MYD88 L265P Mutation Research Articles

High Prevalence of MYD88 and CD79B Mutations in Primary Sinonasal Diffuse Large B-Cell Lymphoma: Identification of an MCD-like Subtype.

Primary sinonasal diffuse large B-cell lymphoma (PSDLBCL) is a rare aggressive lymphoma. Recently, genetic classification using Next Generation Sequencing (NGS) demonstrated that PSDLBCL largely consists of the MCD genotype, which has a poor prognosis mainly driven by MYD88 L265P and CD79B gene abnormalities. This study investigated the prevalence and clinicopathological significance of MYD88 L265P and CD79B Y196 mutations using droplet digital PCR in 55 patients with PSDLBCL, as well as the translocation of BCL2/BCL6/c-Myc with FISH. We found mutations in MYD88 L265P (29/55, 52.7%) and CD79B Y196 (20/55, 36.4%). The MCD-like subtype, defined by the mutation of MYD88 and/or CD79B, was found in 32 out of 55 cases (58.2%). This subtype largely consists of non-GCB type (31/32, 96.9%; P<0.01) and double-expressor cases (20/32, 62.5%; P=0.01) compared with the MYD88/CD79B co-wild type, with BCL6 translocation in a small subset (2/32, 6.3%) and no translocations of BCL2 (0/32) or c-Myc (0/32). The MCD-like subtype tended to relapse in specific sites such as the central nervous system, testis, and/or skin compared with the co-wild type (P=0.03), showing poorer outcomes in overall survival (P=0.02) and progression-free survival (P=0.01). In conclusion, our study highlights a high prevalence of MYD88 and CD79B mutations in PSDLBCL, identifying an aggressive MCD-like subtype with a distinct relapse pattern. This molecular subclassification can be helpful for both prognostic prediction and therapeutic strategy in patients with PSDLBCL.

Impact of chromosomal aberrations detected by chromosome banding analysis in symptomatic Waldenström's macroglobulinemia.

The clinicopathologic features and prognostic impact of MYD88 L265P (MYDL265P) and CXCR4 mutations (CXCR4Mut) have been well reported, although little is known regarding the impact of chromosomal aberrations (CA) detected by chromosome banding analysis (CBA) in symptomatic Waldenström's macroglobulinemia (sWM). Thus, we investigated the clinicopathologic features and prognostic impact in sWM with CAs identified by CBA. We retrospectively analyzed the clinicopathologic results and genomic alterations by droplet digital PCR, fluorescence in situ hybridization (FISH), and CBA using the G-banding method in bone marrow samples from sWM patients collected between April 2010 and March 2024 at our institute. The relationship between CAs and clinicopathologic features was evaluated, as well as the time to next treatment (TTNT). Thirty-five patients were enrolled. The median age was 71 years, and the median hemoglobin level was 10.1g/dL. The median serum IgM and M-protein levels were 3,009mg/dL and 2.95g/dL, respectively. MYDL265P was found in 30/35 patients (85.7%), whereas CXCR4Mut was found in 3/35 patients (8.6%). Deletion 6q identified by FISH in 5/18 patients (28%), and CAs using CBA in 9/34 patients (26%), including 4/34 (12%) complex karyotypes. sWM with CAs had more anemia (p = 0.04) and hypoalbuminemia (p = 0.007), in addition to higher serum M-protein and IgM levels (p = 0.03). With a median follow-up of 73 months, the median TTNT in patients with and without CAs was 27 and 68 months, respectively. CAs with CBA may be associated with clinical aggressiveness and shorter TTNT in sWM.

A Diagnostic Challenge in the Biopsy of a Mammographic Abnormality

Abstract Introduction/Objective Primary breast marginal zone lymphoma (MZL) is an uncommon subtype of extranodal lymphoma, and its diagnosis presents challenges, particularly in distinguishing it from lymphoplasmacytic lymphoma (LPL). MYD88 L265P mutation has been identified in a subset of marginal zone lymphomas, comprising 6-9% of cases. This genetic alteration further complicates the differentiation between MZL and LPL in breast tumors. We present a case highlighting the diagnostic complexities associated with primary breast MZL. Methods/Case Report A 71-year-old female patient underwent mammographic screening, which revealed a suspicious lesion in her left breast. Subsequent PET scan showed a hypermetabolic area consistent with a primary breast lesion. Biopsy histology exhibited a diffuse atypical lymphoid infiltration with varying proportions of neoplastic lymphocytes. Immunohistochemical analysis revealed positivity for CD20, PAX5, BCL2, CD138, MUM1, and negativity for CD3, CD5, Cyclin D1, CD10, CD23, CD34, BCL6, TDT, CD30, GATA3, and CK AE1/3. Additionally, there was lambda light chain restriction, and the Ki67 proliferation rate index was approximately 50%. Fish analysis indicated abnormal BCL6 gene rearrangement. Molecular genetic testing confirmed the presence of MYD88 mutation with c.794T&amp;gt;C p.L265P. Notably, bone marrow biopsy showed no abnormalities, with normal hematopoiesis. Serum electrophoresis and immunofixation detected an IgM monoclonal protein with lambda light chain specificity. Results (if a Case Study enter NA) NA Conclusion The diagnosis of primary breast MZL was favored over LPL due to the absence of common LPL- associated symptoms such as bone marrow involvement. However, the distinction between these entities remains challenging, underscoring the importance of comprehensive diagnostic approaches integrating histopathology, immunohistochemistry, and molecular genetics. Further research is warranted to enhance diagnostic certainty in such cases, particularly as the management of MZL and LPL becomes increasingly specific with the advent of novel therapeutic agents and ongoing clinical trials.

