Triple-hit Lymphoma Research Articles

Diagnostic workup of triple hit lymphoma by fine needle aspiration

Abstract Introduction/Objective Triple-hit lymphomas (THL) represent a rare and aggressive subset of high-grade B-cell lymphomas characterized by a triad of translocations involving MYC, BCL2, and BCL6. Patients with THL face poor outcomes when treated with the standard diffuse large B-cell lymphoma protocol. Thus, intensified regimens have emerged as a frontline strategy for managing these patients. Therefore, accurate diagnosis and molecular subclassification of these lymphomas are essential for guiding therapeutic decisions for these patients. Here, we report a case study detailing the diagnostic assessment of THL utilizing limited diagnostic material obtained through palpation-guided fine-needle aspiration (FNA). Methods/Case Report A 58-year-old male presented with a rapidly enlarging neck mass, accompanied by worsening dysphagia, pain, and fatigue over the course of two months. Laboratory testing showed leukocytosis and imaging studies revealed a partially necrotic, 7.8 x 9.1 x 9.4 cm right jugular nodal mass. The patient was referred to otolaryngology for further evaluation, where FNA of the mass was performed by the cytopathology team. The aspirate was sent for cytology with cell block and flow cytometry. The Romanowsky stain revealed abundant, large, discohesive cells with prominent nucleoli and scant cytoplasm with an abundance of lymphoglandular bodies in the background. Immunohistochemistry (IHC) on the cell block demonstrated BCL-2, BCL-6, and MYC expression, and a Ki-67 proliferation index >95%. Concurrent flow cytometric analyses showed an aberrant B-cell population expressing CD19, CD20, CD22, CD23 and surface lambda light chain. Further fluorescence in situ hybridization (FISH) analysis performed on the cell block confirmed gene rearrangements in BCL2, BCL6 and MYC, thereby classifying this patient’s disease as THL. Results (if a Case Study enter NA) NA Conclusion Diagnosing THL typically demands sufficient tissue for histology, cytology, flow cytometry, IHC, and molecular testing. Herein, we showcase a notable example where the diagnostic evaluation of THL was accomplished using limited material obtained from FNA. This case highlights the efficacy of conducting comprehensive diagnostic studies even when tissue availability is constrained. Utilizing cytologic samples not only streamlines the diagnostic process but also minimizes the need for patients to undergo additional invasive procedures, thus potentially reducing delays in diagnosis and therapeutic intervention.

CODOX-M/IVAC-R versus DA-EPOCH-R in double hit/triple hit lymphoma patients aged 60 years or under.

Intensified chemoimmunotherapy regimens are often used in young patients with double hit and triple hit lymphoma (DHL/THL) despite no survival benefit compared to R-CHOP. Favorable retrospective reports on the application of CODOX-M/IVAC-R are subject to selection bias as only young fit patients can tolerate this treatment. We conducted a retrospective analysis to investigate outcome differences between CODOX-M/IVAC-R and DA-EPOCH-R in DHL/THL patients aged 60 years or younger. 113 patients were identified; CODOX-M/IVAC-R (N=49) and DA-EPOCH-R (N=64). 80% (39/49) achieved complete (CR) after completing CODOX-M/IVAC-R compared to 58% (37/64) with DA-EPOCH-R. The median follow-up was 5.3 years and 3.3 years for the CODOX-M/IVAC-R and DA-EPOCH-R group respectively. CODOX-M/IVAC-R demonstrated superior EFS on univariate (HR=0.54, 95%CI=0.31-0.97) and multivariable analysis adjusted for age, BCL translocation (BCL2 vs BCL6 vs both), IPI score and receipt of ASCT (aHR=0.52, 95%CI=0.29-0.93); however there was no significant influence on OS (aHR=0.92, 95%CI=0.46-1.84). The 1, 2 and 5 years EFS in the CODOX-M/IVAC-R group was 68.3%, 64.1% and 61.5% respectively compared to 52.4%, 48.9% and 39.5% respectively in the DA-EPOCH-R group. Primary refractory disease or relapse occurred in 33% (16/49) of CODOX-M/IVAC-R and 54% (35/64) of DA-EPOCH-R recipients, and produced median OS of 10.3 months and 33.7 months, respectively, indicating poor outcomes in the CODOX-M/IVAC-R subgroup with R/R disease. More patients were able to receive subsequent salvage therapies in the DA-EPOCH-R group. No patients died of regimen toxicity and the rates of CNS relapse and therapy related hematologic neoplasms were similar in both groups.

Molecular Classification of Large B-Cell Lymphoma and High-Grade B-Cell Lymphoma Cases and Association with Outcomes in Morocco

