absence of neurosurgical tissue sampling.

Treatment of Patients with Waldenström Macroglobulinaemia: Clinical practice update from the Myeloma Foundation of Australia Medical and Scientific Advisory Group.

Waldenström Macroglobulinaemia (WM) is an indolent B-cell malignancy characterised by the presence of IgM paraprotein, bone marrow infiltration by clonal small B lymphocytes with plasmacytic differentiation, and the MYD88 L265P mutation in >90% of cases. Traditionally, WM has been treated with chemoimmunotherapy. Recent trials have demonstrated the efficacy and safety of Bruton's Tyrosine Kinase inhibitors (BTKi) in WM, both as monotherapy and in combination with other drugs. There is emerging evidence on use of other agents including BCL2 inhibitors and on treatment of rare presentations of WM. In this update, the Medical and Scientific Advisory Group ofMyeloma Australia review available evidence on treatment of WM since the last publication in 2017 and provide specific recommendations to assist Australian clinicians in the management of this disease. This article is protected by copyright. All rights reserved.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Rapid detection of the MYD88 L265P mutation for pre- and intra-operative diagnosis of primary central nervous system lymphoma.

The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is a disease-specific mutation of primary central nervous system lymphoma (PCNSL) among the central nervous system tumors. Accordingly, this mutation is considered a reliable diagnostic molecular marker of PCNSL. As the intra-operative diagnosis of PCNSL is sometimes difficult to achieve using histological examinations alone, intra-operative detection of the MYD88 L265P mutation could be effective for the accurate diagnosis of PCNSL. Herein, we aimed to develop a novel rapid genotyping system (GeneSoC) using real-time polymerase chain reaction (PCR) based on microfluidic thermal cycling technology. This real-time PCR system shortened the analysis time, which enabled the detection of the MYD88 L265P mutation within 15 min. Rapid detection of the MYD88 L265P mutation was performed intra-operatively using GeneSoC in 24 consecutive cases with suspected malignant brain tumors, including 10 cases with suspected PCNSL before surgery. The MYD88 L265P mutation was detected in eight cases in which tumors were pathologically diagnosed as PCNSL after the operation, while wild-type MYD88 was detected in 16 cases. Although two of the 16 cases with wild-type MYD88 were pathologically diagnosed as PCNSL after the operation, MYD88 L265P could be detected in all eight PCNSL cases harboring MYD88 L265P. The MYD88 L265P mutation could also be detected using cell-free DNA derived from the cerebrospinal fluid of two PCNSL cases. Detection of the MYD88 L265P mutation using GeneSoC might not only improve the accuracy of intra-operative diagnosis of PCNSL but also help the future pre-operative diagnosis through liquid biopsy of cerebrospinal fluid.

Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors.

Inhibitors of phosphatidylinositol 3-kinase (PI3K) and Bruton tyrosine kinase (BTK) represent a recognized option for the treatment of patients affected by indolent B cell lymphomas. However, small molecules as single agents show limited success in their ability in inducing complete responses, with only partial remission achieved in most patients, suggesting the need for combination therapies. IRAK4 is a protein kinase downstream of the Toll-like receptor signaling (TLR), a driver pathway of secondary tumor° resistance in both hematological and solid tumor malignancies. Activation of IRAK4 upon TLRs and IL-1 receptor (IL-1R) stimulation and through the adaptor protein MYD88 initiates a signaling cascade that induces cytokine and survival factor expression mediated by the transcription factor NF-κB. MYD88-L265P encoding mutations occur in diffuse large B-cell lymphomas, in lymphoplasmacytic lymphomas and in few marginal zone lymphomas (MZL). The IRAK4 inhibitor emavusertib (CA-4948) has shown early safety and clinical activity in lymphoma and leukemia patients. In this preclinical study, we assessed emavusertib effectiveness in MZL, both as single agent and in combination with targeted agents, with a particular focus on its capability to overcome resistance to BTK and PI3K inhibitors. We showed that the presence of MYD88 L265P mutation in bona fide MZL cell lines confers sensitivity to the IRAK4 inhibitor emavusertib as single agent. Emavusertib-based combinations improved the sensitivity of MZL cells to BTK and PI3K inhibitors, including cells with a secondary resistance to these agents. Emavusertib exerted its activity via inhibition of NF-κB signaling and induction of apoptosis. Considering the early safety data from clinical trials, our study identifies the IRAK4 inhibitor emavusertib as a novel compound to be explored in trials for patients with MYD88-mutated indolent B cell lymphomas as single agent and as combination partner with BTK or PI3K inhibitors in unselected populations of patients.