Testicular large B-cell lymphoma is genetically similar to PCNSL and distinct from nodal DLBCL.

Testicular large B-cell lymphoma (TLBCL) is an infrequent and aggressive lymphoma arising in an immune-privileged site and has recently been recognized as a distinct entity from diffuse large B-cell lymphoma (DLBCL). We describe the genetic features of TLBCL and compare them with published series of nodal DLBCL and primary large B-cell lymphomas of the CNS (PCNSL). We collected 61 patients with TLBCL. We performed targeted next-generation sequencing, copy number arrays, and fluorescent in situ hybridization to assess chromosomal rearrangements in 40 cases with available material. Seventy percent of the cases showed localized stages. BCL6 rearrangements were detected in 36% of cases, and no concomitant BCL2 and MYC rearrangements were found. TLBCL had fewer copy number alterations (p < 0.04) but more somatic variants (p < 0.02) than nodal DLBCL and had more frequent 18q21.32-q23 (BCL2) gains and 6q and 9p21.3 (CDKN2A/B) deletions. PIM1, MYD88 L265P , CD79B, TBL1XR1, MEF2B, CIITA, EP300, and ETV6 mutations were more frequent in TLBCL, and BCL10 mutations in nodal DLBCL. There were no major genetic differences between TLBCL and PCNSL. Localized or disseminated TLBCL displayed similar genomic profiles. Using LymphGen, the majority of cases were classified as MCD. However, we observed a subgroup of patients classified as BN2, both in localized and disseminated TLBCL, suggesting a degree of genetic heterogeneity in the TLBCL genetic profile. TLBCL has a distinctive genetic profile similar to PCNSL, supporting its recognition as a separate entity from DLBCL and might provide information to devise targeted therapeutic approaches.

Blood and cerebrospinal fluid biomarkers in neuro-oncology.

The purpose of this review is to discuss the value of blood and CSF biomarkers in primary CNS tumors. Several analytes can be assessed with liquid biopsy techniques, including circulating tumor cells, circulating cell-free tumor DNA, circulating cell-free RNA, circulating proteins and metabolites, extracellular vesicles and tumor-educated platelets. Among diffuse gliomas of the adult, ctDNA in blood or CSF has represented the most used analyte, with the detection of molecular alterations such as MGMT promoter, PTEN, EGFRVIII, TERT promoter mutation and IDH R132H mutation. In general, CSF is enriched for ctDNA as compared with plasma. The use of MRI-guided focused ultrasounds to disrupt the blood-brain barrier could enhance the level of biomarkers in both blood and CSF. The detection of MYD88 L265P mutation with digital droplet PCR and the detection of ctDNA with next generation sequencing represent the best tools to diagnose and monitoring CNS lymphomas under treatment. In meningiomas, the low concentration of ctDNA is a limiting factor for the detection of driver mutations, such as NF2, AKTs, SMO, KLF4, TRAF7, SMARCB1, SMARCE1, PTEN, and TERT; an alternative approach could be the isolation of ctDNA through circulating extracellular vesicles. Liquid biopsies are being used extensively for diagnosis and surveillance of diffuse midline gliomas, in particular with the detection of the driver mutation H3K27M. Last, specific methylome patterns in CSF may allow the distinction of glioblastomas from CNS lymphomas or meningiomas. This review summarizes the current knowledge and future perspectives of liquid biopsy of blood and CSF for diagnosis and monitoring of primary CNS tumors.

Investigating the frequency of somatic MYD88 L265P mutation in primary ocular adnexal B cell lymphoma.