Background: High-grade B-cell lymphoma with c-MYC and BCL2 and/or BCL6 rearrangements (HGBL-DHL/THL) is a recently identified category in the most recent World Health Organization (WHO) classification. For all tumors displaying the appearance of diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBL), it is necessary to perform fluorescence in situ hybridization (FISH) in order to achieve an accurate diagnosis. The findings of FISH and immunohistochemistry (IHC) examinations from 50 DLBCL/HGBL samples obtained from Hassan II University Hospital in Fez/Morocco are reported. Methods: This retrospective study included 50 patients diagnosed with DLBCL/HGBL over a period of nine years (2013–2022) and treated with RCHOP chemotherapy protocol. All patients underwent a histological study followed by an immunohistochemical study to confirm the diagnosis and to classify patients according to cell of origin into non-GCB and GCB subtypes; then, a cytogenetic study using FISH was performed to classify patients according to the presence or absence of rearrangements in the c-MYC, BCL2 and BCL6 genes. A comparison was made between the molecular subtypes of DLBCL/HGBL in relation to clinicopathological features and outcomes. Results: Among the 50 cases studied in our population, we found 5 cases of HGBL with DLBCL morphology and 45 cases of DLBCL, which consisted of 13 cases (28.89%) of GCB subtype and 32 cases (71.11%) of non-GCB subtype based on the immunohistochemistry Hans algorithm. After FISH testing of all cases, we found three cases of double-hit lymphoma (DHL) and one case of triple-hit lymphoma (THL). Thus, HGBL-DHL/THL accounted for 8% of the cases. Furthermore, two cases were detected with only one rearrangement in the BCL2 gene and one case harboring a rearrangement in the BCL6 gene. DHL and THL patients and patients with a single rearrangement (BCL2 or BCL6) have a worse prognosis than patients with no rearrangement. Conclusions: DHL and THL are an aggressive entity of HGBL with poorer outcomes in comparison to DLBCL/HGBL NOS. First-line treatment with the RCHOP chemotherapy protocol may not be effective for all aggressive DLBCL cases. More targeted treatment is crucial for better patient outcomes.

MYC/BCL2/BCL6 triple hit lymphoma: Rare finding in cerebrospinal fluid without apparent involvement in bone marrow or peripheral blood

MYC/BCL2/BCL6 triple hit lymphoma: Rare finding in cerebrospinal fluid without apparent involvement in bone marrow or peripheral blood

Molecular Profiling of High-Grade B-Cell Non-Hodgkin Lymphomas and Its Clinicopathologic Correlation in an Indian Tertiary Cancer Care Center

Abstract Introduction Double-expressor and double-hit lymphomas (DHL) are known to be more aggressive and have poor outcomes with standard chemotherapy regimens. Objectives To assess the incidence of DHL and triple-hit lymphomas (THL) and correlate them with clinicopathologic parameters. Materials and Methods Patients who were diagnosed with high-grade lymphomas from April 2021 to September 2022 were prospectively followed up, and details comprising clinical and pathological parameters, including the immunohistochemistry expression status and gene rearrangements of MYC, BCL2, and BCL6, were recorded. Results The incidence of DHL and THL in our study was 16.43%. The separate incidence of the DHL-BCL2, DHL-BCL6, and THL groups was 16.43, 13.69, and 2.73%, respectively. The germinal center B cell subtype of histology was predominantly seen in DHLs. MYC, BCL2, and BCL6 expressions do not correlate well with translocations of these genes. Conclusion Double protein expression cannot be used for screening to decide which patients should undergo fluorescence in situ hybridization, as this would result in missing 4.5% of DHLs.

Abstract 6062: Potent and orally bioavailable BCL6 protein degraders demonstrate efficacy in pre-clinical models of diffuse large B-cell lymphoma (DLBCL)

Abstract The B-cell lymphoma 6 (BCL6) transcription repressor protein is a protooncogene especially in diffuse large B-cell lymphoma (DLBCL), conferring survival, protection, and maintenance of lymphoma cells. BCL6 expression in normal B cells contributes to initiation and maintenance of the germinal centers (GC). Somatic mutations of the BCL6 gene including chromosome translocations have been shown to result in the deregulation of BCL6 expression and contributes to a subgroup of poor prognostic double- and triple-hit lymphomas. To solve this unmet clinical need, we have developed highly specific, potent, and orally bioavailable BCL6 protein degrader HSK43608 that demonstrates potent in vitro and in vivo efficacy in multiple pre-clinical DLBCL models. HSK43608 achieves >90% BCL6 degradation at 10 nM and DC50 of <1 nM in the OCI-Ly1 model following 24 hr treatment. Time-course studies showed >90% BCL6 degradation within 1 hr which could be maintained at 24 hrs. Importantly, medicinal chemistry efforts have resulted in the successful development of new-generation IMIDs to improve the oral bioavailability of BCL6 degrader for in vivo treatment. As expected, concentration of HSK43608 in tumor tissue was much higher than that in plasma in the DLBCL xenograft models. This tumor tissue enrichment of HSK43608 achieved >95% BCL6 degradation 48 hr after last dosing and convincing tumor growth inhibition. It is worth noting that BCL6 knockout mice suffer from myocarditis with eosinophils infiltration in heart muscle. The tissue distribution study demonstrated less HSK43608 in heart than in plasma, partially explained why there was no sign of myocarditis with eosinophils infiltration in the heart of the HSK43608 treated mice. Taking advantage of the new IMIDs structure, HSK43608 barely induce the degradation of the first generation IMIDs targets including GSPT1, SALL4, CK1α, Aiolos and Ikaros, indicating its safety. In summary, these results indicate HSK43608 as a potent and orally bioavailable BCL6 degrader and support further development. Citation Format: Jun Xu, Yangguang Li, Ju Wang, Pingming Tang, Yuting Liao, Chen Zhang, Pangke Yan. Potent and orally bioavailable BCL6 protein degraders demonstrate efficacy in pre-clinical models of diffuse large B-cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6062.