Waldenström Macroglobulinemia and Non-IgM-Type Lymphoplasmacytic Lymphoma Are Genetically Similar

Introduction: Waldenström macroglobulinemia (WM) represents a subset of lymphoplasmacytic lymphoma (LPL) with the immunoglobulin (Ig)M paraprotein. MYD88 L265P and CXCR4 mutations are common mutations in WM patients, and mutations in ARID1A and KMT2D (MLL2) have also been reported. However, little information has been accumulated on genetic changes in LPL with other paraproteins like IgG. Methods: We therefore aimed to evaluate genetic differences between WM and LPL with non-IgM paraprotein (non-IgM-type LPL) using targeted next-generation sequencing (NGS) in 20 Japanese patients (10 with WM, 10 with non-IgM-type LPL). Results: Mutations were detected in ARID1A (10%), CXCR4 (20%), MYD88 (90%), and KMT2D (0%) for WM patients and in ARID1A (10%), CXCR4 (20%), MYD88 (70%), and KMT2D (10%) for non-IgM-type LPL patients. No significant differences were identified. No mutations were detected in NOTCH2, PRDM1, CD274 (PD-L1), PDCD1LG2 (PD-L2), RAG2, MYBBP1A, TP53, or CD79B. Discussion: Mutant allele frequency in MYD88 L265P did not differ significantly between WM and non-IgM-type LPL. Most mutations detected by NGS were subclonal following MYD88 L265P, although one non-IgM-type LPL patient harbored only CXCR4 S338X mutation. Our NGS analyses reveal genetic characteristics in LPL patients and suggest genetic similarities between these two subsets of LPL, WM and non-IgM-type.

CD79B Y196 mutation is a potent predictive marker for favorable response to R-MPV in primary central nervous system lymphoma.

Rituximab, high-dose methotrexate (HD-MTX), procarbazine and vincristine (R-MPV), has significantly prolonged the survival of patients with primary central nervous system lymphoma (PCNSL), but predictive factors for response to R-MPV have not yet been investigated. Herein, we investigated the correlation of MYD88 L265P and CD79B Y196 mutations, which are the most frequently found molecular alterations in PCNSL, with prognosis of patients with PCNSL treated with R-MPV. We investigated the long-term clinical course and status of MYD88 and CD79B genes in 85 patients with PCNSL treated with R-MPV or HD-MTX treatment, and the correlation of these genetic mutations with prognosis. R-MPV achieved an excellent tumor control rate (61.6% and 69.9% of 5-year progression-free and overall survival rates, respectively). While MYD88 L265P mutation had no significant effect on survival, patients with CD79B Y196 mutations exhibited prolonged survival (p < 0.05). However, the association of CD79B Y196 mutation with a better prognosis was not observed in the HD-MTX cohort, which indicated that CD79B Y196 mutation was a predictive marker for a favorable response to R-MPV. Furthermore, we established an all-in-one rapid genotyping system for these genetic mutations. In conclusion, CD79B Y196 mutation is a potent predictive marker for favorable response to R-MPV in PCNSL. The rapid identification of MYD88 L265P and CD79B Y196 mutations can be helpful not only for the accurate molecular diagnosis of PCNSL but also for the prediction of response to R-MPV.

Unique Presentation of Bing-Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia.

Unique Presentation of Bing-Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia.