Ocular adnexal B cell lymphoma is the most common orbital malignancy in adults. Large chromosomal translocations and alterations in cell-signaling pathways were frequently reported in lymphomas. Among the altered pathways, perturbations of NFκB signaling play a significant role in lymphomagenesis. Specifically, the MYD88 L265P mutation, an activator of NFκB signaling, is extensively studied in intraocular lymphoma but not at other sites. Therefore, this study aims to screen the MYD88 L265P mutation in Ocular adnexal B cell lymphoma tumors and assess its clinical significance. Our study of twenty Ocular adnexal B cell lymphoma tumor samples by Allele-Specific Polymerase Chain Reaction identified two samples positive for the MYD88 L265P mutation. Subsequent Sanger sequencing confirmed the presence of the heterozygous mutation in those two samples tested positive in Allele-Specific Polymerase Chain Reaction. A comprehensive review of MYD88 L265P mutation in Ocular adnexal B cell lymphoma revealed variable frequencies, ranging from 0 to 36%. The clinical, pathological, and prognostic features showed no differences between patients with and without the MYD88 L265P mutation. The present study indicates that the MYD88 L265P mutation is relatively infrequent in our cohort, underscoring the need for further validation in additional cohorts.

Clinicopathological and Molecular Characteristics of Rare EBV-associated Diffuse Large B-cell Lymphoma With IRF4 Rearrangement.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) is a rare form of aggressive B-cell lymphoma with limited molecular information reported regarding interferon regulatory factor 4 (IRF4) status. Here, we presented 3 EBV-positive DLBCL cases with IRF4 rearrangement (EBV+DLBCL-IRF4-R) verified by fluorescence in situ hybridization (FISH). Three patients, including 1 male and 2 females (median age: 64y; range: 45 to 68y), had normal immune function. During a median follow-up of 12 months (range: 0 to 24 mo), 2 patients succumbed to the disease, and 1 patient achieved complete response. Three tumors were present in the mediastinum, stomach, and thalamus, respectively. All three tumors exhibited DLBCL morphology and were identified as the non-germinal center B-cell subtype, with EBV-encoded small RNA positivity ranging from 70% to 80%. RNA sequencing was able to identify RHOH and IGH as fusion partners of IRF4 in two cases. No MYC and BCL2 rearrangements were detected in 3 cases by FISH and RNA sequencing. Next-generation sequencing revealed a low mutation burden, and only IRF4 was recurrently mutated in two EBV+DLBCL-IRF4-R cases. Using the LymphGen 2.0 classifier, 1 case was classified as the MCD (including MYD88L265P and CD79B mutations) subtype. We report rare EBV+DLBCL-IRF4-R that may enhance our understanding of the diverse spectrum of large B-cell lymphoma.

Vitreoretinal large B- cell lymphoma (VR- LBCL): Clinical and pathological features and treatment outcomes

ContextVitreoretinal large B- cell lymphoma (VR- LBCL) is a type of non- Hodgkin lymphoma confined to the eye and central nervous system (CNS). The clinical manifestations of intraocular lymphoma can precede, occur simultaneously with, or follow disease at CNS sites. It differs from other forms of extra-nodal lymphoma; in that it does not involve systemic sites other than CNS. ObjectivesTo analyse the clinical and pathological features, and treatment outcomes of a cohort of patients diagnosed with vitreoretinal lymphoma (VRL) in Royal Victoria Eye and Ear Hospital, Ireland between 2010 and 2024. MethodRetrospective review of medical records and pathology specimens of patients with ocular involvement in VR- LBCL over 14-year period and a review of the literature. ResultsEight patients were included. All of them underwent pars plana vitrectomy and were confirmed to have VR- LBCL. The median age at diagnosis was 71 years. Three were men and five were women. Six had bilateral disease and two unilateral. Four of four patients had MYD88 L265P mutation present. Four patients showed a high interleukin-10 (IL-10) to interleukins-6 (IL-6) ratio in keeping with the diagnosis of VRL. Three patients had primary CNS lymphoma with subsequent eye involvement, despite systemic chemotherapy treatment. Of the five patients who presented with ocular lymphoma, two patients had CNS involvement after primary vitreoretinal lymphoma was diagnosed. Of those, one was initially treated with local intravitreal chemotherapy. Three patients had no CNS recurrence. At the time of this study, seven patients of eight are alive, four are disease free and two are on a first- line local chemotherapy treatment. One underwent treatment for CNS relapse. One patient died of the disease before commencing targeted therapy. ConclusionThis case series demonstrated excellent treatment outcomes for seven patients, alive at the time of the study. Both local radiotherapy and intravitreal chemotherapy achieved good ocular control with acceptable side effects and no significant difference in visual outcome. VRL is a difficult diagnosis and vitreous cytology should be prioritised in cases of vitritis unresponsive to treatment. Analysis of MYD88 L265P mutation and IL- 10: IL- 6 ratio >1 are useful adjuncts in the diagnosis of VR- LBCL, particularly in cases where limited vitreous material makes cytological evaluation challenging.