Do Double-Expressor High-Grade B-Cell Lymphomas Really Need Intensified Treatment? A Report from the Real-Life Series of High-Grade B-Cell Lymphomas Treated with Different Therapeutic Protocols at the Institute of Oncology Ljubljana.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements are known for their aggressive clinical course and so are the ones with MYC and BCL2 protein overexpression. The optimal therapy for these lymphomas remains to be elucidated. A retrospective analysis of all diffuse large B-cell lymphomas and high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements diagnosed between 2017 and 2021 at the Institute of Oncology Ljubljana, Slovenia, has been performed. Only patients with double-expressor lymphoma (DEL), double-hit lymphoma (DHL), or triple-hit lymphoma (THL) were included. Demographic and clinical parameters were assessed, as well as progression-free survival (PFS) and overall survival (OS). In total, 161 cases out of 309 (161/309; 52,1%) were classified as DEL. Sixteen patients had DHL, MYC/BCL2 rearrangement was observed in eleven patients, and MYC/BCL6 rearrangement was observed in five patients. Five patients were diagnosed with THL. Out of 154 patients (according to inclusion/exclusion criteria) included in further evaluation, one-hundred and thirty-five patients had double-expressor lymphoma (DEL), sixteen patients had DHL, and three patients had THL. In total, 169 patients were treated with R-CHOP, 10 with R-CHOP and intermediate-dose methotrexate, 19 with R-DA-EPOCH, and 16 with other regimens. The median follow-up was 22 months. The 5-year OS for the whole DEL group was 57.1% (95% CI 45.9-68.3%) and the 5-year PFS was 76.5% (95% CI 72.6-80.4%). The log-rank test disclosed no differences in survival between treatment groups (p = 0.712) while the high-risk international prognostic index (IPI) carried a significantly higher risk of death (HR 7.68, 95% CI 2.32-25.49, p = 0.001). The 5-year OS for DHL patients was 32.4% (95% CI 16.6-48.2%) while all three TH patients were deceased or lost to follow-up. Our analyses of real-life data disclose that the R-CHOP protocol with CNS prophylaxis is a successful and curative treatment for a substantial proportion of DEL patients.

MYC rearrangements in HIV-associated large B-cell lymphomas: EUROMYC, a European retrospective study

AbstractLarge B-cell lymphoma (LBCL) carrying MYC rearrangement, alone or together with BCL2 and/or BCL6 translocations, have shown a poor prognosis when treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in the HIV population. Scanty data are available on the prevalence and prognostic impact of MYC rearrangements in HIV-associated LBCL. We conducted a retrospective study to evaluate the clinical effect of MYC rearrangement in HIV-associated LBCL. We evaluated clinical characteristics, treatment received, and outcome of LBCL in patients with HIV with MYC rearrangement (MYC+) and without MYC rearrangement (MYC–). A total of 155 patients with HIV who had received fluorescence in situ hybridization analysis for MYC were enrolled in 11 European centers: 43 with MYC+ and 112 MYC–. Among patients with MYC, 10 had double-/triple-hit lymphomas, and 33 had isolated MYC rearrangement (single-hit lymphoma). Patients with MYC+ had more frequently advanced stage, >2 extranodal site at presentation, and higher proliferative index. There were no significant differences in overall survival and progression-free survival (PFS) between the 2 groups. However, patients with MYC+ received more frequently intensive chemotherapy (iCT) (44%) than (R)CHOP alone (35%) or infusional treatment (DA-EPOCH-R and R-CDE) (19%). Among patients with MYC+, those who received iCT achieved a better outcome than patients who received nonintensive treatment (complete remission, 84% vs 52%; P = .028; 5-year PFS, 66% vs 36%; P = .021). Our retrospective results suggest that HIV-associated LBCL with MYC+ could be considered for an intensive therapeutic approach whenever possible, whereas (R)CHOP seems to give inferior results in this subset of patients in terms of complete remission and PFS.

Diving Deep into Diagnosis: Unveiling Section High Grade B-cell Lymphoma

The term “lymphoma” comprises a heterogeneous group of biologically and clinically distinct neoplasms that arise from cells in the lymphoid organs. Based on the identification of Reed-Sternberg cells (RS cells), it is divided into Hodgkin and NonHodgkin’s Lymphoma (NHL). Lymphadenopathy is a common presenting feature of lymphoma. Extranodal involvement may show gastrointestinal, cutaneous, neurological, or other symptoms. Hereby, the authors present a case report of 76-year-old male with non-Hodgkin’s lymphoma whose symptoms were masked by the abdominal pain of emphysematous pyelonephritis. Although the infection was treated, the patient continued to complain of dull, aching, persistent abdominal pain. As a result, the patient underwent additional radiological examinations, including a contrast-enhanced Computed Tomography (CT) scan of the abdomenpelvis, which disclosed multiple lymphadenopathies. Hence, an endoscopic ultrasound-guided biopsy was performed from the lymph node between the portal vein and inferior vena cava, revealed High-grade B-cell Lymphoma (HGBL). The prognosis for HGBL with double- and triple-hit lymphomas is extremely bad. The patient that came to us had an early diagnosis of HGBL, which led to a favourable prognosis, in contrast to most cases of the disease, which usually manifest late in the course and are linked with a very grave prognosis.

A Prospective, Exploratory, Phase II Study of the Xpo-1 Inhibitor Selinexor in Combination with RCHOP in Double/Triple-Hit Large B-Cell Lymphoma