Detection of Cell Free Tumor DNA in Plasma of Patients with Large B-Cell Lymphoma of the Sanctuary Sites By Digital Droplet PCR

Background: Primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL) are rare mature B-cell lymphoproliferative diseases that arise at immunological sanctuary sites. Cytomorphology (CM) and flow cytometry (FCM) analysis of spinal fluid is diagnostic in about 15% of PCNSL. In the remaining patients, a tumor biopsy is necessary, which carries the risk of complications. Response assessment in PCNSL and PTL relies on imaging studies. However, MRI scans and 18F-FDG PET-scans often cannot differentiate between active tumor and scar tissue. Hence, there is a need for non-invasive tools to aid in diagnosis and help in response assessment. PCNSL and PTL are characterized by a high incidence of hotspot mutations in MYD88 and CD79B. Detection of these mutations in cell free DNA (cfDNA) might aid in diagnosis and follow-up of disease activity in patients with PCNSL and PTL. Digital droplet PCR (ddPCR) offers a robust inexpensive tool with a fast turnaround time allowing sensitive detection of such hotspot mutations. Methods: Plasma was available for a total of 26 patients (20 PCNSL and 6 PTL) in the OncoLifes biobank of the University Medical Center Groningen. All patients gave informed consent. Corresponding tumor samples were available for 17 cases (14 PCNSL and 3 PTL). A ddPCR was performed for MYD88 L265P and in case of sufficient cfDNA also for CD79B Y196X (Bio-Rad Laboratories, Hercules, USA). The ddPCR Assay for CD79B Y196X targets the mutation Y196H c.586T>C, Y196D c.586T>G, Y196S c.587A>C, Y196C c.587A>G, and Y196F c.587A>T. The cut off value for the allele frequency (AF) used was ≥0.5%. Results: The MYD88 L265P and CD79B Y196X mutations were detected in 9/14 (64.2%) and 2/13 (15.4%) of the PCNSL tissue samples. The two CD79B Y196X positive tissue cases had a concomitant MYD88 L256P. The MYD88 L265P mutation was detected in 2/20 (10%) cfDNA samples. The CD79B Y196X mutation was detected in 1/19 (5.2%) cfDNA samples; remarkably, this mutation was not detected in the corresponding tissue sample. In PTL, MYD88 L265P mutations were detected in 3/3 of tumor tissues and a CD79B Y196X mutation was detected in 1/2 of tumor tissues, which co-occurred with the MYD88 L265P. In PTL, 2/6 (33.3%) and 1/6 (16.6%) cfDNA samples were positive for MYD88 L265P and CD79B Y196X. Overall, hotspot MYD88 and/or CD79B mutations were detected in cfDNA in 5 /26 cases (18.4%) (Figure 1). Figure 1. ddPCR results of MYD88 and CD79BConclusion: We explored the feasibility of using a combination of two common hotspot mutations in MYD88 and CD79B in PCNSL and PTL plasma samples to aid in diagnosis and disease monitoring. The data presented show that the value of ddPCR for hotspot mutations in plasma of PCNSL and PTL patients is limited. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Progression Risk and Long-Term Survival Trends of 915 Patients with Asymptomatic IgM Monoclonal Gammopathy

