MYD88 Mutations Research Articles

Identification of MYC-Driven High-Grade B-Cell Lymphoma Using Deep Learning-Based Whole Slide Image Analysis

Introduction High-grade B-cell lymphoma (HGBL) is an aggressive lymphoma that often harbors MYC rearrangements ( MYC-R) and molecular signatures attributed to aberrant MYC activation (Olszewski et al. Blood 2022). Identifying and classifying HGBL is challenging; current classification systems recognize diffuse large B cell lymphoma/HGBL with MYC and BCL2 rearrangements ( MYC-R/ BCL2-R; double-hit; defined molecularly) and HGBL-not otherwise specified (defined morphologically) in which MYC-R occur in up to 45% of cases and the double-hit signature (DHITsig) occurs in 54% of cases (Alaggio et al. Leukemia 2022, Campo et al. Blood 2022, Olszewski et al. Blood 2022). Existing methods for molecular classification, such as fluorescence in situ hybridization (FISH), are expensive, time-consuming and not widely available, and morphological classification is subjective and associated with high inter-reader variability (Natkunam et al. Histopathology 2023). Therefore, we applied a deep-learning approach to identify MYC-driven HGBL from whole-slide images (WSIs) of ubiquitously available hematoxylin and eosin-stained slides. Methods The model was trained on a real-world data set containing 600 WSIs from multiple sites using MYC-R status determined by FISH as a label. The model was evaluated on an independent test set comprised of 287 WSIs from the GOYA study (ClinicalTrials.gov identifier: NCT01287741). WSIs were scanned with a Roche Ventana DP200 scanner at 0.25 μm/px. Model architecture extracted cytological features from single cells and architectural features from larger tissue regions, enabling the quantification of high-grade morphology characterized by monomorphic sheets of dense cells with round, intermediately sized nuclei and finely dispersed chromatin. Model predictions were compared with MYC-R and BCL2-R with the immunoglobulin heavy chain locus (IGH), gene expression signatures (including DHITsig and cell of origin [COO]), gene mutation signatures obtained by next generation sequencing, computationally derived morphological measurements, and morphological evaluation by hematopathologists. MCD ( MYD88/CD79B mutations) and A53 ( TP53 mutations) genetic subtypes were determined by LymphGen. Statistical comparisons were made using Kendall's tau-b (τb) correlation for continuous variables and Fisher's exact test with conditional maximum likelihood estimated odds ratios (ORs) for categorical variables. Results In the test set, the model distinguished between cases with and without MYC-R (IGH) with an area under the receiver operating characteristic curve of 0.85, sensitivity of 0.95, specificity of 0.59, positive predictive value of 0.16, and negative predictive value of 0.99. The model correctly classified 21/22 MYC-R (IGH) cases and all four MYC-R/ BCL2-R (IGH) double-hit cases. Of DHITsig cases, the model classified 25/33 as MYC-driven HGBL. Model scores correlated with MYC expression (τb = 0.25; p < 0.0001) and Ki67 expression (τb = 0.15, p < 0.0001) ( Figure 1A). Tumors classified as MYC-driven HGBL were enriched for the non-germinal center B-cell-like COO (OR 2.0; p < 0.01) and the MCD genetic subtype (OR 1.9; p < 0.05), and depleted for the A53 genetic subtype (OR 0.3; p < 0.001). Additionally, enrichment was observed for MCD hallmark mutations in MYD88 (OR 1.9; p < 0.05), CD79b (OR 4.6; p < 0.001) and CDKN2A (OR 3.6; p < 0.0001) ( Figure 1A). Cell density (τb = 0.30; p < 0.0001), mean nuclei roundness (τb = 0.15; p < 0.001), coefficient of variation of nucleus roundness (τb = −0.19; p < 0.0001) and size (τb = −0.29; p < 0.0001), and mean nuclear pixel intensity (τb = −0.36; p < 0.0001) were associated with positive model predictions ( Figure 1A) corresponding to high-grade morphology. WSIs labeled as having high-grade cytomorphology by hematopathologists also had high model scores (p = 0.07; OR 3.5). Conclusions Our deep learning model, trained using MYC-R status, identified MYC-driven HGBL by enriching for cases with MYC-R, MYC overexpression, molecular gene expression profiles associated with aberrant MYC activation and cell proliferation, and high-grade morphology. The model also identified molecular characteristics other than MYC-R, such as MCD genetic subtype, associated with increased MYC expression (Schmitz et al. N Engl J Med 2018, Lacy et al. Blood 2020, Varano et al. Nature 2017), which converge on a common phenotype and high-grade morphology ( Figure 1B).

Mutational Landscape of Bone Marrow CD19 and CD138 Cells in Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathy of Undetermined Significance (IgM MGUS)

Background Waldenström's Macroglobulinemia (WM) is an incurable B-cell neoplasm characterized by serum monoclonal immunoglobulin M (IgM) and clonal lymphoplasmacytic cells infiltrating the bone marrow (BM). Smoldering WM (sWM) is the asymptomatic/indolent form with a high risk of progressing to symptomatic WM requiring treatment whereas IgM monoclonal gammopathy of undetermined significance (IgM MGUS) is an early precursor stage of WM. Despite recurrent and activating mutations, including MYD88, CXCR4, ARID1A, KMT2D, and CD79B have been identified, the genetic basis for WM and the risk of progression of IgM MGUS to WM remain to be fully elucidated. Methods Based on the mutational profiling of WM and our previous wide transcriptome analyses on BM CD19+ and CD138+ cells of patients with WM and IgM MGUS, we decided to investigate the mutation status of these patients as follows: WM (n=10), sWM (n=7), and IgM MGUS (n=5), by performing high throughput targeted AmpliSeq next generation sequencing on 117 target genes potentially involved in B-cell lymphomagenesis and in the risk of progression of IgM MGUS to WM (Trojani A. et al. Cancers, 2021). Results We identified genetic aberrations in multiple genes, including previously described classic mutations in MYD88 (L260P alias L265P) found in 82.3% (14/17) of WM/sWM patients and in 60% (3/5) of IgM MGUS subjects. The CXCR4 mutation (S338Ter) was detected in 23.5% of CD19+ cells of WM patients, whereas it was absent in IgM MGUS subjects. Interestingly, we also identified new mutated genes, including WNK2 somatic mutations affecting 47% of WM patients, for which a recurrent allelic variant (V1635Ter) was observed, and BCL9 with a recurrent frameshift variant (P516Lfs) identified in 29.4% of WM patients exclusively in the CD19 cell fraction. The BCL9 (P516Lfs) variant was also detected in the B cells of one IgM MGUS patient. Moreover, sequencing evaluation revealed recurrently frameshift or missense mutations involving NFKB2 (L473Afs), PTPN13 (P1546Tfs), CARD11 (S622del), KMT2C (I823T), and ATM in WM and IgM MGUS patients (Figure 1). Of interest, KMT2C (also known as MLL3) exhibited a somatic hypermutated phenotype in both CD19+ and CD138+ cells of WM and IgM MGUS samples. The KMT2C mutations were not mutually exclusive with those involving KMT2D in our cohort of patients. Conclusion The present genomic analysis revealed new insights into the mutational landscape of Waldenström Macroglobulinemia, providing evidence of the recurrence of some variants in subjects with IgM MGUS as well. Notably, we uncovered new genetic mutations with somatic variants recurrently observed (n≥3) in KMT2C, WNK2, BCL9, PTPN13, CARD11 and NFKB2 genes. These recurring somatic events provide a genomic basis for future research and a better understanding of the WM pathogenesis as well as the risk of transformation of IgM MGUS to WM.