Background Double-hit (DH) lymphoma or triple-hit (TH) lymphoma, according to 2016 WHO Classification Criteria, is recognized as a distinct subset of non-Hodgkin lymphoma. The optimal treatment for DH/TH lymphoma remains unclear, although outcomes are inadequate with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) alone. Selinexor is a first-in-class, oral therapeutic that forms a slowly reversible covalent bond with cytoplasm by exportin 1 (XPO1/CRM1), leading to its inactivation. The U.S. Food and Drug Administration has approved selinexor monotherapy for relapsed/refractory DLBCL. Previous studies also suggested that selinexor was used to treat 5 patients with relapsed/refractory DH lymphoma, and 1 patient achieved CR and 2 achieved PR. Here, we report on the efficacy and safety of selinexor in combination with RCHOP as first-line treatment in patients with DH or TH lymphoma. Methods This prospective, phase 2, exploratory study was conducted in Zhejiang Cancer Hospital. The primary endpoint of the study was to evaluate the objective response rate (ORR), including complete remission (CR) rate and partial remission (PR) rate. In addition, diagnostic tumor tissue was prepared to conduct NGS analysis, and dynamic peripheral blood samples were collected before treatment, after 4 cycles when achieving CR from first-line chemotherapy, and progression of disease, to conduct ctDNA analysis. The initial dose of each drug in this regimen is as follows (repeat every 21 days, 6 cycles in total): rituximab: 375mg/m 2, d0; vindesine 4mg, d1; epirubicin 75mg/m 2, d1 (or liposomal doxubicin 35mg/m 2, d1); cyclophosphamide: 750mg/m 2, d1; prednisone: 100mg, d1-5; selinexor 60mg orally once a week. AEs were assessed according to the National Cancer Institute Common Terminology Criteria (CTCAE, version 4.0). Results From May, 2022, 8 patients were enrolled in the study, including 5 females and 3 males. All patients had received at least 2 cycles of selinexor with RCHOP. A full summary of patient baseline characteristics and response to chemotherapy was shown in Table 1. Four patients have completed full cycles of chemotherapy, while 2 patients are still undergoing treatment, and 2 patients suspended subsequent chemotherapy due to rejection of antitumor treatment. A total of 34 cycles of chemotherapy have been completed. Best responses after induction as assessed by positron emission tomography/CT were 5 CRs and 3 PRs. Two patients received ASCT as consolidation therapy after achieving CR. CNS prophylaxis with intrathecal methotrexate was administered to all patients for 2-6 cycles. No patient experienced disease progression, and no patient had CNS involvement during follow-up period (Figure 1A). All patients were assessable for toxicity. All patients in the study received prophylactic granulocyte colony-stimulating factor after each cycle of chemotherapy. The most common hematologic toxicities included anemia (15/34, 44.1%), neutropenia (20/34, 58.8%), thrombocytopenia (13/34, 38.2%), leukopenia (10/34, 29.4%). Other nonhematologic toxicities included fever (6/34, 17.6%), nausea (16/34, 47.1%), vomiting (13/34, 38.2%), fatigue (20/34, 58.9%), and infection (5/34, 14.7%). NGS was conducted successfully in 3 patients, while another 5 patients could not provide NGS data due to insufficient tumor tissue. 139 somatic alterations were detected in 3 patients, with a median of 30 mutations per sample. BCL6/IGLL5/TMSB4X were the most frequently mutated gene in 100% of patients followed by 66.7% who had BTG2/ETS1/EZH2/MYC/NFKBIE/OSBPL10/SOCS1/TNFRSF14 mutations. TP53 mutations were detected in 33.3% of the patient (Figure 1B). In 71.4% (5/7) of the cases, we were able to detect a total of 143 variants in the ctDNA from pretreatment serum samples. 80 variants were detected in both tumor tissue and peripheral blood samples. 4 variants were detected only in the ctDNA, and 59 were detected only in the tissue biopsy. Serial ctDNA monitoring results showed that 100% (8/8) reduction to zero in ctDNA after 4 cycles of chemotherapy. Conclusions Selinexor combined with RCHOP may be an effective first-line regimen for patients with DH/TH lymphoma. Dynamic testing of ctDNA demonstrated deep remission to chemotherapy. Based on the encouraging results of the current study, a large scale, multicenter trial is needed to further investigate this promising regimen.

The Impact of Induction Regimens on Clinical Outcomes in Double and Triple-Hit Lymphoma: A Meta-Analysis of Comparative Studies

Background: Double and triple hit lymphomas (DTHL) are aggressive high-grade lymphomas that harbor translocations of MYC and BCL2 and/or BCL6. They constitute 6-14% of all aggressive B-cell lymphoma and have been associated with poor clinical outcomes. Though there is no established standard therapy for DTHL, induction chemotherapy vacillates between R-CHOP-like regimens, R-EPOCH, or more dose-intense regimens (DI) such as R-hyperCVAD/MA and CODOX-M/IVAC with or without consolidation with stem cell transplant. Currently, there are no randomized trials establishing a standard induction regimen. Furthermore, several retrospective studies reported conflicting results regarding the comparative benefits of these various induction approaches. Therefore, we conducted this meta-analysis to evaluate the impact of induction chemotherapy regimens on the clinical outcomes of DTHL. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language, diagnosis of DTHL, comparative studies of induction chemotherapy regimens, and studies that reported overall (OS) and progression-free (PFS) survival. Studies that reported the aggregate outcome of mixed samples of DTHL and other lymphomas were excluded. A meta-analysis using the fixed effects and random effects models was conducted. Results: Seven retrospective comparative studies with a total of 1433 patients were included. The median age was 62 years, and the median follow-up was 27.5 months. DI regimens were associated with better PFS than R-CHOP-like regimens (HR = 0.81, 95%CI 0.66-0.99, p = 0.039) but without improved OS. R-EPOCH, including R-DA-EPOCH, regimens were associated with better PFS than R-CHOP-like regimens (HR = 0.62, 95%CI 0.43-0.91, p = 0.013) but without improved OS. R-HyperCVAD/MA regimen was associated with better PFS compared to R-CHOP-like regimens (HR = 0.64, 95%CI 0.47-0.87, p = 0.005) but without improved OS. Conclusions: This is the first and largest meta-analysis to show that induction DI regimens, in general, and R-EPOCH and R-HyperCVAD/MA regimens, in particular, are associated with better PFS than R-CHOP-like regimens in patients with DTHL. However, this advantage did not translate into a survival benefit.

