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.

Utility of clinical, laboratory, and lymph node MYD88 L265P mutation in risk assessment of diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma and is characterized by heterogeneity in biology and clinical behavior. Mutations in the myeloid differentiation primary response 88 (MYD88) are found in different lymphoproliferative disorders and are associated with variable clinical and prognostic impact.AimTo investigate the frequency of MYD88 L265P mutation and its clinical impact in a cohort of Egyptian DLBCL patients.MethodsFFPE lymph node samples from 87 DLBCL patients (46 males / 41 females; median age, 58 years) were included and analyzed for MYD88 L265P by an allele-specific PCR.ResultsMYD88 L265P mutations were found in 52 patients (59.8%) out of 87 DLBCL cases. Patients with L265 mutation were significantly younger than non-mutated patients (p = 0.022). None of the patients with the L265P mutation showed a significant association with the clinical parameters of DLBCL. Interestingly, MYD88 L265 mutated patients were found to be significantly correlated with HCV infection (p = 0.037). The median follow-up time across the entire cohort was 26 months. Univariate analysis showed that overall survival (OS) was affected by gender, LDH level, and CNS-IPI scoring (p = 0.048, 0.008, and 0.046, respectively), while disease-free survival (DFS) was affected by B symptoms and LDH level (p = < 0.000 and 0.02, respectively). However, the MYD88 mutation status and other prognostic factors showed no association with OS or DFS.ConclusionsOur findings indicate a high frequency of MYD88 L265P mutations in our study population and not associated with prognostic markers or the outcome of the disease.

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.

Intraparenchymal low-grade B-cell lymphomas of the central nervous system: Clinicopathologic and molecular analysis of three cases and a review of the literature

Primary central nervous system (CNS) lymphomas represent 1 % of all non-Hodgkin lymphomas, with diffuse large B-cell lymphomas as the prevailing subtype. Low-grade B-cell lymphomas are exceptional with only 24 marginal zone B-cell lymphomas (EMZL) and 1 follicular lymphoma (FL) previously reported so far. While their molecular profiles are studied elsewhere, data on primary intraparenchymal CNS cases remain limited. The objective of the present study is to contribute new cases of primary intraprenchymal low-grade B-cell lymphomas in the CNS and characterize their mutational profile. We conducted a comprehensive review of cases and a literature review to identify similar instances. Clinical, imaging, histological, immunohistochemical, and molecular characteristics were analyzed. Diagnoses were established according to established criteria. We present three novel cases of intraparenchymal CNS low-grade B-cell lymphomas. One case of intraparenchymal EMZL exhibited plasmacytic differentiation, while another lacked a plasma cell component. The third case was diagnosed as FL. The L265P mutation of MYD88 was absent in all cases. Next generation sequencing revealed pathogenic mutations in SPEN (Glu1970ValfsTer64) and ARID1A (Pro1355LeufsTer118) genes in one EMZL case. In conclusion, intraparenchymal CNS low-grade B-cell lymphomas are rare, with few reported cases. Our findings expand knowledge on their clinical and molecular features. We present the first molecular profile of primary CNS intraparenchymal EMZL, underscoring the need for further research to understand their biology and optimize treatment strategies.

In Silico Screening, Molecular Dynamics Simulation and Binding Free Energy Identify Single-Point Mutations That Destabilize p53 and Reduce Binding to DNA.

Considering p53's pivotal role as a tumor suppressor protein, proactive identification and characterization of potentially harmful p53 mutations are crucial before they appear in the population. To address this, four computational prediction tools-SIFT, Polyphen-2, PhD-SNP, and MutPred2-utilizing sequence-based and machine-learning algorithms, were employed to identify potentially deleterious p53 nsSNPs (nonsynonymous single nucleotide polymorphisms) that may impact p53 structure, dynamics, and binding with DNA. These computational methods identified three variants, namely, C141Y, C238S, and L265P, as detrimental to p53 stability. Furthermore, molecular dynamics (MD) simulations revealed that all three variants exhibited heightened structural flexibility compared to the native protein, especially the C141Y and L265P mutations. Consequently, due to the altered structure of mutant p53, the DNA-binding affinity of all three variants decreased by approximately 1.8 to 9.7 times compared to wild-type p53 binding with DNA (14 μM). Notably, the L265P mutation exhibited an approximately ten-fold greater reduction in binding affinity. Consequently, the presence of the L265P mutation in p53 could pose a substantial risk to humans. Given that p53 regulates abnormal tumor growth, this research carries significant implications for surveillance efforts and the development of anticancer therapies.

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.

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.

The Molecular Landscape of Primary CNS Lymphomas (PCNSLs) in Children and Young Adults.