BACKGROUND: Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with a serum monoclonal IgM. Early asymptomatic stages include IgM monoclonal gammopathy of undetermined significance (MGUS) and smoldering WM (SWM). While most IgM MGUS patients (pts) will not progress during follow-up, a greater proportion of SWM pts will do. Most prognostic models identify high-risk SWM; however, they fail to characterize IgM MGUS and low-risk SWM. Moreover, data is scarce regarding how IgM MGUS and SWM survival trends have evolved during the last decades. We wondered whether inclusion of new biomarkers could improve progression risk models using the largest cohort of IgM MGUS and SWM reported. Here, we describe a comprehensive proposal to model progression risk and give more insight into the survival trends of IgM MGUS and SWM using retrospective data from 13 Spanish hospitals. METHODS: Diagnostic criteria were defined by the Second International Workshop. MYD88 L265P was analyzed by allele-specific or digital droplet polymerase chain reaction. Progression free survival (PFS) was calculated from the date of diagnosis to last follow-up or disease progression (symptomatic WM, other non-Hodgkin lymphoma or AL amyloidosis). Overall survival (OS) was calculated up to death from any cause. To find the best model, we first used a backward stepwise regression with meaningful variables as continuous. Also, a logistic and lasso regression (least shrinkage and selection operator) selected important variables that predicted progression at 10 years. We then categorized continuous data according to Liu and Youden methods. Relative survival (RS) was calculated using the Ederer II method leveraging the Human Mortality Database. RESULTS: 915 pts (462 IgM MGUS and 453 SWM) were included. Median age at diagnosis was 70 years. 102 (11%) pts were diagnosed before the year 2000. MYD88 L265P (available in 465 pts) was detected in 107 (54%) IgM MGUS and 217 (81%) SWM pts. After a median follow-up of 6 years, disease progression was documented in 250 pts (16% in IgM MGUS and 39% in SWM). The incidence rate of progression per 100 person-years was 2.9 (95% confidence interval [CI] 2.4 - 3.7) and 7.0 (95% CI 6.1 - 8.2) in IgM MGUS and SWM, respectively. Continuous (serum IgM, IgG, IgA, albumin, β2-microglobulin, bone marrow infiltration [BM]) and categorical (MYD88 L265P) candidates to clinical significance were fitted into a backward stepwise regression. Low IgG (hazard ratio [HR] 0.92; p=0.03), high IgM (HR 1.03; p<0.01), high BM infiltration (HR 1.02; p=0.01), and low albumin (HR 0.37; p<0.01) were selected. As the median follow-up of our series was 6 years, we also performed lasso to select predictors of progression at 10 years. Here, all previous variables were selected plus low IgA. We then chose the 4 best predictors between the two methods (BM infiltration, IgM, decreased IgG/IgA, and albumin). To fit into an easy-to-use model, we categorized continuous data as meaningful for clinicians. Albumin (<3.5 g/L) and IgG/IgA (<6.8/0.6 g/L) were categorized below the normal values; BM infiltration (≥ 20%) and IgM (≥ 10 g/L) cutpoints were established as previously described. 553 pts were introduced in the final model. Each variable was assigned a point so that low (0-1), intermediate (2) and high-risk (3-4) groups were able to discriminate pts. 84% of IgM MGUS pts were categorized as low-risk, while 57% of SWM were intermediate or high-risk. Regarding OS, there were no differences between IgM MGUS and SWM; however, each stage clearly differed from that of series of symptomatic WM pts from the same hospitals (p<0.01) (Figure 1A). Regarding survival trends, IgM MGUS had a RS slightly higher than that of the matched general population during the first 10 years of diagnosis, with a subsequent decline especially at 20 years. SWM had a RS lower than the general population, with a difference observed from the first 10-15 years of diagnosis (Figure 1B). CONCLUSION: We developed a new single ready-to-use risk model accounting for both IgM MGUS and SWM. Most IgM MGUS pts were categorized in the low-risk group, while SWM accounted for the vast majority of intermediate and high-risk groups. There were no differences in OS between IgM MGUS and SWM. However, RS of IgM MGUS was higher in the first 10 years from diagnosis, probably due to both early access to public health and follow-up, while SWM showed a constant decreasing RS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Rapid Genotyping Assay for CSF Accelerates Diagnosis and Treatment Initiation for CNS Malignancies