Chimeric Antigen Receptor T-Cell Therapy in Elderly Patients with Relapsed or Refractory Large B-Cell Lymphoma: A Multicenter Study

Background: Anti-CD19 chimeric antigen receptor T-cell (CART) therapy is a standard of care in patients (pts) with relapsed or refractory (RR) large B-cell lymphoma (LBCL). However, it remains underutilized in elderly pts due to limited efficacy and safety data. We therefore evaluated real world outcomes of CART in such pts. Methods: Seven academic centers participated in this retrospective study. Pts aged ≥65 years with RR LBCL who received a commercial CART product were included. Baseline characteristics, treatment data, CART toxicity, and clinical outcomes were collected. Progression-free survival (PFS; time from infusion to disease progression or death) and overall survival (OS; time from infusion to death from any cause) were analyzed using Kaplan-Meier method. Cumulative incidences of non-relapse mortality (NRM) and disease relapse/progression were estimated accounting for competing risks. Statistical analyses were performed in JMP v15 and XLSTAT v2022.1.2. Results: 209 pts were identified. Baseline characteristics and treatment pattern are shown in Table. Median age at infusion was 71 years (range 65-89), and 25 (12%) were ≥80 years. 131 (63%) were male, 188 (90%) were white, and 27 (13%) had ECOG PS of ≥2. The median HCT-CI was 1 (range 0-6), and 39 (22%) pts had a score of ≥3. 165 (84%) had stage III-IV disease, 131 (70%) had extranodal involvement, 24 (18%) had bulky disease, and 24 (11%) had high grade B-cell lymphoma (HGBL; double- or triple-hit lymphoma, n=15; HGBL, NOS, n=9). The median number of prior lines of therapy was 2 (range 1-8), and 99 (48%) pts had ≥3 prior lines of therapy, 45 (22%) had prior bendamustine, and 44 (21%) had prior stem cell transplant (SCT; auto-SCT, n=41; allo-SCT, n=2; both auto- and allo-SCT, n=1). Bridging therapy was given to 100 (52%) pts; among those, 57 had chemotherapy, 20 had radiotherapy, 6 had both chemotherapy and radiotherapy, 10 had lenalidomide-based regimen, and 7 had CD20 antibody and/or corticosteroids. Axicabtagene ciloleucel (axi-cel) was most frequently used (63%, n=131), followed by lisocabtagene maraleucel (liso-cel) (24%, n=49) and tisagenlecleucel (tisa-cel) (13%, n=27). Cytokine release syndrome (CRS) occurred in 75% of pts, and a minority (6%) had grade ≥3. Immune effector cell associated neurotoxicity syndrome (ICANS) occurred in 55%, and 29% had grade ≥3. The management of CRS and ICANS included corticosteroids in 101 (49%) pts, tocilizumab in 116 (56%) pts, and other biologic agents (anakinra, siltuximab, basiliximab) in 8 (4%) pts. Day 30 response was evaluable in 195 pts with an objective response rate (ORR) of 84%, and a complete response rate (CR) of 52%. 82% of the responders continued to have a response at 3 month follow up. The ORR/CR rates were 86%/53% for axi-cel, 64%/44% for tisa-cel, and 89%/52% for liso-cel, respectively. At a median follow up of 19.1 months (95% CI 16.1-23.6) after infusion, the median PFS was 6.7 months (95% CI 5.1-11.5), with 6- and 12-month PFS rates of 54% (95% CI 47-61) and 43% (95% CI 35-50), respectively. The median OS was 16.5 months (95% CI 13.2-26.7), and the 6-and 12-month OS rates were 70% (95% CI 64-77) and 61% (95% CI 54-68), respectively. The NRM rate was 17% (95% CI 11-24) at 1 year, primarily due to infections including 5 COVID-19 related deaths. In univariate analysis, ECOG PS ≥2, bulky disease, elevated LDH, and the need for bridging therapy were associated with inferior PFS and OS. No significant difference in PFS and OS was seen based on age at infusion with a median PFS of 6.4 vs 9.6 vs 6.4 vs 6.0 months ( P=0.93) (Figure) and OS of 16.4 vs 19.2 vs 28.5 vs 12.7 months ( P=0.85) in age groups ≥65-69 vs ≥70-74 vs ≥75-79 vs ≥80-89. In addition, there was no statistically significant difference in PFS and OS based on type of CART with a median PFS of 6.4 vs 5.6 vs 12.7 months ( P=0.16) and OS of 15.1 vs vs 16.1 vs NR months ( P=0.24) in pts treated with axi-cel vs tisa-cel vs liso-cel. Conclusions: In this large multicenter study of elderly pts with RR LBCL, CART therapy demonstrated favorable efficacy and safety profile comparable to that in pivotal registration trials. Survival outcomes appeared similar regardless of patient age, including those aged ≥80 years, and type of CART product used. Similar to younger pts, high tumor burden and poor performance status at CART infusion were associated with inferior survival. This analysis confirms that older pts should not be excluded from receiving CART therapy solely due to chronological age.