Waldenström Macroglobulinemia - A State-of-the-Art Review: Part 1: Epidemiology, Pathogenesis, Clinicopathologic Characteristics, Differential Diagnosis, Risk Stratification, and Clinical Problems.

Waldenström macroglobulinemia (WM) is an infrequent variant of lymphoma, classified as a B-cell malignancy identified by the presence of IgM paraprotein, infiltration of clonal, small lymphoplasmacytic B cells in the bone marrow, and the MYD88 L265P mutation, which is observed in over 90% of cases. The direct invasion of the malignant cells into tissues like lymph nodes and spleen, along with the immune response related to IgM, can also lead to various health complications, such as cytopenias, hyperviscosity, peripheral neuropathy, amyloidosis, and Bing-Neel syndrome. Chemoimmunotherapy has historically been considered the preferred treatment for WM, wherein the combination of rituximab and nucleoside analogs, alkylating drugs, or proteasome inhibitors has exhibited notable efficacy in inhibiting tumor growth. Recent studies have provided evidence that Bruton Tyrosine Kinase inhibitors (BTKI), either used independently or in conjunction with other drugs, have been shown to be effective and safe in the treatment of WM. The disease is considered to be non-curable, with a median life expectancy of 10 to 12 years.

Transient Bacillary Layer Detachment During the Disease Course of Primary Vitreoretinal Lymphoma

ABSTRACT Purpose To report the clinical course and the retinal imaging features of a case of cytology-proven primary vitreoretinal lymphoma (PVRL) presenting with a transient bacillary layer detachment (BALAD) during the disease course. Methods Observational case report. Results A 50 year-old woman was referred to us with a 2-month history of vitritis in both eyes, poorly responding to oral prednisolone. After discontinuation of oral prednisolone, worsening of vitritis and the appearance of multiple creamy-like subretinal infiltrates in the mid-peripheral retina of both eyes, along with the exclusion of common causes of intermediate/posterior uveitis, made us consider PVRL. Aqueous humor sampling detected MYD88 L265P mutation, and subsequent diagnostic pars plana vitrectomy in the left eye yielded a positive cytology for large B cell lymphoma consistent with PVRL. During the disease course, optical coherence tomography of the macula showed a BALAD in the right eye, which resolved during follow-up. Conclusion Our case indicates that BALAD is a possible rare manifestation of PVRL, and this should be considered in the differential diagnosis process in order to avoid diagnostic delays.

Simplified Risk Stratification Model for Patients With Waldenström Macroglobulinemia.

Patients with Waldenström macroglobulinemia (WM) have disparate outcomes. Newer therapies have emerged since the development of International Prognostic Scoring System, and MYD88L265P mutation is now frequently assessed at diagnosis, warranting reexamination of the prognostic parameters. We reviewed records of 889 treatment-naïve patients with active WM, consecutively seen between January 01, 1996, and December 31, 2017, to identify clinical predictors of overall survival (OS) in univariate analyses. Patients with complete data for the parameters significant on the univariate analyses (n = 341) were included in a multivariable analysis to derive a prognostic model, subsequently validated in a multi-institutional cohort. In the derivation cohort (n = 341), age (hazard ratio [HR], 1.9 [95% CI, 1.2 to 2.1]; P = .0009), serum lactate dehydrogenase (LDH) above upper limit of normal (HR, 2.3 [95% CI, 1.3 to 4.5]; P = .007), and serum albumin <3.5 g/dL (HR, 1.5 [95% CI, 0.99 to 2.3]; P = .056) were independently prognostic. By assigning a score of 1 point each to albumin <3.5 g/dL (HR, 1.5) and age 66-75 years (HR 1.4) and 2 points for age >75 years (HR, 2.6) or elevated LDH (HR, 2.3), four groups with distinct outcomes were observed on the basis of the composite scores. Five-year OS was 93% for the low-risk (score 0), 82% for low-intermediate risk (score 1), 69% for intermediate-risk (score 2), and 55% for the high-risk (score ≥3; P < .0001) groups. In the validation cohort (N = 335), the model maintained its prognostic value, with a 5-year OS of 93%, 90%, 75%, and 57% for the four groups, respectively (P < .0001). Modified Staging System for WM (MSS-WM), utilizing age, albumin, and LDH is a simple, clinically useful, and externally validated prognostic model that reliably risk-stratifies patients with symptomatic WM into four groups with distinct prognosis.