The overlapping clinical and radiographic features of CNS neoplasms, combined with low yields of conventional CSF studies, complicate diagnostic workflows and delay treatment initiation. Neurosurgical biopsies of high-risk lesions are frequently required, and decisions regarding extent of surgical resection are challenging. In order to address these challenges, we previously developed the Targeted Rapid Sequencing (TetRS) assay to detect highly recurrent point mutations characteristic of CNS neoplasms from CSF, within 80 minutes of sample collection. This qPCR-based panel offers parallel detection of MYD88, TERT promoter, IDH1/2, BRAF and H3F3A point mutations. In this study, we describe the clinical performance and real-time utilization of this assay in a large, single-center prospective patient sample. We asked whether rapid point mutation detection from CSF liquid biopsies can (1) accelerate times to diagnosis and (2) provide a basis for safely initiating disease-directed therapies in the absence of solid tissue sampling. We collected CSF from 98 patients with new CNS lesions suspicious for neoplasms over an 18-month period. Patient enrollment, selection of specific assay variants, and utilization of results in clinical decision-making were at the discretion of providing physicians. IRB approval was obtained. In this diverse cohort, 40 of 98 patients ultimately established a diagnosis of a CNS neoplasm. A point mutation was detected by TetRS in 15 out of 40 patients with a neoplasm (37.5%); this included cases of primary and secondary CNS lymphoma (PCNSL), glioblastoma, IDH-mutant brainstem glioma and H3K27M-mutant diffuse midline glioma. In time-to-event analyses, we found that detection of a mutation by TetRS accelerated the return of diagnostic information from CSF sampling when compared to contemporaneous and historical control cases in which a mutation was not detected (median 0.5 vs 10.5 days, P<0.01). We found multiple cases in which mutation detection by TetRS helped to avoid neurosurgical biopsy. In one representative case, detection of the MYD88 L265P mutation established the diagnosis of PCNSL; methotrexate and rituximab were initiated within 3 days of CSF sampling. In this patient, TetRS results over serial CSF samplings correlated with treatment response and detecting disease recurrence. In another representative case, a lesion initially thought to be an H3K27M-mutant diffuse midline glioma on clinical and radiographic grounds was instead diagnosed as an IDH-mutant brainstem glioma based on TetRS detection of the IDH1 R132G mutation in CSF. This enabled rapid initiation of fractionated radiation, as well as targeted IDH inhibitor therapy; the patient experienced clinical and radiographic response over 10 months of follow-up. TetRS thus represents a novel, targeted point mutation panel that can rapidly and specifically diagnose a broad array of CNS neoplasms. We demonstrate the potential of this novel tool to accelerate diagnosis, follow treatment response and detect recurrence in selected patients without solid tissue sampling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Autochthonous Mouse Models of Serve As Preclinical Tools to Investigate Pathogenesis and Treatment of MCD/C5 DLBCL

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Both, biologically and clinically, DLBCL represents a highly heterogeneous disease. Based on gene expression profiling DLBCL is subdivided into germinal center B cell (GCB)-like and non-GCB- or activated B cell (ABC)-like DLBCL, a classifier which distinguishes DLBCL sub-entities displaying distinct biological features and clinical response to frontline anthracycline-based chemo-immune therapy. Plasmablasts have long been regarded as the cell of origin (COO) of ABC DLBCL, however, recent evidence points towards aberrant memory B cells (MB) as the true COO of these lymphomas. Several recent studies attempted to give structure to the genetic heterogeneity observed in DLBCL by clustering cases based on their mutational profiles. Clusters MCD by Schmitz et al. and C5 by Chapuy et al. are amongst the clusters enriched for ABC-DLBCL cases with particularly inferior prognosis. Both clusters are defined by mutations in MYD88 and CD79B as well as high levels of BCL2 expression and recurring mutations in transcriptional regulators of the plasmacytic differentiation process (PRDM1, SPIB). We previously reported an autochthonous mouse model of DLBCL mimicking hallmark mutations of MCD/C5 DLBCL: Combination of Myd88 p.L252P (the murine orthologue of human MYD88 L265P) and overexpression of BCL2 leads to the generation of ABC-DLBCL-like lymphomas in the Myd88p.L252P/wt;R26LSL.BCL2/wt;Cd19Cre/wt (MBC) mouse line. MBC Lymphoma cells show a plasmablastic immunophenotype with expression of CD138 and lack of expression of B220/CD19. In an attempt to refine our genetically-engineered mouse model, we incorporated other key alterations of MCD/C5 DLBCL in the MBC background: B cell specific loss of PRDM1, overexpression of SPIB and hallmark mutations in CD79B. Young Prdm1fl/fl;Myd88p.L252P/wt;R26LSL.BCL2/wt;Cd19Cre/wt (PPMBC) and Myd88p.L252P/wt;R26LSL.BCL2/LSL.Spib;Cd19Cre/wt (SMBC) compound animals display a strong reduction of the CD138+ population compared to control, accompanied by a decrease in serum immunoglobulins, in agreement with a block in plasma cell differentiation mediated by these additional genetic alterations. In contrast, cells with a marker profile of age-associated B cells (ABC, B220+/CD19+/CD21-/CD23-) as well as Memory B cells (MB, B220+/IgD-/Fas-/CD38+) accumulate markedly in all lymphoma lines, but particularly in PPMBC animals, compared to control. This is in line with recent studies hinting at ABC/ MB as the cells of origin of ABC-DLBCL. While MBC lymphomas are largely CD138+ and represent a plasmablastic/-cytic differentiation stage, PPMBC tumors express B-cell markers B220 and CD19 and lack expression of CD138. Additionally, PPMBC lymphomas frequently show a marker profile reminiscent of memory B cells, while CD21/CD23 - negativity suggests a relation to age-associated B cells, a population enlarged also in the pre-malignant setting. PPMBC tumors furthermore show an enrichment for B-cell receptor (BCR), NFkB and PI3K signaling gene signatures, when compared to MBC lesions. These signs of enhanced B cell receptor signaling translate into an increased sensitivity towards inhibition of Bruton's Tyrosine Kinase (BTK) by ibrutinib. The sensitivity of ibrutinib is further enhanced in lymphomas additionally expressing Cd79b p.Y195H, both in the Prdm1-deficient and the Prdm1-proficient model systems. We have established and characterized novel refined autochthonous mouse models of MCD/C5 DLBCL which serve as a preclinical tool to test novel therapeutic strategies in this sub-entity and pave the way to a deeper understanding of molecular mechanisms of pathogenesis, treatment response and resistance in highly aggressive DLBCL.