High-Dose Methotrexate, Ibrutinib and Temozolomide (MIT) for the Treatment of Newly Diagnosed Primary Central Nervous System Lymphoma: A Multi-Center Prospective Phase II Study

Abstract Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive subtype of B-cell lymphoma with poor survival. Genetic alterations often occur in the chronic active B-cell receptor signaling which mediate the response to BTK inhibition (BTKi) in PCNSL. However, the efficacy of traditional high-dose methotrexate-based regimen as a first-line treatment was suboptimal with a short effective remission time and low response rate. Therefore, we initiated this phase II trial to investigate the efficacy of novel induction combination of high-dose methotrexate (HD-MTX) plus ibrutinib and temozolomide (MIT), in the treatment of newly diagnosed PCNSL for the first time (ClinicalTrials.gov identifier: NCT04514393). Methods: Patients aged 18-75 years, newly diagnosed PCNSL and at least one measurable or evaluable lesion were enrolled. The MIT induction treatment included six cycles of HD-MTX (3.5 g/m 2, every three weeks), ibrutinib (560 mg/d, after HD-MTX clearance), and temozolomide (150 mg/m2 d1-d5, every three weeks). After the completion of the six cycles of MIT induction treatment, ASCT consolidation (only for patients < 65 years) or daily ibrutinib maintenance (560 mg/d) were administered up to two years, or until disease progression, intolerable toxicity, or death. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every two cycles of MIT treatment. The primary objective of this study was to evaluate the overall response rate (ORR) of MIT induction treatment. The safety profile of MIT was also investigated. At the same time, next generation sequencing (NGS) of baseline tissues and dynamic monitoring with CSF/ plasma ctDNA were performed by Nanjing Geneseeq Technology Inc. Results: Between July 2021 and October 2022, thirty-three patients were enrolled in this open-label, multi-center phase II prospective clinical study. The median age was 56 years (range: 27-75) and 17 of them were male. Eye involvement occurred in 5 patients (15%). Twenty-five patients (76%) had multiple cerebral lesions. The best ORR was 94% and complete response rate was 73%. The median follow-up was 17.5 months (range: 5.8-21.3 months) for survivors. Twenty-three patients underwent ibrutinib maintenance after 6 cycles of induction therapy. The swimmer plot of the 33 patients was shown in Figure 1. The 1-year progression-free survival was 66.4% (95%CI: 58.1-74.7%) and overall survival was 90.5% (95%CI: 85.2-95.8%) (Figure 2). Treatment-related toxicities were acceptable, with grade 3-4 neutropenia occurring in 9%, thrombocytopenia in 6%, subdural hematoma (SH) in 6%, and atrial fibrillation in 3%. The 2 patients with SH recovered following discontinuation of ibrutinib. No fungal infections and treatment-related deaths were observed. NGS of Tissue/CSF was tested in 30 patients, 19 (63%) of whom were classified as MCD subtype, 2 (7%) as BN2 subtype. The mutational profiles of baseline tissue and/or CSF samples revealed that PIM1 and MYD88 mutations were present in 75.9% and 65.5% of the entire cohort followed by BTG2 (55.2%) and CD79B (51.7%). Patients with dynamic CSF ctDNA monitoring during MIT treatment were analyzed (Figure 1). The consistence of ctDNA clearance in CSF/plasma samples and imaging complete remission was observed. Patients with clearance of ctDNA in the CSF after 2 cycles of MIT treatment achieved better PFS. Conclusions: The novel induction treatment of MIT achieved encouraging responses in newly diagnosed PCNSL patients with acceptable toxicities. The early clearance of ctDNA in CSF may be related to favorable survival. Disclosures No relevant conflicts of interest to declare.

CD58 Genetic Alterations and Its Contribution to Upregulation of PD-L1 and IDO Via LYN/CD22/SHP1 Axis in DLBCL