Survival and CNS Relapse in Patients with CD5-Positive Diffuse Large B-Cell Lymphoma: A Multi-Institutional Observational Study in Japan

Background: CD5-positive diffuse large B-cell lymphoma (CD5+ DLBCL) is usually characterized by activated B-cell type, poor prognosis, and frequent central nervous system (CNS) relapse. A sandwich therapy of dose-adjusted (DA)-EPOCH-R combined with high-dose methotrexate (HD-MTX) (DA-EPOCH-R/HD-MTX) showed excellent efficacy in a phase II study for newly diagnosed stage II-IV CD5+ DLBCL, including 2-year (yr) overall survival (OS), progression-free survival (PFS), and CNS relapse rates of 89%, 79%, and 9%, respectively (Miyazaki K, et al. Haematologica, 2020). It has been introduced in practice in Japan; however, the current status of treatment and the prognosis in patients with CD5+ DLBCL are unclear. Methods: We retrospectively analyzed the prognosis and CNS relapse rate of consecutive patients with untreated CD5+ DLBCL who were diagnosed at 30 hospitals in Japan from January 2016 to December 2021. Patients who were diagnosed with double-/triple-hit lymphoma, those with CNS involvement at diagnosis, and those who had not received any anthracycline-containing chemotherapy with rituximab (R-chemo) were excluded. We identified risk factors for OS using Cox proportional hazards method. The outcomes in the stage II-IV patients who received DA-EPOCH-R/HD-MTX were compared with those of the phase II study. To evaluate the efficacy of CNS prophylaxis, we divided patients into four groups: HD-MTX alone, intrathecal administration of MTX alone (IT MTX), both HD-MTX and IT MTX (HD/IT MTX), and no CNS prophylaxis. Results: Among the413 enrolled patients, 20 were excluded due to ineligible diagnosis or insufficient clinical information, 18 had CNS involvement at diagnosis, and 27 did not receive R-chemo. As a result, 348 patients were eligible and showed the following features: median age, 71 yrs (range, 23-92); male sex, 54%; stage II-IV, 90%; stage III or IV, 73%; ECOG performance status (PS) > 1, 30%; > 1 extranodal site, 42%; elevated serum lactate dehydrogenase (sLDH) level, 71%; high-intermediate or high International Prognostic Index (IPI), 63%; high CNS-IPI, 41%; and B symptoms, 29%. According to Hans's criteria, the nongerminal center B-cell type accounted for 67% of the 276 cases examined. Among the 348 patients, 62 (18%) received DA-EPOCH-R/HD-MTX, and 286 (82%) were treated with other R-chemo, such as R-CHOP (n = 254). The median age was 63.5 yrs (range, 29-75) in the DA-EPOCH-R/HD-MTX group and 72 yrs (range, 23-92) in the other R-chemo group. The DA-EPOCH-R/HD-MTX group included significantly more patients aged ≤ 60 yrs ( P < 0.001), although it tended to have more patients with ECOG PS > 1 than the other R-chemo group ( P = 0.069). Patients who received IT MTX made up 16% of the DA-EPOCH-R/HD-MTX group and 31% in the other R-chemo group. HD-MTX was used in 100% of patients in the DA-EPOCH-R/HD-MTX group and 17% of patients in the other R-chemo group. Details of CNS prophylaxis were HD-MTX alone in 84 patients (24%), IT MTX alone in 70 (20%), both HD/IT MTX in 28 (8%), and no CNS prophylaxis in 166 (48%). At a median follow-up of 43 months, the 2-yr OS, PFS, and CNS relapse rates of patients with stage II-IV disease in the DA-EPOCH-R/HD-MTX group were 87% (95% CI, 73-94%), 78% (95% CI, 63-87%), and 7.3% (95% CI, 2.8-18.2%), respectively. There were no significant differences in OS or PFS between stage I and stage II-IV patients in the DA-EPOCH-R/HD-MTX group. In all 348 patients with stage I-IV, both the OS ( P = 0.044) and PFS ( P = 0.043) in the DA-EPOCH-R/HD-MTX group were longer than those in the other R-chemo group. The 2-yr OS in these groups were 89% and 78%, and their 2-yr PFS were 78% and 64%, respectively. Multivariate analysis identified elevated sLDH ( P = 0.005), > 1 extranodal involvement ( P < 0.001), no IT MTX ( P = 0.005), and no DA-EPOCH-R/HD-MTX ( P = 0.005) as independent risk factors for OS. The 2-yr CNS relapse rate was 6.5% in the DA-EPOCH-R/HD-MTX group (n = 4) and 10.0% in the other R-chemo group. There were no significant differences in the CNS relapse rate between the two groups ( P = 0.54). The 2-yr CNS relapse rates of HD-MTX, IT MTX alone, both HD/IT MTX, and no prophylaxis were 9.8%, 9.1%, 3.7% (n = 1), and 10.3%, respectively. Conclusions: Our results confirmed favorable survival in the phase II study of DA-EPOCH-R/HD-MTX in clinical settings. DA-EPOCH-R/HD-MTX and IT MTX were associated with longer OS of patients with CD5+ DLBCL in our study cohort, warranting further investigation.

RCHOP Plus High-Dose Methotrexate As First-Line Therapy in Large B-Cell Lymphoma with Testis Involvement