Anti-Myelin-associated Glycoprotein Neuropathy

Anti-myelin-associated glycoprotein (MAG) neuropathy, which occurs secondary to immunoglobulin (Ig)M paraproteinemia such as monoclonal gammopathy of undetermined significance, is characterized by slow progression, sensory or sensorimotor disturbances, and ataxia. The estimated prevalence of this neuropathy in Japan is 0.28 per 100,000 population with male preponderance. This neuropathy is diagnosed based on the detection of M protein and anti-MAG antibodies in patients' serum. Nerve conduction studies show prolonged distal latency, and histopathological evaluation of sural nerve biopsies shows widely spaced myelin on electron microscopy. Usually, immunotherapy, including administration of intravenous Ig and corticosteroids, is ineffective, and rituximab is beneficial in approximately 50% of patients. Novel therapies, such as administration of Bruton's tyrosine kinase inhibitors are expected to benefit patients with the MYD88L265P mutation.

Mutation landscape in Chinese nodal diffuse large B-cell lymphoma by targeted next generation sequencing and their relationship with clinicopathological characteristics

BackgroundDiffuse large B-cell lymphoma (DLBCL), an aggressive and heterogenic malignant entity, is still a challenging clinical problem, since around one-third of patients are not cured with primary treatment. Next-generation sequencing (NGS) technologies have revealed common genetic mutations in DLBCL. We devised an NGS multi-gene panel to discover genetic features of Chinese nodal DLBCL patients and provide reference information for panel-based NGS detection in clinical laboratories.MethodsA panel of 116 DLBCL genes was designed based on the literature and related databases. We analyzed 96 Chinese nodal DLBCL biopsy specimens through targeted sequencing.ResultsThe most frequently mutated genes were KMT2D (30%), PIM1 (26%), SOCS1 (24%), MYD88 (21%), BTG1 (20%), HIST1H1E (18%), CD79B (18%), SPEN (17%), and KMT2C (16%). SPEN (17%) and DDX3X (6%) mutations were highly prevalent in our study than in Western studies. Thirty-three patients (34%) were assigned as genetic classification by the LymphGen algorithm, including 12 cases MCD, five BN2, seven EZB, seven ST2, and two EZB/ST2 complex. MYD88 L265P mutation, TP53 and BCL2 pathogenic mutations were unfavorable prognostic biomarkers in DLBCL.ConclusionsThis study presents the mutation landscape in Chinese nodal DLBCL, highlights the genetic heterogeneity of DLBCL and shows the role of panel-based NGS to prediction of prognosis and potential molecular targeted therapy in DLBCL. More precise genetic classification needs further investigations.

Detection of circulating tumor DNA in plasma of patients with primary CNS lymphoma by digital droplet PCR

BackgroundPrimary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL.MethodsWe analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively.ResultsMYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196.ConclusionThe low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.

Bruton Tyrosine Kinase Inhibition: an Effective Strategy to Manage Waldenström Macroglobulinemia.

The treatment of Waldenström macroglobulinemia (WM) has evolved over the past decade. With the seminal discoveries of MYD88 and CXCR warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) mutations in WM cells, our understanding of the disease biology and treatment has improved. The development of a new class of agents, Bruton tyrosine kinase inhibitors (BTKi), has substantially impacted the treatment paradigm of WM. Herein, we review the current and emerging BTKi and the evidence for their use in WM. Clinical trials have established the role of covalent BTKi in the treatment of WM. Their efficacy is compromised among patients who harbor CXCR4WHIM mutation or MYD88WT genotype. The development of BTKC481 mutation-mediated resistance to covalent BTKi may lead to disease refractoriness. Novel, non-covalent, next-generation BTKi are emerging, and preliminary results of the early phase clinical trials show promising activity in WM, even among patients refractory to a covalent BTKi. Covalent BTK inhibitors have demonstrated meaningful outcomes in treatment-naïve (TN) and relapsed refractory (R/R) WM, particularly among those harboring the MYD88L265P mutation. The next-generation BTKi demonstrate improved selectivity, resulting in a more favorable toxicity profile. In WM, BTKi are administered until progression or the development of intolerable toxicity. Consequently, the potential for acquired resistance, the emergence of cumulative toxicities, and treatment-related financial burden are critical challenges associated with the continuous therapy approach. By circumventing BTK C481 mutations that alter the binding site to covalent BTKi, the non-covalent BTKi serve as alternative agents in the event of acquired resistance. Head-to-head comparative trials with the conventional chemoimmunotherapies are lacking. The findings of the RAINBOW trial (NCT046152), comparing the dexamethasone, rituximab, and cyclophosphamide (DRC) regimen to the first-generation, ibrutinib are awaited, but more studies are needed to draw definitive conclusions on the comparative efficacy of chemoimmunotherapy and BTKi. Complete response is elusive with BTKi, and combination regimens to improve upon the efficacy and limit the treatment duration are also under evaluation in WM.

Nomogram for predicting survival of patients with diffuse large B-cell lymphoma.