Abstract Background: Recurrent abnormalities of immune surveillance-related genes play a crucial role in DLBCL progression. Prior studies have shown that CD58, a key adhesion molecule that acts as a ligand for the T-cell costimulatory molecule CD2, is frequently mutated or deleted in certain hematological malignancies. Aberrant expression of CD58 may result in tumor cells evading immune surveillance mechanisms mediated by T/NK cells. Downregulation or loss of CD58 is linked to resistance to ICB therapy in melanoma and CAR-T therapy in B-cell malignancies. Nevertheless, the role of CD58 in cancer is not yet well understood. Thus, the aim of this study was to comprehensively characterize CD58 genetic alterations in DLBCL, unveil the multiple roles of CD58 in anti-tumor immunity, and provide insights for potential therapeutic strategies. Methods: Comprehensive analysis of the genetic characteristics of CD58 were performed through targeted deep sequencing (n=176), whole exome sequencing (n=38), and RNA-sequencing (n=162) in patients with de novo DLBCL. To investigate the mechanistic impacts of CD58 alterations on co-inhibitory molecules expression and immune cell function, we performed bulk and single-cell RNA-sequencing analysis of tumor samples and conducted co-IP, flow cytometry and co-culture assays in vitro. Results: We identified that CD58 mutagenesis rate was 9.1%, with 88.9% of the identified mutations resulting in truncated protein or impairment of its normal function. 61.1% mutations occurred in exon 2, which may disrupt CD58-CD2 binding. Co-mutation analysis revealed higher frequencies of KMT2D, CD79B and MYD88 mutations in patients with CD58 mutations(p < 0.05) (Fig 1A). The CD58 copy number loss rate was 44.7% (Fig 1B). Both mutations and copy number loss led to a mild decrease in mRNA expression of CD58. Patients with CD58 mutation, copy number loss or low expression exhibited lower complete response rates following first-line R-CHOP therapy, as well as significantly poorer PFS and OS. To reveal the profound impact of CD58 on the immune microenvironment of DLBCL, we performed bulk and single-cell RNA sequencing and revealed that increased NK cells, activated CD4 +/CD8 + T cells, as well as the weaker exhaustion status of CD8 + T cells, correlated with CD58 high expression. By co-culturing assays, we identified that CD58 downregulation or mutation in DLBCL cells suppressed T cell proliferation and CAR-T cell-mediated killing. Interestingly, blocking CD58-CD2 signaling with a CD2 antibody did not fully eliminate the inhibitory effect, indicating that CD58 alterations could trigger additional immunosuppressive mechanisms. We further revealed that CD58 downregulation or mutation in DLBCL cells resulted in elevated expression of PD-L1 and IDO. This suggested that CD58 may have a dual effect on T cells, whereby its downregulation or mutation may lead to a lack of CD2 co-stimulatory signaling, as well as an increase in PD-L1 and IDO inhibitory signaling. We found that CD58 downregulation or mutation caused JAK2/STAT1 phosphorylation, which in turn promoted PD-L1 and IDO expression. Co-IP identified that CD58 can interact with Lyn, which phosphorylated ITIM of the inhibitory receptor CD22, leading to recruitment and activation of the inhibitory phosphatase SHP1. CD58 downregulation suppressed the negative regulation of the LYN/CD22/SHP1 axis, thereby promoting the activation of JAK2/STAT1 and upregulating PD-L1 and IDO expression. To overcome the dual effects induced by CD58 alterations, anti-PD-L1 antibody or IDO inhibitor and recombinant CD58-Fc chimera protein were added alone or in combination to the co-culture cells. We showed that the combination regimens reversed the inhibitory effect of CD58-downregulated or mutated DLBCL cells on T cell proliferation and restored sensitivity to CAR-T cell-mediated killing (Fig 1C, D). Conclusions: Our study comprehensively characterized CD58 genetic alterations in DLBCL. We demonstrated that CD58 downregulation or mutation led to upregulation of PD-L1 and IDO expression mainly by regulating the LYN/CD22/SHP1 axis. Moreover, we explored strategies to directly activate CD2 co-stimulatory signaling or in combination with immune checkpoint inhibitors to address the diverse effects of CD58 alterations. Our findings provide novel insights for individualized therapy for DLBCL patients with CD58 mutation or deletion.

Indolent Lymphoma: High CR and VGPR Rate with Fixed Duration Bendamustine, Rituximab and Acalabrutinib in Waldenstroms Macroglobulinaemia (BRAWM)

Background: Waldenström's macroglobulinaemia (WM) is an uncommon lymphoproliferative disorder. Although many options are available, an optimal first-line therapy for WM has not been defined. We postulated that combining bendamustine and rituximab (BR) with a next generation BTK inhibitor would result in deeper responses as measured by complete response (CR) and very good partial response (VGPR) rates, and provide a longer duration of response. Objectives: The primary objective of this trial is to document the CR and VGPR rates Methods: The BRAWM clinical trial combines BR with acalabrutinib in a one-year, fixed duration treatment course including six cycles of BR and 12 months of acalabrutinib (6 months of monotherapy). This ongoing trial is taking place at 9 clinical sites across Canada and 44 patients have been enrolled as of July 1, 2023, with a recruitment goal of 59. Results: Median age of participants was 68; 34 were male; 3 participants were low risk, 21 intermediate and 20 high risk. 27 participants completed combination therapy, 17 completed monotherapy and 7 were followed-up at 18 months, and 3 at 24 months after first dose. Table 1 shows the observed Clinical Responses using 6 th IWWM; 2 participants improved from a VGPR to a CR between cycles 7 and 12, both of whom have not reached month 18 for assessment. The interim primary endpoint of combined CR and VGPR rate is 13/17 (77%), with no participants having progressed at this interim analysis. 3 participants discontinued treatment early: 1 at cycle 7 (with a VGPR) who experienced an adverse event requiring treatment; 1 at cycle 3 with inter-current illness, and 1 at cycle 2 for personal reasons not related to treatment. The total observed Treatment Related Adverse Events (TRAEs) in combination therapy was 188 amongst 35 participants, and n= 24 in 10 participants during monotherapy. There were 24 Grade 3/4 TRAEs (combination n= 20, monotherapy n= 4). Total observed Serious Adverse Events was 18, and 7 TRSAEs 7; 6 during combination therapy (3 febrile neutropenia and 1 for each fever, allergic reaction and bowel obstruction) and 1 pneumonia during monotherapy. Grade 3/4 TRAEs resulted in 29 dose interruptions in 13 participants, and 2 dose reductions. Mutational analysis of participants with adequate sample for assessment (n=35), showed 33 with MYD88 mutation, 10 with a CXCR4 mutation, and 1 with a TP53 mutation. Minimal residual disease (MRD) analysis of Peripheral Blood (PB) and Bone Marrow, using next generation sequencing, is underway. Initial review shows that most evaluable patients (those with samples at two or more time-points) have become MRD undetectable in PB. Uni- and multi-variate analyses of variables that may be associated with outcomes is underway. Conclusions: Bendamustine, rituximab and acalabrutinib front-line therapy for WM is safe and well tolerated and initial clinical results show that this treatment induces a high percentage of CR + VGPRs. Table 1: Clinical Responses using 6 th IWWM. *The 2 participants observed to have a CR at month 12 have not reached month 18 for progression or survival follow-up.

Ibrutinib and Rituximab Versus Fludarabine, Cyclophosphamide, and Rituximab for Patients in NCRI FLAIR Study with Previously Untreated CLL: Assessment of Mutational Landscape of Patients at Baseline and Prognostic Impact