Primary testicular lymphoma (PTL) and diffuse large B-cell lymphoma with secondary testicular involvement (DLBCL-T) are rare subtypes of lymphomas associated with poor prognosis and a high risk of central nervous system (CNS) relapse. The optimal CNS prophylaxis strategy for these subtypes remains uncertain. High-dose methotrexate (HD-MTX) is debated for CNS prophylaxis, but its efficacy in this setting is unknown. This study aimed to retrospectively analyze the use of HD-MTX in combination with chemotherapy (R HD-MTX CHOP) as frontline treatment for PTL and DLBCL-T and to compare the biological characteristics of these two forms of testicular involvement. Data from patients who received R HD-MTX CHOP as first-line treatment for DLBCL-T or PTL between 2010 and 2020 were collected retrospectively. The study included 15 patients, 5 with PTL and 10 with DLBCL-T. The overall response rate was 86%, with 73% of patients achieving complete remission. Treatment-related adverse events were generally manageable, with febrile neutropenia being the most common. Two patients experienced CNS relapse, both from the DLBCL-T subgroup, with a median time of 10.7 months from diagnosis to CNS relapse. None of the PTL patients receiving HD-MTX experienced relapse during the median follow-up of 6.08 years (Figure 1A), compared to 8/10 DLBCL-T (p=0.007). All five PTL cases had a non-germinal center phenotype, while 7 out of 10 DLBCL-T cases were non-germinal center. One DLBCL-T case exhibited high-grade lymphoma. Myc expression was positive in 3 out of 10 patients assessed, of whom all were double expressors. Bcl-2 expression was positive in all 13 assessable patients, while Bcl-6 expression was positive in 6 patients. Myc, Bcl-2, and Bcl-6 rearrangements were assessed in 10 patients and were present in 1 patient who was found to have triple-hit lymphoma. No other patient had double-hit lymphoma or any significant rearrangement. Polysomy of Myc, Bcl-2, and/or Bcl-6 was found in 2 patients. PDL1 expression was observed in one out of six patients analyzed, while no PD1 expression was detected. In 8 out of 15 patients with available tissue material, next-generation sequencing using a panel targeting 43 lymphoid genes was performed. Among these 8 patients, 87% exhibited gain-of-function mutations in MYD88 (MYD88 L265P), and 62% had missense mutations in CD79B at position Y196. Multiple mutations in the PIM1 gene, a known target of aberrant somatic hypermutation, were also observed in 7 patients. Non-recurrent mutations were detected in various genes, including PRDM1, CD58, B2M, CREBBP, CIITA, CARD11, CDKN2A, EP300, CCDND3, FOXO1, IRF4, NFKBIE, and TCF3 (Figure 1B). Ig rearrangement was analyzed in 4/9 patients with available DNA. All 4 were clonal, with distinct but mutated VH segments (sequence identity between 81 and 89%). This study suggests that HD-MTX in combination with RCHOP chemotherapy is highly effective in preventing CNS relapse in PTL but less so in DLBCL-T. DLBCL-T had a higher risk of CNS relapse and poorer outcomes compared to PTL. Further studies are needed to determine the optimal timing and sequence of HD-MTX in DLBCL-T, as well as alternative treatment approaches. PTL and DLBCL-T have distinct clinical presentations and responses to frontline chemotherapy with HD-MTX prophylaxis. PTL may benefit from HD-MTX in preventing relapse, while alternative treatments should be explored for DLBCL-T. Future studies should consider PTL and DLBCL-T as separate entities to better understand their characteristics and optimize treatment strategies.

A Genomic Approach for High-Grade B Cell Lymphoma with MYC and BCL2 and / or BCL6 Rearrangements: Two Clusters Stood out on Next Generation Sequencing Results

Introduction High-grade B cell lymphomas with double or triple hit rearrangement (HGBL DH/TH), which appeared on the 4th revised edition WHO classification of lymphoid neoplasms, are aggressive mature B-cell lymphomas with high-grade cytology (Burkitt-like, blastoid or DLBCL-like) that have a MYC (8q24) rearrangement in combination with a BCL2 (18q21) and / or a BCL6 (3q27) rearrangement. There are currently no treatment guidelines for this subtype, and the usual immunochemotherapy leads to few complete remissions. While the NGS technology has been used in diffuse large B cell lymphoma, there has been no specific study on additional genetic events which would predict the clinical outcome in HGBL DH/TH. Here, we report our preliminary results using a targeted DNA capture panel approach (NGS) to identify subgroups with different clinical outcomes among HGBL DH/TH. Methods We conducted a retrospective study on 44 patients diagnosed with DH or TH HGBL and treated at Centre Hospitalier Lyon Sud or Centre Léon Bérard starting in 2013. We collected the medical information regarding the treatment, time to response to treatment or relapse, progression free survival, and overall survival. They all benefited from a DNA-targeted NGS analysis covering 54 genes. Results The cohort included 20 females and 24 males, with a median age of 65 (31-87). Among them, 22 patients were MYC/ BCL2 DH (50%), 12 were MYC/ BCL6 DH (27,3%) and 10 were TH (22,7%). Eleven patients had B symptoms (25%). The Ann Arbor stage was high (III-IV) in 97.5%. The WHO Performance Status was low (0-1) for most of the patients (70%). The majority of them presented with an IPI above to 2 (82,5%) and an elevated LDH level (80%). The Ki67 index was above 70% (33/42) in 78,5% of the cases and above 90% (19/42) in nearly half of them. The major finding of the NGS analysis was the identification of 2 groups based on the TP53/ CDKN2A_p14ARF mutational status. The CDKN2A locus encodes for 2 alternative open reading frames leading to 2 unrelated proteins p14ARF and p16INK4A. P14ARF is involved in the regulation of the TP53 pathway whereas p16INK4A is not. We therefor defined 2 groups : the first group included patients with either TP53 or CDKN2A_p14ARF-only alterations (group A, n=10) while the second group had no alteration in these two coding sequences (group B, n=34) although 12 patients had a CDKN2A_p16INK4A loss or mutation affecting both CDKN2A transcripts. Most of TP53 mutations were missense mutations mostly in TP53 DNA-binding domain and 2 cases appeared to be single hit mutations with an allele frequency below 50%. The patient with a p14ARF only mutation had a start codon mutation p.(M1*) with an allele frequency of 96% without CDKN2A copy number loss. The median overall survival (OS) (Figure 1) was 6,2 months for group A, and 55,5 months for group B (p = 0,0076). The median follow-up time was 14 months (7,5 months in the group A, 18 in group B). Only 2 patients in group A survived and are still in complete remission, one was DH BCL6 and the other DH BCL2 Conclusion Our preliminary results indicate a strong negative prognostic impact of either CDKN2A_p14ARF or TP53 gene alterations on the OS of HGBL DH/TH patients. The biological basis for the difference between groups would be in favour of the alteration of the p14ARF-TP53 tumor suppressive pathway as major contributor of chemoresistance. A more detailed analysis is on-going to further identify mutation profiles of double hit and triple hit lymphomas and compare our finding with existing data. Our results confirm the more favourable outcome of DH BCL6 patients (data not shown), which is consistent with the 5 th revised OMS classification that removed that specific entity. Our results also indicate that routine NGS analysis of HGBL DH/TH patients offers new perspectives for patient stratification. Regarding their survival, patients with TP53 pathway alterations should probably not be treated with the standard RCHOP chemo-immunotherapy. Instead, a more intensive immunochemotherapy regimen or novel strategies such as first-line bispecific antibodies or CARTCells might be considered.