Pembrowm: Results of a Multi-Centre Phase II Trial Investigating the Safety and Efficacy of Rituximab and Pembrolizumab in Relapsed/Refractory Waldenström's Macroglobulinaemia

Background Waldenström's macroglobulinemia (WM) is a rare low grade B-cell NHL, with generally excellent outcomes underpinned by treatment with Rituximab combination therapies and covalent BTK inhibitors (cBTKi). However, these options are not curative and there are very little data to guide treatment options or a clear understanding of prognosis of relapsed/refractory (R/R) WM post cBTKi. PD-1 inhibition has been shown to be highly effective in classical Hodgkin lymphoma, and soluble PD-1 ligands have been implicated in T-cell regulatory function in the WM microenvironment. There is therefore a clear rationale to investigate the efficacy of PD-1 blockade in WM. Aim To determine the safety, tolerability and efficacy of pembrolizumab in combination with rituximab for the treatment of relapsed/refractory WM following prior cBTKi exposure. Methods PembroWM is a phase II, non-randomised, single arm, open label NCRI UK-wide study (NCT03630042) enrolling patients (pts) with R/R WM who had received 1 or more prior lines of treatment, who were not refractory to rituximab based therapy (progressing on or relapsing within 6 months of a rituximab containing regimen). Eligible pts accross 6 sites received rituximab 375mg/m2 weekly for 4 weeks, then 3 monthly (total of 8 doses) and pembrolizumab 200mg IV on a 3-weekly cycle, for a maximum of 18 cycles. The primary end point was the percentage of pts achieving an overall response rate (ORR) of greater than 25% reduction in serum IgM at 24 weeks post treatment. Secondary end points included safety and tolerability, response rates (CR/VGPR) , progression free survival (PFS) and overall survival (OS) at 1 year. Disease response was assessed with serum paraprotein/immunofixation, BM and CT imaging at 24 weeks and 1 year using the International Working Group for WM response criteria. The trial required recruitment of 42 patients with a 55% response rate to meet the primary end point. The trial closed early due to low recruitment rate with a total of 17 patients with a revised response requirement of 9 patients to meet primary end point. Results 17 patients were registered with median age of 70 years (range 43-84), 83% were male, 94% had a PS of 0-1, and the median number of lines of previous treatment was 3 (range 1-6). The majority of patients had a cBTKi as their last treatment prior to enrollment (only 2 patients were BTK naïve). 8 patients were positive for the MYD88 L265P mutation, and 4 patients had mutations in BTKC481. Median IgM levels were 40 g/L (range 2.5-72g/L) with 53% of patients exhibiting WM cells in bone marrow and extra nodal disease in 6%. Median follow up is 15.1 months (0.5 - 24.9). ORR at 24 weeks post treatment was 47.1% (60% CI 34.5-60%) with 6% attaining VGPR, 18% PR, 18% MR and in 6% response was unavailable but there was evidence of MR at week 12. 47% were non-responders; 35% progressed, 12% had stable disease and one patient withdrew during cycle 1 so was non-evaluable. ORR at 52 weeks post treatment was 36.4% (95% CI 10.9-69.2%) with 9% obtaining a VGPR, 27% PR and nil achieving CR. Median PFS was 12.6 months (95% CI 3-n/a) and median OS was not reached. 5 patients completed 18 cycles of treatment. 6 progressed, 3 withdrew due to AE, 2 withdrew consent and one withdrew to an unrelated clinician decision. The AE leading to withdrawal were immune thrombocytopenia (one), and infection in the other two (including one COVID-19 infection) The majority of adverse events were grade 1-2 with the commonest events involving anaemia (29%), fever (29%), infusion related reactions (35%) and raised creatinine (29%)., There were only 2 grade 3 infections. 1 patient experienced an embolic stroke and 1 patient died from respiratory failure; both events were considered unrelated to treatment. Conclusions PEMBROWM is the first study to investigate checkpoint inhibition in WM, and demonstrates the safety and tolerability of the combination of pembrolizumab and rituximab, with encouraging efficacy in a heavily pre-treated population. The recruitment challenges highlighted the combination of reduction in research activity during the COVID-19 pandemic, the difficulties of recruiting to an investigator-led study in a rare indication and the timing of availability/effectiveness of cBTKi in WM in the United Kingdom. A randomised study would be required to evaluate the additional benefit that PD-1 blockade is adding to rituximab monotherapy in a highly relapsed/refractory patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disparities in Somatic Mutations and Outcomes in Primary Central Nervous System Lymphoma Comparing Patients of Hispanic and Non-Hispanic Ethnicity