Introduction: Recent studies using next generation sequencing have revealed recurrent mutated genes in CLL are associated with clinical outcome. We assessed the baseline mutational profile of 771 patients from FLAIR treated with either fludarabine, cyclophosphamide and rituximab (FCR) or ibrutinib and rituximab (IR). The prognostic impact of individual gene mutations on disease progression was investigated. Method: FLAIR is a phase III, multicentre, randomised, controlled, open, parallel group trial for previously untreated CLL requiring therapy according to IWCLL criteria. Patients with >20% chromosome 17p deletion were excluded. Patients were randomised to FCR or IR. Somatic hypermutation (SHM) status was determined by PCR amplification of IGHV-IGHD-IGHJ gene rearrangements using IGHV leader/FR1 primers. Bidirectional Sanger sequencing was analysed using IMGT V-Quest and the ARResT/AssignSubsets tool. Extracted DNA was sequenced using an Illumina MiSeq and analysed using an in-house pipeline. Amplicon based targeted sequencing of 33 recurrently mutated genes in lymphoid malignancies were performed in parallel. Detected variants were reported down to minimum variant allele fractions of 3-5% and coverage of 100X. Low level variants were confirmed by repeat sequencing. Results: 771 patients were randomly assigned to receive FCR (385) or IR (386). IGHV gene SHM status was available for 728/771 (94.4%): 388 (53.3%) IGHV unmutated (>98% of nucleotide identity to germline), 294 (40.4%) IGHV mutated and 46 (6.3%) stereotyped subset #2 (n=28 IGHV mutated, n=18 IGHV unmutated). Gene mutations were assessed in 767/771 (99.5%) patients at baseline and mutations (>1) were detected in 480/767 (62.6%). Mutation frequencies ranged from 0.1-18.8% with mutations in SF3B1 (18.8%), ATM (14.5%), NOTCH1 (10.0%), MYD88 (6.1%), POT1 (5.7%), TP53 (5.0%), BRAF (4.4%) and RPS15 (4.0%) being the most frequent. No detectable mutations at baseline were found in 287/767 (37.4%) and there was no significant difference in PFS or OS compared to patients with >1 mutation. A significantly shorter PFS and OS was observed for TP53 mutated patients compared to wildtype (HR 2.23 [95% CI 1.31-3.79]; p=0.003 and HR 2.43 [95% CI 1.04-5.66]; p=0.039 respectively). When further subdivided by treatment, a trend for shorter PFS was observed in TP53 mutated patients in both arms compared to wildtype, but significance was only achieved for FCR (HR 2.48 [95% CI 1.25-4.91]; p=0.009) and not IR (HR 2.27 [95% CI 0.97-5.30]; p=0.059). A significantly shorter PFS was observed for FCR compared to IR in patients with ATM mutations at baseline (HR 0.35 [95% CI 0.16-0.80]; p=0.012) and RSP15 (HR 0.19 [95% CI 0.04-0.96]; p=0.044). A trend for shorter PFS was also observed in NOTCH1 mutated patients treated with FCR however, this was not found to be significant when compared to wildtype or NOTCH1 mutated IR patients. CREBBP mutation at baseline (n=19; 2.5%) was associated with shorter PFS (HR 2.63 [95% CI 1.29-5.35]; p=0.008) and OS (HR 4.37 [95% CI 1.75-10.94]; p=0.002) compared to wild type (Fig). When further subdivided by treatment arm, shorter PFS and OS was also observed for CREBBP mutated patients treated with FCR compared to wildtype (n=11; HR 4.37 [95% CI 1.75-10.94]; p=0.002 and HR 10.14 [95% CI 3.83-26.83]; p<0.001 respectively). A similar trend was not observed with IR (n=8; PFS HR 0.79 [CI 0.11-5.74]; p=0.819 and OS HR NE [95% CI NE-NE]; p=0.988). CREBBP mutated patients treated with FCR had a shorter PFS compared to IR (HR 0.1 [95% CI 0.01-0.86]; p=0.036). The PFS and OS for IGHV subset #2 was similar to IGHV mutated patients (Fig). In FCR treated patients there was no difference between mutated (n=13) and unmutated subset #2 (n=7) but with IR there was a trend that IGHV mutated subset #2 patients (n=15) had better PFS and OS than their IGHV unmutated counterparts. Conclusions: Our study confirms that TP53 mutations result in shorter PFS and OS in CLL. We demonstrate improved PFS when patients with ATM or RPS15 mutations are treated with IR compared to FCR. Mutations in the CREBBP gene have recently been described as a novel candidate driver in CLL. We report a significant improvement in disease progression in CREBBP mutated patients treated with IR when compared to FCR. In contrast to previous studies in frontline CLL IGHV subset #2 patients had a similar outcome to good risk IGHV mutated patients.

Post-Hoc Analysis of the Final, Three-Year Follow-up Results of a Phase I/II Study on Tirabrutinib (ONO-4059) in Patients with Relapsed or Refractory Primary Central Nervous System Lymphoma

[Background] Tirabrutinib (TIR; ONO-4059) is a second-generation inhibitor of Bruton's tyrosine kinase with high kinase selectivity. Based on the results of study ONO-4059-02 in patients with relapsed or refractory (r/r) primary central nervous system lymphoma (PCNSL) (Phase I/II study, jRCT: 2080223590), TIR was approved in Japan, Korea, and Taiwan. We have reported the results of 3-year follow-up (median follow-up: 37.1 months) of study ONO-4059-02, in which the overall response rate (ORR), the primary endpoint, was 63.6% and median duration of response was 9.2 months. The 3-year PFS and OS rates were 13.9% and 56.7%, respectively (Asai et al. ASCO 2023, Abstract 7548). Several clinical questions still remained to be addressed, including the proportion of patients who received subsequent treatment after discontinuation of TIR, and their treatment types; the characteristics of patients with higher efficacy of TIR; and the impact of long-term TIR treatment on Karnofsky Performance Status (KPS) and the Quality of Life (QoL). Here, we report the results of post-hoc analyses of the 37-month follow-up data of study ONO-4059-02. [Methods] In study ONO-4059-02, 44 patients with r/r PCNSL, age ≥20, and KPS≥70 were treated with TIR once daily at doses of 320 mg (n=20), 480 mg (n=7), or 480 mg (n=17) under a fasted condition. TIR was administered in 28-day cycles, and treatment was continued until disease progression or clinically unacceptable toxicity was observed. We assessed the number of patients who received subsequent treatment after discontinuation of TIR and their treatment types. ORR was assessed in patient subgroups according to their characteristics. In addition, patient characteristics associated with 12-month PFS rates were identified using univariate/multivariate logistic regression analysis. The time course of KPS and QoL during the TIR treatment was also assessed. QoL was evaluated using the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, EORTC QLQ-BN20, and EuroQol 5 dimensions. [Results] Of the 44 patients, 6 patients were treated for 3 years. At the time of last visit, 2 patients (4.5%) continued the TIR treatment, 10 (22.7%) discontinued the TIR treatment but had not received subsequent treatment, and 32 (72.7%) received subsequent treatment after discontinuation of TIR. The subsequent treatments included systemic therapy, radiotherapy, hematopoietic stem cell transplantation, and surgery. Major subsequent treatments were MTX-based therapy (n=18) and radiotherapy (n=10) with the 3-year OS rate of 61.1% and 40.0%, respectively. The subgroup analyses of ORR by patient characteristics showed that response to TIR was observed in all patient subgroups; ORR was low in those refractory to the treatment just before TIR (33.3%), those receiving ≥ 4 previous treatments (33.3%), those receiving hematopoietic stem cell transplantation (28.6%), those with high tumor burden (42.9%), and those having mutations in CD79B and MYD88 (40.0%). The univariate/multivariate analyses for the 12-month PFS rate identified that baseline KPS 90-100 was significantly associated with long-term response (odds ratio 5.99, P=0.0289). The median baseline KPS was 80.0 (range: 70~100) and was maintained during the TIR treatment (max: 49 cycles). QoL scores were generally maintained. [Conclusion] The majority of patients were able to receive subsequent treatment, including MTX, after TIR. TIR responses were observed in all patient subpopulations, and the baseline KPS was suggested to be associated with long-term response. KPS and QoL were generally maintained during the long-term observation period.