The international prognostic index (IPI) system has been widely used to predict prognosis in diffuse large B-cell lymphoma (DLBCL). However, this system categorizes DLBCL patients into four risk groups, and cannot optimize individualized prognosis. In addition, other clinicopathological factors, such as molecular aberrations, are not incorporated into the system. To partly overcome these weak points, we developed nomograms to predict individual patient survival. We also incorporated MYD88L265P and CD79BY196 mutations into the nomograms since these mutations are associated with a worse prognosis and their signaling pathways have been highlighted as a therapeutic target. We analyzed 302 DLBCL cases for which multivariate analysis by Cox proportional hazard regression was performed. Nomograms for progression-free survival (PFS) and overall survival (OS) were constructed and assessed by a concordance index (C-index). The nomograms were also evaluated using an open external dataset (n = 187). The MYD88L265P and/or CD79BY196 (MYD88/CD79B) mutation was detected in 62/302 patients. The nomograms incorporating IPI factors exhibited a C-index of 0.738 for PFS and a C-index of 0.765 for OS. The nomograms incorporating IPI factors and the MYD88/CD79B mutation showed a C-index of 0.745 for PFS and a C-index of 0.769 for OS. The nomograms we created were evaluated using an external dataset and were well validated. The present nomograms incorporating IPI factors and the MYD88/CD79B mutation have sufficient discrimination ability, and may effectively predict prognosis in DLBCL patients. The prognostic models we have presented here may help clinicians personalize prognostic assessments and clinical decisions.

PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay: A highly sensitive method for detection of MYD88 L265P mutation.

Agarose gel-based conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays are currently used for sensitive detection and quantification of MYD88 L265P mutation. Visual inspection of an agarose gel can often be ambiguous. We propose a new allele-specific quantification PCR (AS-qPCR) assay, PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay, that uses Intercalating Nucleic Acid (INA®) technology for increased affinity and specificity. This study compares PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay with conventional AS-PCR. We included a total of 102 peripheral and bone marrow blood samples from 94 patients with a lymphoproliferative disorder. Droplet digital PCR (ddPCR) was used as a third method in case of discrepancy. A positive percent agreement of 100% (95% CI 0.92-1.0) and a negative percent agreement of 98% (95% CI 0.90-1.0) were found between the conventional AS-PCR and the AS-qPCR methods. Including the ddPCR results to validate the discrepant cases, the sensitivity and specificity of PlentiPlex™ MYD88 Waldenström lymphoma qPCR Assay were 1.0 (95% CI 0.97-1.0) and 1.0 (95% CI 0.96-1.0), respectively. Our data demonstrate that PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay is a fast, highly sensitive, and specific method for the detection of MYD88 L265P compared with conventional AS-PCR.

Abstract C139: The IRAK4 inhibitor emavusertib (CA-4948) synergizes with second generation BTK inhibitors acalabrutinib and zanubrutinib in MYD88-L265P mutated lymphoma cell lines

Abstract Background. IRAK4 is a protein kinase downstream the Toll-like receptor (TLR), activated through the adaptor protein MYD88, crucial for the signaling cascade that leads to NF-κB-mediated cytokine and survival factor expression. Somatic mutations in the gene coding for the adaptor protein MYD88 are common in lymphoid tumors. In particular, the MYD88-L265P mutations, known to sustain NF-κB activation, is specifically observed in a fraction of the activated B cell (ABC) diffuse large B-cell lymphomas (DLBCL), in the vast majority of lymphoplasmacytic lymphoma (LPL), and in rare marginal zone lymphomas (MZLs). So far, no success has been achieved to directly inhibit MYD88. Emavusertib (CA-4948) is an IRAK4 inhibitor, which has shown preclinical activity in ABC DLBCL and MZL models, especially in cases bearing the MYD88-L265P. We and others have shown synergism when combining emavusertib with the first generation BTK inhibitor ibrutinib. Clinical trials are on-going exploring emavusertib as single agent or in combination in patients with hematological cancers or solid tumors. Since ibrutinib is associated with possible toxicities that can outbalance its clinical efficacy, here, we have tested the combination of emavusertib with acalabrutinib and zanubrutinib, two 2nd generation BTK inhibitors that have shown improved safety profile in clinical trials. Methods. Cell lines were exposed to five increasing concentrations of each compound as single agent or in combination for 72 h, followed by MTT assay. The beneficial effect of the combinations compared to the single agents was considered both as synergism according to the Chou-Talalay combination index and as potency and efficacy according to the MuSyC algorithm. Results. Cell lines bearing the MYD88-L265P mutation and derived from ABC DLBCL (OCI-Ly-10, TMD8, HBL1) or MZL/LPL (Karpas-1718) were exposed to emavusertib, acalabrutinib or zanubrutinib as single agent or in combination. Synergism, defined as a median Chou-Talalay index &amp;lt; 0.9, was observed for both the combination of emavusertib plus acalabrutinib and for the combination of emavusertib plus zanubrutinib in all four cell lines. Based on the MuSyC algorithm, the synergistic activity of emavusertib was mainly due to an improved efficacy (i.e., maximal effect) rather than improved potency (i.e., minimal active dose) of the BTK inhibitors. Conclusions. In MYD88-L265P mutated lymphoma cell lines, synergism is observed combining the IRAK4 inhibitor emavusertib with the 2nd generation BTK inhibitors acalabrutinib and zanubrutinib. Our data sustain further clinical exploration of the combination of IRAK4 and BTK inhibitors for lymphoma patients. Citation Format: Francesca Guidetti, Alberto J. Arribas, Eleonora Cannas, Emanuele Zucca, Anastasios Stathis, Reinhard von Roemeling, Francesco Bertoni. The IRAK4 inhibitor emavusertib (CA-4948) synergizes with second generation BTK inhibitors acalabrutinib and zanubrutinib in MYD88-L265P mutated lymphoma cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C139.