Pediatric brain tumors are often noted to be different from their adult counterparts in terms of molecular features. Primary CNS lymphomas (PCNSLs) are mostly found in elderly adults and are uncommon in children and teenagers. There has only been scanty information about the molecular features of PCNSLs at a young age. We examined PCNSLs in 34 young patients aged between 7 and 39 years for gene rearrangements of BCl2, BCL6, CCND1, IRF4, IGH, IGL, IGK, and MYC, homozygous deletions (HD) of CDKN2A, and HLA by FISH. Sequencing was performed using WES, panel target sequencing, or Sanger sequencing due to the small amount of available tissues. The median OS was 97.5 months and longer than that for older patients with PCNSLs. Overall, only 14 instances of gene rearrangement were found (5%), and patients with any gene rearrangement were significantly older (p = 0.029). CDKN2A HD was associated with a shorter OS (p < 0.001). Only 10/31 (32%) showed MYD88 mutations, which were not prognostically significant, and only three of them were L265P mutations. CARD11 mutations were found in 8/24 (33%) cases only. Immunophenotypically, the cases were predominantly GCB, in contrast to older adults (61%). In summary, we showed that molecular findings identified in the PCNSLs of the older patients were only sparingly present in pediatric and young adult patients.

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.

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 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.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

CD19-Targeting CAR T-Cell Therapy in Transformed Waldenström Macroglobulinemia/Lymphoplasmacytic Lymphoma: A Descar-T and US Collaborative Study

Introduction: Patients with histological transformation (HT) of Waldenström macroglobulinemia (WM) who relapse or are refractory (R/R) have a poor prognosis with standard chemoimmunotherapy, notably patients who are unable to undergo high dose chemotherapy (HCT) and autologous stem cell transplantation (ASCT) or who relapse after HCT/ASCT. CD19-targeted chimeric antigen receptor (CAR) T-cell therapies can lead to durable responses in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and thus may represent a promising therapeutic option in R/R transformed WM (tWM); however, data from pivotal trials is lacking. This study aimed to evaluate the efficacy and safety of anti-CD19 CAR T-cells in real-word patients with R/R tWM. Methods: We retrospectively identified patients with biopsy-proven transformed WM/lymphoplasmacytic lymphoma treated with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) in the French DESCAR-T registry (NCT04328298) and 2 US centers. The primary endpoint was best complete response rate (CRR). Secondary outcomes were best overall response rate (ORR), progression-free survival (PFS), overall survival (OS) and safety. Responses were assessed according to the Lugano 2014 criteria at 1 month, 3 months and 6 months. Subsequent assessments were per institutional pratice. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to the ASTCT 2019 consensus criteria. Hematological toxicity and infections were graded according to the NCI CTCAE (version 5.0). Results: Between June 2017 and May 2023, 22 patients (18 from DECAR-T centers and 4 from US centers) with R/R tWM were treated with anti-CD19 CAR T-cells (13 axi-cel and 9 tisa-cel). Median age at WM diagnosis was 58 years (range 35-72). MYD88 L265P and CXCR4 mutations were present in 85% (11/13) and 29% (2/7) of patients with available data, respectively. Patients received a median of 1 prior line of treatment (range, 0-4) for WM and 2 prior lines (range, 1-4) for HT. Eight patients had received prior ASCT (2 for WM and 6 for HT) and 1 patient allogeneic SCT. The median time from WM to HT diagnosis was 4.5 years (range, 0-32) and 26 months (range, 3-90) from HT diagnosis to CAR T infusion. Of note, CNS involvement was present in 3 patients at DLBCL relapse before CAR T-cell therapy. At time of infusion, the median age was 66 years (range, 41-82) and 8 patients (36%) were older than 70 years. Median Eastern Cooperative Oncology Group (ECOG) was 1 (range, 0-2). Bridging therapy was administered to 16 patients (73%). Following bridging therapy, 6 patients had progressive disease (PD), 4 stable disease (SD) and 6 were responders (2 complete responses (CR) and 4 partial responses (PR)). After CAR T-cell infusion, best CRR was 82% and best ORR was 95%. The ORR/CRR was 95%/77% at 1 month, 76%/71% at 3 months and 68%/68% at 6 months. Seventeen patients (78%) experienced CRS, including 2 grade 3 (all after axi-cel), and 9 ICANS occurred (41%), including 1 grade 3 and 1 grade 4 (all after axi-cel). Eight patients (36%) presented infections, including 3 grade 3 and 1 grade 4. Neutropenia was reported in 78% (grade 3-4 = 68%), thrombocytopenia in 86% (grade 3-4 = 45%) and anemia in 86% (grade 3-4 = 32%) of patients. After a median follow-up of 17 months (range, 1-47), the 1-year PFS and OS were 70% and 84% respectively (figure). Five deaths were reported, 4 due to progressive disease and 1 from SARS-CoV-2 infection. Conclusion: This study shows a high efficacy of anti-CD19 CAR T-cell therapy in R/R tWM with no unexpected toxicity. Longer follow-up is needed to confirm the long-term efficacy of CAR T-cells in tWM.