Establishment of a Dynamic Ctdna Monitoring System to Predict the Prognosis of CAR-T Cell Therapy in R/R B-NHL Patients

Introduction: The objective of this study is to establish a prediction system using circulating tumor DNA (ctDNA) to determine the effectiveness of CAR-T cell therapy in patients with relapsed or refractory B-cell non-Hodgkin lymphoma (R/R B-NHL). Furthermore, the study aims to compare the mutation characteristics between different therapeutic groups to provide guidance for the prevention and follow-up treatment of patients who fail CAR-T cell therapy. Methods: In this part of the study, a total of 79 (192 serum samples) R/R B-NHL patients with CAR-T cell therapy were collected for ctDNA detection (187 lymphoma-related gene panel), including 62 samples before treatment (T0), 69 samples at 1 week (T1) and 62 samples at 4 weeks (T2) after reinfusion. Differences of categorical variables were tested using the chi-square test or Fisher's exact test, and differences in survival events between different groups were compared using Kaplan-Meier analysis and log-rank test. Results: The results of Kaplan-Meier analysis showed that patients with ctDNA mutation genes >10 before CAR-T cell therapy had poorer OS (1-year OS rate: 0% vs 74.9%, 2-year OS rate: 0% vs 68%, P<0.001) and PFS (1-year PFS rate: 0% vs 52.5%, 2-year PFS rate: 0% vs 36.6%, P=0.0056). Patients with MYD88, FAT1 and BTG2 mutation before CAR-T cell therapy had poorer OS, while patients with MUC16 mutation had better OS. The CR rate in patients with TP53 mutation before CAR-T cell treatment was significantly lower than that of patients without TP53 mutation (33.3% vs 68.1%, P=0.02). However, the TP53 mutation status before treatment did not have an impact on the long-term survival of patients. All patients with TP53 mutations at 4 weeks after CAR-T cell therapy failed to achieve CR, and OS was poorer (1-year OS rate: 37.5% vs 66.4%; 2-year OS rate: 12.5% vs 56.3%, P=0.0023). For CR patients, patients with BCR mutation at 4 weeks after treatment had poorer OS (2-year OS rate: 40.9% vs 76.1%, P=0.035). At one week after CAR-T cell therapy(Figure 1), patients without mutations of CDKN2A, CBLB, APC, SPEN, KMT2D, CARD11, FOXO1 and PDGFRB, were more likely to achieve CR (76.6% vs 28.6%, P<0.001), and had better OS (1-year OS rate: 81.5% vs 38.9%, 2-year OS rate: 62.2% vs 5%, P<0.001) and PFS (1-year PFS rate: 67.2% vs 0%, P<0.001). Conclusions: In this study, we evaluated the characteristics of gene mutation between different therapeutic groups, and a gene set was screened to predict the efficacy of CAR-T cell therapy in R/R B-NHL patients, helping clinicians accurately evaluate the efficacy and assisting in decision-making of treatment options.

Advances in Treatment of Waldenström Macroglobulinemia.

The discovery of recurring somatic mutations, in particular MYD88 and CXCR4 mutations, in Waldenström macroglobulinemia (WM), a rare B-cell lymphoproliferative disorder, led in the last decade to the development of several therapeutic agents with high efficacy. This review aims to provide an overview of available treatments in WM and novel agents, focusing on studies published over recent years. There is no international consensus on the best first-line option in treatment-naïve patients. Randomized clinical trials are rare in WM and there has been no prospective comparison of chemoimmunotherapy and BTK inhibitors in the frontline setting. Chemoimmunotherapy and BTK inhibitors, the two feasible and most widely used treatments in first-line treatment, represent very different options in terms of duration of therapy, route of administration, cost, and adverse effect. In addition to tumor genotype and patient comorbidities, choice of therapy in WM should take into account these parameters. Results of ongoing and future clinical trials evaluating fixed-duration combinations with BTK inhibitors and novel agents are awaited.

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

Prognostic impact of MYD88 and TP53 mutations in diffuse large B Cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of lymphoma. It is a highly heterogeneous lymphoid neoplasm, with variations in gene expression profiles and genetic alterations. MYD88 and TP53 genes are common to be expressed and mutated in DLBCL patients with controversy regarding their role in prognosis and survival. This study aims to determine the predictive and prognostic role of MYD88 and TP53 gene mutation in DLBCL. A prospective cohort study was conducted on 50 patients who were diagnosed with DLBCL and 30 healthy individuals to assess the sensitivity and specificity of MYD88 and TP53 genetic mutations. MYD88 and TP53 gene mutations were more sensitive, specific, and accurate in predicting overall mortality and disease progression in comparison with the international prognostic index. Mutant MYD88 and TP53 showed their prognostic importance for worse objective response rates and survival outcomes. Both mutant MYD88 and TP53 were associated with worse ORR. There was a significant statistical difference for both MYD88 and TP53 with regard to 2-year PFS and 2-year OS rate. Hence, both mutant MYD88 and TP53 can be used in predicting disease progression and overall mortality.

Copy Number Variations and Gene Mutations Identified by Multiplex Ligation-Dependent Probe Amplification in Romanian Chronic Lymphocytic Leukemia Patients.