Abstract B174: The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma

Abstract Background: The SRC family kinase Hematopoietic Cell Kinase (HCK) is upregulated in cycle–quiescent and chemotherapy-resistant leukemic stem cells (LSCs), and inhibition of HCK reduces the survival and self-renewal capacity of LSCs. As such, targeting HCK has emerged as a potential therapeutic strategy in acute myeloid leukemia (AML). In lymphoma, HCK phosphorylates and activates Bruton Tyrosine Kinase (BTK) downstream of Toll-like receptor (TLR) or the IL6 receptor. In conjunction with BTK, HCK engages essential pro-survival signaling through the NF-kB, mTOR, AKT and STAT pathways. Meanwhile, in lymphoma, HCK can take over pro-survival signaling to confer resistance to BTK inhibition. A dual HCK/BTK inhibitor thus provides a promising therapeutic approach to target leukemias and lymphomas. Methods: TTX-114 was identified through a structure-based medicinal chemistry campaign and characterized in cells using Western Blot and Cell Titer Glow for signaling and viability read-outs, respectively. For in vivo efficacy analysis, NSG newborn mice were irradiated prior to bone-marrow reconstitution with patient-derived primary AML LCSs. Mice were treated orally with TTX-114 after AML was established. Results: Here, we describe TTX-114, a non-covalent HCK/BTK inhibitor with anti-tumor activity in leukemia and lymphoma. In biochemical kinase profiling TTX-114 displayed sub-nM activity against BTK with a KD = 0.58 nM and the SRC-kinase cluster with a KD = 0.23nM for HCK. By inhibiting HCK and BTK activity in cells, TTX-114 prevented autophosphorylation of HCK at Y411 and BTK at Y223 in a dose-dependent manner in human peripheral blood mononuclear cells (PBMCs), enabling a starting point for clinical demonstration of target engagement. TTX-114 had anti-tumor activity across a larger panel of leukemia and lymphoma cell lines, with diffuse large B cell lymphoma (DLBCL) cell lines being among the most sensitive (IC50, ≤4 nM). In marginal zone lymphoma (MZL), the activity was slightly higher in the presence of MYD88 L265P mutation. In vivo, TTX-114 treatment significantly reduced the tumor burden in a dose and time-dependent manner in AML-derived PDX mice. A systematic pharmacokinetic/pharmacodynamic (PK/PD) study identified distinct PK driver for efficacy and tolerability, respectively enabling a well-tolerated dosing strategy for TTX-114 which induced complete response in AML-xenograft bearing mice with no impact on body weight. Conclusion: TTX-114 is an orally bioavailable, non-covalent HCK/BTK inhibitor which prevents HCK over-activation in cancer cells and provides a new targeted therapy for leukemias and lymphomas. Citation Format: Ulrike Rauh, Michael Serrano-Wu, Virendar Kaushik, Sabine Pilari, Ikuko Ogahara, Akiko Kaneko, Alberto J Arribas, Eleonora Cannas, Franceso Bertoni, Nathalie R Javidi-Sharifi, Matthew S Davids, Takehiro Fukami, Yoriko Saito, Fumihiko Ishikawa, Anthony Letai, Brian Hubbard. The non-covalent HCK/BTK inhibitor TTX-114 displays potent in vitro and in vivo anti-tumor activity in leukemia and lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B174.