Background: Primary central nervous system lymphoma (PCNSL) is characterized by frequent MYD88 L265P activating mutations, biallelic CDKN2A loss, and mutations in CD79B, PIM1, and CARD11. However, the extent to which mutations vary by ethnicity is unknown. The purpose of this study is to describe differences in genetic mutations and survival between PCNSL patients of different ethnicities. Methods: We retrospectively analyzed patients ≥ 18 years with PCNSL diagnosed between January 2011- December 2020 and treated at UCSF-Fresno and University of Oklahoma. Next generation sequencing was performed on Formalin Fixed Paraffin Embedded tissue. We examined mutations in 434 genes by DNA analysis and 1408 genes by RNA analysis. Sequencing was performed using an Illumina NextSeq 550 system platform. The clinical data including age, sex, ethnicity, race, ECOG performance status, number and location of lesions, HIV status, LDH, protein in the CSF, and treatment regimens were reviewed. Treatment response was assessed by MRI using the international consensus group response criteria. Comparison of patient characteristics and rate of genetic alterations between Hispanic and Non-Hispanic patients was done using the Fisher exact test or Chi-square test. Time-to-event analyses were conducted using the Kaplan-Meier method and were compared by a log-rank test. Univariate analyses were performed via the Cox proportional hazards model. Analysis was performed with STATA version 16.0. Results: Among the 51 patients analyzed, the median age was 61. 54% were males and 43% were Hispanic. 13% had HIV, 27% had deep brain involvement, and 58% had non-germinal center (non-GC) type per Hans Criteria. 81% of Hispanic patients had non-GC vs 41% non-Hispanic (P=0.006). There was no statistically significant difference in age, performance status, HIV status, deep brain involvement, or CSF involvement between Hispanic non-Hispanic patients. 90% of patients received systemic chemotherapy for initial treatment, with 41% receiving high dose Methotrexate, Rituximab and Temozolomide. No patients received autologous stem cell transplant in first remission. 90% Hispanic Vs 89% Non-Hispanic received systemic chemotherapy (P=0.447). 29 patients (56%) achieved complete response (CR)/unconfirmed complete response (CRu). 2-year progression-free survival (PFS) was 67.4%, and 2-year overall survival (OS) was 55% for all patients. 177 genetic alterations were detected with the most common being MYD88, CD79B, TET2, PIM1, ETV6 and TP53 (table). Hispanic patients, compared with non-Hispanic patients, had a lower rate of MYD88 mutation (40.9% vs 72.4%, P=0.024) and TP53 mutation (0.0% vs 24.1%, P=0.001). 2-year PFS in Hispanics was 40% vs 30% in non-Hispanics (P=0.526) and 2-year OS in Hispanics was 0.63% vs 0.33% in non-Hispanics (P=0.05). In univariate analysis, age and genetic alterations (MYD88, CD79B, TET2, PIM1 and ETV6) were not associated with significant differences in 2-year PFS or OS. Performance status (ECOG ≤ 2), negative CSF cytology, and treatment with multi-agent chemotherapy rather than high dose Methotrexate alone were associated with better 2-yr OS. Conclusions: We identified highly recurrent genetic alterations in PCNSL. Our data suggest that some heterogeneity in the most frequent mutations in PCNSL may be related to ethnicity. Hispanic patients in our study have better 2-year OS; however, the data is limited by the retrospective analysis and the small patient number. Further studies on a larger patient population are warranted to evaluate genetic heterogeneity in different ethnic groups and effect of ethnicity and socio- economic status on outcomes in patients with PCNSL Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BIOM-58. ROLE OF CD79B Y196 MUTATION IN PCNSL AS A POTENT PREDICTIVE MARKER FOR FAVORABLE RESPONSE TO R-MPV TREATMENT AND DEVELOPMENT OF RAPID ALL-IN-ONE GENOTYPING SYSTEM FOR MOLECULAR MARKERS OF PCNSL