Chronic lymphocytic leukemia (CLL) is known for its wide-ranging clinical and genetic diversity. The study aimed to assess the associations between copy number variations (CNVs) and various biological and clinical features, as well as the survival rates of CLL patients and to evaluate the effectiveness of the multiplex ligation-dependent probe amplification (MLPA) technique in CLL patients.DNA was extracted from 110 patients, and MLPA was performed. Mutations in NOTCH1, SF3B1, and MYD88 were also analyzed. A total of 52 patients showed at least one CNV, 26 had at least one somatic mutation, and 10 presented both, CNVs, and somatic mutations. The most commonly identified CNVs were del(114.3), del(11q22.3), and dup(12q23.2). Other CNVs identified included del(17p13.1), del(14q32.33), dup(10q23.31), and del(19p13.2). One patient was identified with concomitant trisomy 12, 13, and 19. NOTCH1 and SF3B1 mutations were found in 13 patients each, either alone or in combination with other mutations or CNVs, while MYD88 mutation was identified in one patient. Forty-two patients had normal results. Associations between the investigated CNVs and gene mutations and patients' overall survival were found. The presence of NOTCH1 and SF3B1 mutations or the combination of NOTCH1 mutation and CNVs significantly influenced the survival of patients with CLL. Both mutations are frequently associated with different CNVs. Del(13q) is associated with the longest survival rate, while the shortest survival is found in patients with del(17p). Even if MLPA has constraints, it may be used as the primary routine analysis in patients with CLL.

Molecular characteristics in Chinese with chronic lymphocytic leukemia by next-generation sequencing: A single-center retrospective analysis.

Although the prevalence of Asian chronic lymphocytic leukemia (CLL) patients is not as high as that of Caucasians, there are more atypical CLLs in Asia whose genetic characteristics and their clinical significance are distinct and remain unclear. A retrospective analysis of 85 CLL samples in our center was conducted from 2019 to 2022. We used next-generation sequencing with a 172 gene panel to explore the multi-gene mutational data and the mutational status of immunoglobulin heavy variable (IGHV) gene. MYD88 (20.0%) was the most frequently mutated gene, much higher than in Europe, followed in order by TP53 (18.8%), NOTCH1 (14.1%), IGLL5 (11.8%), and DNMT3A (8.2%). In addition, the incidence of ATM and SF3B1 mutations was relatively lower in our centre compared to Europe. Mutated (M)-IGHV patients were more likely to have a cooccurrence of MYD88 mutation, while complex karyotype and DNMT3A mutation were more common in the unmutated (U)-IGHV group. MYD88 mutated CLL was characterized by prevalence in young males in high-risk staging, with isolated 13q deletion and concomitant mutation of IGLL5. CLL patients with MYD88 and TP53 mutation showed an unfavorable prognosis. These results would be valuable in helping to understand the characteristics and significance of cytogenetic genetics in Chinese patients with CLL.

Molecular associations of response to the new-generation BTK inhibitor zanubrutinib in marginal zone lymphoma.

Using tissue whole exome sequencing (WES) and circulating tumor cell-free DNA (ctDNA), this Australasian Leukaemia & Lymphoma Group translational study sought to characterize primary and acquired molecular determinants of response and resistance of marginal zone lymphoma (MZL) to zanubrutinib for patients treated in the MAGNOLIA clinical trial. WES was performed on baseline tumor samples obtained from 18 patients. For 7 patients, ctDNA sequence was interrogated using a bespoke hybrid-capture next-generation sequencing assay for 48 targeted genes. Somatic mutations were correlated with objective response data and survival analysis using Fisher exact test and Kaplan-Meier (log-rank) method, respectively. Baseline WES identified mutations in 33 of 48 (69%) prioritized genes. NF-κB, NOTCH, or B-cell receptor (BCR) pathway genes were implicated in samples from 16 of 18 patients (89%). KMT2D mutations (n= 11) were most common, followed by FAT1 (n= 9), NOTCH1, NOTCH2, TNFAIP3 (n= 5), and MYD88 (n= 4) mutations. MYD88 or TNFAIP3 mutations correlated with improved progression-free survival (PFS). KMT2D mutations trended to worse PFS. Acquired resistance mutations PLCG2 (R665W/R742P) and BTK (C481Y/C481F) were detected in 2 patients whose disease progressed. A BTK E41K noncatalytic activating mutation was identified before treatment in 1 patient who was zanubrutinib-refractory. MYD88, TNFAIP3, and KMT2D mutations correlate with PFS in patients with relapsed/refractory MZL treated with zanubrutinib. Detection of acquired BTK and PLCG2 mutations in ctDNA while on therapy is feasible and may herald clinical disease progression. This trial was registered at https://anzctr.org.au/ as #ACTRN12619000024145.

Waldenström macroglobulinemia: a review of pathogenesis, current treatment, and future prospects.

Waldenström macroglobulinemia (WM) is a chronic B-cell lymphoproliferative disorder characterized by lymphoplasmacytic cell overgrowth in the bone marrow and increased secretion of IgM immunoglobulins into the serum. Patients with WM have a variety of clinical outcomes, including long-term survival but inevitable recurrence. Recent advances in disease knowledge, including molecular and genetic principles with the discovery of MYD88 and CXCR4 mutations, have rapidly increased patient-tolerable treatment options. WM patients may benefit from chemotherapy regimens that include rituximab-based regimens, alkylating drugs, proteasome inhibitors, monoclonal antibodies, and drugs targeting Bruton tyrosine kinase inhibitors. In light of these advancements, patients can now receive treatment customized to their specific clinical characteristics, focusing on enhancing the depth and durability of their response while limiting the adverse effects. Despite the rapidly developing therapeutic armament against WM, a lack of high-quality evidence from extensive phase 3 trials remains a significant challenge in the research. We believe clinical outcomes will keep improving when new medicines are introduced while preserving efficacy and minimizing toxicity.

Primary testicular lymphoma demonstrates overexpression of the Wilms tumor 1 gene and different mRNA and miRNA expression profiles compared to nodal diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) shows a high degree of clinical and biological heterogeneity. Primary testicular lymphoma (PTL) is an extranodal variant of DLBCL associated with a higher risk of recurrence, including contralateral testicles and central nervous system sanctuary sites. Several molecular aberrations, including somatic mutation of MYD88, CD79B, and upregulation of NF-kB, PDL-1, and PDL-2, are thought to contribute to the pathogenesis and poor prognosis of PTL. However, additional biomarkers are needed that may improve the prognosis and help understand the PTL biology and lead to new therapeutic targets. RNA from diagnostic tissue biopsies of the PTL-ABC subtype and matched nodal DLBCL-ABC subtype patients was evaluated by mRNA and miRNA expression. Screening of 730 essential oncogenic genes was performed, and their epigenetic connections were examined using the nCounter PAN-cancer pathway, and Human miRNA assays with the nCounter System (NanoString Technologies). PTL and nodal DLBCL patients were comparable in age, gender, and putative cell of origin (p>0.05). Wilms tumor 1 (WT1) expression in PTL exceeded that in nodal DLBCL (>6-fold; p=0.01, FDR <0.01) and WT1 associated pathway genes THBS4, PTPN5, PLA2G2A, and IFNA17 were upregulated in PTL (>2.0-fold, p<0.01, FDR <0.01). Additionally, miRNAs targeting WT1 (hsa15a-5p, hsa-miR-16-5p, has-miR-361-5p, has-miR-27b-3p, has-miR-199a-5p, has-miR-199b-5p, has-miR-132-3p, and hsa-miR-128-3p) showed higher expression in PTL compared to nodal DLBCL (≥2.0-fold; FDR 0.01). Lower expression of BMP7, LAMB3, GAS1, MMP7, and LAMC2 (>2.0-fold, p<0.01) was observed in PTL compared to nodal DLBCL. This research revealed higher WT1 expression in PTL relative to nodal DLBCL, suggesting that a specific miRNA subset may target WT1 expression and impact the PI3k/Akt pathway in PTL. Further investigation is needed to explore WT1's biological role in PTL and its potential as a therapeutic target.