The Incidence, Clinical Application and Prognostic Significance of MYD88 and CXCR4 Mutation in Chinese Patients with Lymphoplasmacytic Lymphoma/ Waldenström Macroglobulinemia

Introduction Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) is a rare B-cell lymphoma with prevalent somatic mutations, including MYD88 and CXCR4. However, their mutation rates in Chinese LPL/WM and the comparison of different detection methods remain unclear. Moreover, the clinical implications of MYD88 and CXCR4 mutations in the immunochemotherapy and targeted therapy eras need exploration. Methods This study included 458 LPL/WM patients (418 WM, 40 non-IgM LPL) from December 2014 to June 2023. Four methods, such as Sanger sequencing, next-generation sequencing (NGS), allele-specific quantitative polymerase chain reaction (AS-PCR), and droplet digital polymerase chain reaction (ddPCR), were used to detect MYD88L265P and CXCR4 mutations. Demographic data, clinical characteristics, tumor burden, cytogenetic abnormalities, treatment regimens, and survival data were collected. Results A total of 458 patients were assessed for MYD88 L265P mutation, of whom 266 were tested by Sanger sequencing, 300 by NGS, 370 for AS-PCR, 251 for ddPCR. The MYD88 L265P mutation was found in 398/458 (86.9%) of the patients. AS-PCR and ddPCR showed the highest sensitivity (97.2% and 96.5%, respectively), outperforming Sanger sequencing and NGS, which had higher false-negative rates in low tumor load patients. A high false-negative rate for MYD88 by Sanger sequencing and NGS was observed in patients with tumor burden less than 10% (40.0% and 21.9% respectively). There was no significant difference in the MYD88 mutation rate detected by ddPCR and ASPCR among patients with different tumor burden groups (P=0.402, P=0.252). MYD88 mutation was detected in a high incidence of 86.7% and 85.5% in less than 1% infiltrated WM tumor cells specimens by ddPCR and ASPCR. Thus, ddPCR and ASPCR assay were effective and accurate enough for un-sorted low infiltrated WM specimens. CXCR4 mutation testing was performed in 428 patients of LPL/WM, comprising 311 with Sanger sequencing, 300 with NGS, and 371 with AS-PCR. Overall, 31.8% (136/428) patients were identified as having the CXCR4 mutation. NGS exhibited the highest sensitivity (79.2%) among the three detection means. Among the 458 patients of LPL/WM, 60 (13.1%) were classified as MYD88 wild-type patients. MYD88 wild-type patients had a significantly lower hemoglobin levels (median 83g/L vs. 92g/L, P=0.044), lower proportion of males (56.7% vs. 72.5%, P=0.012) and a significantly higher proportion elevated β2-macroglobulin (81.5% vs. 68.5%, P=0.027), and lactic dehydrogenase (32.2% vs. 11.1%, P&amp;lt;0.001). However, there were no significant differences in either PFS or OS between MYD88 L265P wild-type and MYD88 L265P mutation group (PFS: median 35.7 months vs. 52.9 months, P=0.40; OS: 5-year rate 72.0% vs. 81.8%, P=0.62). MYD88 wild-type patients had worse PFS and post-BTKi failure-free survival under immunochemotherapy (median 34.3 months vs. 58.4 months, P=0.03), and BTKi regimens (median: 26.2 months vs. 45.2 months, P=0.02), respectively. Patients with CXCR4 mutation tended to be older (median 64 vs. 60, P=0.001), had lower hemoglobin levels (median 83g/L vs. 94g/L, P=0.008), and higher baseline BM involvement (by FCM, median 12.6% vs. 6.2%, P&amp;lt;0.001) compared with CXCR4 wild-type patients. The proportion of patients with thrombocytopenia (43.7% vs. 20.6%, P&amp;lt;0.001), serum IgM level more than 40g/L (47.4% vs. 34.6%, P=0.019) was significantly higher, the proportion of patients with low risk profile of IPSSWM (17.2% vs. 30.5%, P=0.007) was significantly lower in mutation group. Notably, CXCR4 mutation group had significantly worse survival compared with the wild-type group (PFS: median 40.5 months vs. 62.5 months, P=0.024; OS: median 103.8 months vs. not reached, P=0.022). Meanwhile, patients with CXCR4 mutation experienced inferior post-BTKi failure-free survival (median 30.4 months vs. 57.6 months, P=.04) and post-BTKi overall survival (3-year rate 71.9% vs. 84.9%, P=0.03) in BTKi therapy groups. Conclusions This study sheds light on MYD88 and CXCR4 mutations in Chinese WM/LPL based on a large cohort. AS-PCR and ddPCR were highly sensitive for MYD88 mutation detection, especially in low infiltrated WM. NGS was the most sensitive method for detecting CXCR4 mutation. MYD88 mutation had prognostic significance in BTKi-based therapy, while CXCR4 mutation indicated higher tumor burden and inferior survival in BTKi-based therapy.