Prospective Evaluation of Minimal Residual Disease in Waldenström Macroglobulinemia across Different Tissues and Treatments: Results of the “BIO-WM” Trial of the Fondazione Italiana Linfomi (FIL)

Introduction. MYD88 L265P is the hallmark mutation in Waldenström Macroglobulinemia (WM) and is becoming increasingly important in the management of IgM-gammopathies, due to its role as prognostic and predictive biomarker. Recently, MYD88 L265P detection by allele-specific quantitative PCR was proposed as reliable minimal residual disease (MRD) marker in bone marrow (BM) samples (Varettoni, Hematol Oncol 2022). Novel, more sensitive, techniques as droplet digital PCR (ddPCR) might extend the feasibility of MRD detection in WM also in non-invasive tissues, as peripheral blood (PB) or plasmatic cell-free (cf) DNA. This was a secondary endpoint of the multicenter, observational “BIOWM” (NCT03521596) trial, sponsored by the Fondazione Italiana Linfomi (FIL) and the International WM Foundation/Leukemia and Lymphoma Society. From 2018 to 2020 this trial enrolled 300 consecutive patients with primary diagnosis of WM or IgM-MGUS and a systematic biobanking was performed. Here are presented the first results of the MRD study on the patient subset who received frontline treatment, with the aim of driving correlations with clinical response, type of therapy received and outcome prediction. Methods. Paired BM, PB and plasma samples were collected for each patient at baseline (T0) and follow-up and MYD88 L265P detection was performed by ddPCR in all specimens (Drandi, Haematologica 2018). As of today, 59/300 patients (58 WM and 1 IgM-MGUS) received frontline treatment and MRD levels after therapy (T2) were detected by ddPCR: actually, T0-T2 paired samples were available in 49 (BM), 56 (PB) and 48 (cfDNA) cases, respectively. Moreover, MRD was evaluated also by 8-color multiparametric flow cytometry (MFC) in 23 BM and 24 PB samples with a minimum of 10 million cells acquired. Results. Median age of the 59 treated patients was 68 years (24-85), 32% were female, median IgM value was 2420 mg/dL (101-8840), median Hb 10.5 g/dL (8 -17) and IPSS-WM was high in 35% and intermediate in 46%. MYD88 L265P mutation rate was 94% (46/49) in BM, 80% (45/56) in PB and 90% (43/48) in cfDNA samples, respectively. Treatment was started because of anemia in 32% cases, lymphadenopathy or splenomegaly in 34%, hyperviscosity in 20%, other reasons in 13%, and the only MGUS patient required treatment for anti-myelin-associated glycoprotein polyneuropathy. Thirty-one out of 59 patients received bendamustine-rituximab (BR), 23/59 dexamethasone, rituximab and cyclophosphamide (DRC) or DRC-like regimen and 5/59 a single agent therapy (namely, 3 rituximab, 1 cyclophosphamide, 1 ibrutinib). Overall response rate was 90% (87% for BR and 96% for DRC), with 19% CR, 29% VGPR and 35% PR in BR and 4% CR, 0% VGPR and 91% PR in DRC, respectively. Overall, MRD negativity after treatment was 30% (14/46) in BM, 89% (40/45) in PB and 54% (23/43) in cfDNA, respectively. Moreover, among patients still MRD positive after treatment, median MYD88 L265Ptumor burden shrinkage was about 2 Logs in BM (5.5E-03 vs 2.1E-01) and around 1 Log in cfDNA (5.9E-03 vs 3.7E-02) and in PB samples (3.6E-03 vs 2.5E-02), respectively (Figure 1). Interestingly, the MRD shrinkage in BM was deeper among patients receiving BR (48% MRD negative with a median MYD88 L265P decrease of more than 2 Logs among patients still MRD positive) vs patients receiving DRC (10% MRD negative with a median decrease of 1 Log), Figure 2. Similar trends in favor of BR in inducing MRD negativity were reported in PB (96% vs 88%) and cfDNA (70% vs 35%), respectively. MRD results by MFC were overall in line with ddPCR results: namely 27% of patients reached MRD negativity in BM and 69% in PB. The median follow-up for the whole series was 41 months, resulting in a 3-years PFS of 71% and 3-years OS of 89%, respectively. Interestingly, despite the current limited follow-up for such an indolent disease, MRD positivity by ddPCR in PB predicted a dismal clinical outcome if compared with MRD negative patients (3-years PFS 40% vs 73%, p=0.038). Conclusions. To the best of our knowledge, this is the first report of prospective MRD evaluation in a WM clinical trial. Our preliminary results suggest that MYD88 L265P monitoringby ddPCR is a suitable target for MRD analysis, in the contest of common immunochemotherapeutic schedules. Moreover, different levels of disease persistence were described across BM, PB and cfDNA, with MRD monitoring in non-invasive tissues showing promising predictive value for outcome discrimination.