Indolent lymphoma: Bendamustine, rituximab and acalabrutinib in Waldenstroms Macroglobulinemia (BRAWM)

Background: Waldenström’s macroglobulinaemia (WM) is an uncommon lymphoproliferative disorder. Many options are available, however, an optimal first-line therapy for WM has not be defined. We postulated that combining bendamustine and rituximab (BR) with a next generation BTK inhibitor would result in deeper responses as measured by complete response (CR) and very good partial response (VGPR) rates, and provide a longer duration of response. Objectives: The primary objective of this trial is to document the CR and VGPR rates Methods: The BRAWM clinical trial combines BR with acalabrutinib in a fixed duration treatment course including six cycles of BR and 12 months of acalabrutinib. This trial is taking place at 8 clinical sites across Canada and 33 patients have been enrolled, with a recruitment goal of 59. Results: A pre-defined interim analysis of the first 30 enrolled patients showed; median age of patients was 66; 25 were male; two patients were low risk, 14, intermediate and 15 high risk. Seventeen patients completed combination therapy, 9 completed monotherapy and 2 were followed-up at 18 months (6 months post therapy). Clinical results to date: Two patients discontinued treatment early; 1 at cycle 7 (with a VGPR) but experienced an adverse event requiring treatment; 1 at cycle 3 with possible disease progression. There were 186 treatment related adverse events (TRAEs) among 25 of 30 participants; 5 participants did not experience a TRAE; 163 of these occurred during combination therapy; 23 during monotherapy in 7 of 17 participants, where 10 participants did not experience a TRAE. During combination therapy, 16 of the 163 TRAE’s were grade 3; neutropenia (n = 8), including 2 that were febrile neutropenia, n = 1 for each: atrial fibrillation, transaminitis, cellulitis, fatigue and pulmonary emphysema. An additional five were also considered serious and included febrile neutropenia (n = 2), and n = 1 for each fever, allergic reaction and bowel obstruction. During monotherapy, there were no serious TRAEs. There were 2 grade 3 events during monotherapy in two different patients; decreased neutrophil count, and syncope. There were 21 dose interruptions in 11 participants, all but one of whom returned to regular dosage. Of assessed patients, 20/20 have MyD88 mutations, 4/20 have a CXCR4 mutation, and none had a TP53 mutation. Minimal residual disease (MRD) analysis using next generation sequencing of the IgV regions will be reported. Conclusions: Bendamustine, rituximab and acalabrutinib front-line therapy for WM is safe and well tolerated and initial clinical results show that this treatment induces a high percentage of VGPRs. The research was funded by: AstraZeneca Keywords: Combination Therapies, Indolent non-Hodgkin lymphoma Conflicts of interests pertinent to the abstract. N. L. Berinstein Consultant or advisory role: AstraZeneca Research funding: AstraZeneca, Merck, IMV N. Forward Honoraria: AstraZeneca, AbbVie, BeiGene, Celgene/BMS, IMV, Kite, Janssen, Pfizer, Roche, Servier Research funding: Astellas, AstraZeneca, IMV, Merk, MorphoSys, Seattle Genetics, Roche Other remuneration: Speaker Fees: Pfizer, BeiGene, AstraZeneca M. Shafey Consultant or advisory role: Jansen, Roche Canada, Kite/Gilead, Novartis, BeiGene, Incyte, Abbvie, BMS, AstraZeneca A. Nikonova Consultant or advisory role: Forus, Janssen, Astra Zeneca, Apotex, Incyte Educational grants: Janssen D. MacDonald Honoraria: Abbvie, Astra Zeneca, Beigene, BMS, Incyte, Kite Gilead, Roche, and Seattle Genetics D. Villa Consultant or advisory role: AZ, BeiGene, Janssen, Roche, Kite/Gilead, Merck, BMS/Celgene, ONO Pharmaceuticals. Honoraria: AZ, BeiGene, Janssen, Roche, Kite/Gilead, Merck, BMS/Celgene, ONO Pharmaceuticals. Research funding: Roche, AstraZeneca I. Sandhu Honoraria: Celgene/BMS, Kite/Gilead, Janssen, Sanofi, FORUS, Pfizer M. Aljama Consultant or advisory role: Jansen, Sanofi, Pfizer, Beigene J. Larouche Consultant or advisory role: Incyte, Gilead Research funding: Incyte, Astra-Zeneca, Genmab