Abstract Although high-dose methotrexate (HD-MTX)-based chemotherapy, such as rituximab, HD-MTX, procarbazine and vincristine (R-MPV) regimen, has significantly prolonged the survival of patients with primary central nervous system lymphoma (PCNSL), predictive factors for response to R-MPV have not yet been investigated. Then, we investigated the correlation of MYD88 L265P and CD79B Y196 mutations, which are the most frequently found molecular alterations in PCNSL, with prognosis of patients with PCNSL treated with R-MPV. 47 and 21 patients with PCNSL treated with R-MPV and HD-MTX respectively were included, and correlation of their MYD88 and CD79B status with prognosis was analyzed. R-MPV achieved an excellent tumor control rate (61.6% and 69.9% of 5-year progression-free and overall survival rates, respectively). Among the 47 patients, 36 harbored MYD88 L265P or CD79B Y196 mutations. While the MYD88 L265P mutation had no significant effect on survival, patients with CD79B Y196 mutations exhibited prolonged survival (P &amp;lt; 0.05). However, the association of the CD79B Y196 mutation with a better prognosis was not observed in the HD-MTX cohort, which indicated that the CD79B Y196 mutation was a predictive marker for a favorable response to R-MPV. Furthermore, we established an all-in-one rapid genotyping system for these genetic mutations. This system enabled the detection of these genetic mutations in a small amount of biopsy samples within 90 min. In conclusion, we revealed that the CD79B Y196 mutation is associated with a good response to R-MPV, and the rapid and accurate genotyping system for MYD88 L265P and CD79B Y196 mutations that we developed in this study will enable reliable rapid molecular diagnosis and early prediction of response to R-MPV, which might help to determine the best treatment strategy for PCNSL.

EXTH-93. THE IRAK-4 INHIBITOR EMAVUSERTIB (CA-4948) FOR THE TREATMENT OF PRIMARY CNS LYMPHOMA

Abstract BACKGROUND PCNSL is an aggressive brain tumor accounting for 3% of all CNS malignancies and is associated with poor prognosis. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients cannot tolerate this intensive therapeutic intervention. Novel treatments are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK-4 that demonstrates clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. HYPOTHESIS: Emavusertib has anti-tumor activity against preclinical PCNSL. METHODS Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Survival responses in preclinical PCNSL models were measured in response emavusertib treatment, along with in vivo tumor proliferation and downstream biomarker expression by multi-parameter IHC. RESULTS Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We show that emavusertib can achieve therapeutically relevant concentrations in the brain and CSF. Emavusertib impairs tumor cell proliferation in vitro and in vivo, and shows dose-dependent single-agent activity in preclinical PCNSL models. We confirm decreased ERK1/2, MAPK and NF-κB activation, indicative of downstream IRAK-4 inhibition. CONCLUSIONS Our data supports that emavusertib demonstrates anti-tumor activity in the CNS space, warranting further preclinical and clinical evaluation of this agent for the treatment of PCNSL.