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 highly prevalent somatic mutations including MYD88, and CXCR4. The mutation rates of LPL/WM in China and the direct comparison among different deletion means are still not clear. Methods: 387 patients with LPL/WM were included in this study. Tumor cells were collected from 357 un-sorted BM 28 PB and 2 lymph nodes (FFPE). MYD88L265P mutation were detected by Sanger sequencing (FGS), NGS, allele-specific quantitative polymerase chain reaction (ASPCR), and droplet digital polymerase chain reaction (ddPCR). CXCR4 mutation were detected by Sanger sequencing, NGS, and ASPCR. Results: 386 patients were assessed for MYD88L265P mutation, of whom 263 were tested by FGS, 235 by NGS, 311 for AS-PCR, 118 for ddPCR. The MYD88L265P mutation was found in 88.6% of the patients. ASPCR and ddPCR had the highest sensitivity of 97.1% and 96.8%. FGS and NGS failed to detected the mutation in low tumor load. A high false-negative rate (FNR) for MYD88 by FGS and NGS was observed in patients with tumor load less than 10% (FNR: 35.5% and 20%). There was no significant difference in the MYD88 mutation rate detected by ddPCR and ASPCR among patients with different tumor load groups (p = 0.794). MYD88 mutation was detected in a high incidence of 88.5% in less than 1% infiltrated WM tumor cells specimens by ddPCR and ASPCR. CXCR4 mutation testing was performed in 358 patients of LPL/WM, comprising 301 with FGS, 236 with NGS, and 311 with ASPCR. Overall, 31.2% patients were identified as having the CXCR4 mutation. NGS exhibited the highest sensitivity among the three detection means. MYD88 wild-type patients had a significantly lower Hb levels (median 79 g/L vs. 90 g/L, p = 0.015), lower proportion of males (54.5% vs. 51.8%, p = 0.012) and a significantly higher proportion elevated β2-MG (84.2% vs. 65.6%, p = 0.021), and LDH (30.2% vs. 11.9%, p = 0.001). However, there were no significant differences in either PFS or OS between MYD88L265P wild-type and MYD88L265P mutation group (p = 0.35, p = 0.42). Patients with CXCR4 mutation tended to be older (median 64 vs. 60, p < 0.001) and displayed lower hemoglobin level (median 81 g/L vs. 93 g/L, p = 0.008), higher serum IgM level (median 36.1 g/L vs. 31.6 g/L, p < 0.001), and higher baseline BM involvement (by FCM, median 15.1% vs. 7.1%, p < 0.001) compared with CXCR4 wild-type patients. Notably, CXCR4 mutation group had significantly worse survival compared with the wild-type group (p = 0.03, p = 0.02). The research was funded by: National Nature Science Foundation of China (81970187, 82170193, 81920108006, and 81900203) and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2022-I2M-1-022, 2021-I2M-C&T-B-081) Keywords: diagnostic and prognostic biomarkers, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

EXPLORING DIFFERENTIAL MUTATIONAL DISTRIBUTION BY TARGETED SEQUENCING IN A R‐CHOP TREATED COHORT OF DIFFUSE LARGE B‐CELL LYMPHOMA PATIENTS

Background: Diffuse large B-cell lymphoma (DLBCL) harbors striking clinical and molecular heterogeneity, which continues to encumber risk stratification with a consequently high rate of refractory/relapsed patients (pts). Though the genomic landscape of DLBCL has been comprehensively uncovered, the search for eligible molecular biomarkers continues. Exploratively, we set out to retrospectively evaluate differential mutational distributions in a DLBCL cohort using targeted sequencing. Patients and method: A cohort of 105 de novo DLBCL pts, uniformly treated with first-line R-CHOP, were studied. Thirty pts relapsed, of which 70% had early relapses (<2 years after completing therapy), while 30% had late relapses (≥2 years). Targeted next-generation sequencing (90 gene panel) was applied on archival paraffin-embedded diagnostic samples and, if available, first relapse samples. Results: In total, 118 samples were sequenced, each displaying a median of 12 (range 1–46) putative somatic protein-coding mutations. Germinal center B-cell (GCB) DLBCL (n = 60) displayed a differential somatic signature with enrichment of EZH2, TNFRSF14, BCL2, ACTB, SOCS1, and FAS mutations (Fisher's Exact, p < 10-4), whereas mutations in CDKN2A, PRDM1, MYD88, and CD79B were significantly enriched in non-GCB pts (p < 10-4, n = 44). Survival analysis confirmed inferior overall survival (OS) in non-GCB DLBCL (5-year OS, log-rank (Mantel-Cox), p = 0.01, hazard ratio (HR) 1.7). Pts with double-hit biology (DHB), defined as either MYC and BCL2 and/or BCL6 fluorescent in situ hybridized positive and/or with MYC and BCL2 immunohistochemical double expression (n = 32), did not display a significant mutational signature or differential OS. However, a subgroup of DHB pts characterized by mutations in MYC, PRDM1, or IRF4 (n = 15) had markedly inferior OS (5-year OS, log-rank, p = 0.0003, HR 3.34) compared to negative non-DHB pts (n = 55) or negative non-DHB/DHB pts (p = 0.0017, HR 2.7, n = 72). Noticeably, ACTB mutations were only present in non-DHB pts. The mutational distribution at diagnosis did not differ between pts with relapse and in sustained remission. However, in the relapse group, GNA13 mutation at diagnosis was more frequent in early relapses than late relapses (43% vs. 0%, Fisher’s Exact without correction, p = 0.03). The presence of GNA13 in the entire cohort showed a trend towards inferior survival. Conclusion: Differential distribution with significant mutational signatures were expectedly found in GCB versus non-GCB, but not in subgroups with DHB or relapse. Interestingly, mutations in MYC, PRDM1 or IRF4 identified a distinct subset of DHB pts with inferior survival. Furthermore, GNA13 mutations were significantly enriched in early relapse pts. These preliminary findings call for validation of GNA13 as a potential marker of early relapse as well as the prognostic value of MYC, PRDM1 and IRF4 mutations in DHB DLBCL. The research was funded by: The Danish Cancer Society and The Vissing Foundation Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, tumor biology and heterogeneity Conflicts of interests pertinent to the abstract T. S. Larsen Consultant or advisory role Roche, Gilead, Novartis, Celgene/BMS Research funding: Genentech

Real-World Data Analysis of Survival Outcomes and Central Nervous System Relapses in Testicular Diffuse Large B Cell Lymphoma.

Primary testicular lymphoma is a rare type of non-Hodgkin lymphoma, mostly of the diffuse large B cell lymphoma (DLBCL). Although a consensus on standard treatment has been established, unresolved issues remain, such as recurrence in the central nervous system (CNS). We retrospectively analyzed the clinical characteristics and survival outcomes of 65 testicular DLBCL patients according to clinical settings and treatment modalities. The median age of the patients in our study was 65 years, and two-thirds of them had disease limited to one testis. There was no right or left lateralization of testicular involvement. Over a median follow-up of 53.9 months (95% confidence interval 34.0-73.7 months), patients with stage I disease and a low international prognostic index score showed better survival outcomes than those in other categories. Orchiectomy, six cycles of chemotherapy, and radiation therapy (RT) to the contralateral testis demonstrated survival benefits, whereas CNS prophylaxis therapy did not reduce CNS recurrence. During the follow-up period, the survival curves showed continuous decline, mostly due to disease progression. CNS recurrence was observed in 15% of patients, and parenchymal involvement was dominant. However, no factors were associated with CNS recurrence in our analyses. Although our molecular analyses were performed in a small number of patients, MYD88, CD79B, and PIM1 mutations were frequent. In our study, treatment with orchiectomy, six cycles of immunochemotherapy, and contralateral RT was effective. However, because CNS prophylaxis is an essential part of testicular DLBCL management, better treatment strategies than intrathecal therapy are required.