Key Prognostic Factors in Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma: An Evidence Based Systematic Literature and Medical Review

Introduction: A multi-center multi-country retrospective cohort study (ORCHID [NCT05338879]), using real-world data (RWD) from electronic medical records (EMRs) and research databases, for patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) treated with standard of care is being conducted to contextualize treatment outcomes in a single-arm trial. A systematic literature review (SLR) was carried out to identify baseline prognostic factors that will be evaluated for imbalances between cohorts in the single-arm trial and RWD study. This SLR was followed by an evidence-based clinical expert review of prognostic variables and ranking in order of importance for the ORCHID study as described herein. Methods:The approach and methods of the SLR (International Prospective Register of Systematic Reviews [PROSPERO] CRD42022307557) were informed by guidelines set forth by the Cochrane Handbook for Systematic Reviews of Interventions (Higgins et. al. 2021) and PRISMA (Page et. al. BMJ 2021) and described previously (Hampp et. al. ISPOR 2023). Following the conduct of the SLR, the identified potential prognostic variables were evaluated by the ORCHID study team to remove treatment-specific and outcome variables, determine their availability in the single-arm trial and ORCHID RWD study, and develop a questionnaire. In the questionnaire, prognostic variables were grouped by type of variable - clinical, laboratory data, molecular characteristic, or treatment characteristic. Each prognostic variable was reviewed by an international panel consisting of 3 clinical experts in the field of lymphoma who categorized the prognostic impact on treatment response and survival on a 5-point scale ranging from “Very high importance” to “Not important”. The clinicians were asked to consider possible correlation among the variables, possible effect modifiers, specific variable definitions (e.g., early chemoimmunotherapy failure), and whether there were any other prognostically important variables not captured in the questionnaire. For each variable, the clinical experts categorized the availability within routine care on a 3-point scale ranging from “Readily available” to “Limited availability”. The results of the 3 expert completed questionnaires were reviewed and the top 10 highest ranking variables were determined by summing the clinicians' categorization of prognostic impact and considering variable availability in the event of a tie. Individual interviews with each clinical expert were held to clarify variables and definitions, discuss discrepant categorization, and rank the prognostic variables from 1-10. Following the 3 interviews, the ranking of each variable was summed across the 3 experts to determine the final ranking. Results: Across the 46 studies included, the SLR identified 36 prognostic factors, including patient demographics and clinical characteristics, disease and treatment characteristics, and laboratory data. Seven clinical outcomes with statistically significant associations were identified (most commonly, overall survival [OS], progression-free survival [PFS], and overall response rate [ORR]). No additional prognostic factors were identified by the clinical experts. The final ranked list of the 10 most important prognostic variables in descending order of importance includes: early chemoimmunotherapy failure, Eastern Cooperative Oncology Group (ECOG) performance status, refractory to last line of therapy (LoT), number of prior LoTs, double or triple-hit lymphoma, age at start of LoT, Internal Prognostic Index (IPI) risk classification, Ann Arbor disease stage, serum lactate dehydrogenase (LDH), and Deauville score. Conclusion:SLR-based identification of prognostic factors combined with expert clinical review provides an unbiased evaluation and ranking of the level of evidence to assist in selecting prognostic factors to collect for the ORCHID real-world cohort study. The appropriate selection of prognostic factors is critical for valid outcome estimation. These factors will be used for the evaluation of balance with the single-arm trial and for adjustment in a propensity score model. With this approach, the prognostic factors are chosen a priori to enable balancing of baseline covariates prior to outcome estimation.

Preliminary Results of Nathali-01: A First-in-Human Phase I/IIa Study of UCART20x22, a Dual Allogeneic CAR-T Cell Product Targeting CD20 and CD22, in Relapsed or Refractory (R/R) Non-Hodgkin Lymphoma (NHL)

Background: UCART20x22 is a dual targeted allogeneic CAR T-cell product candidate generated from healthy donor T-cells. Donor-derived T-cells are transduced with a bicistronic lentiviral vector to express CARs targeting CD20 and CD22. TALEN ® gene editing technology is used to inactivate the TRAC and CD52 genes to minimize graft-versus-host disease (GvHD) and allow for the use of alemtuzumab (CLLS52, an anti-CD52 monoclonal antibody) in the lymphodepletion (LD) regimen, respectively. UCART20x22 previously demonstrated robust and sustained activity in vitro and in vivo (Aranda-Orgilles B, et al. Cancer Immunol Res 2023). Here we present the initial preliminary results from the NatHaLi-01 trial (NCT05607420), the first-in-human Phase 1/2a dose-finding and expansion study evaluating UCART20x22 in R/R B-cell NHL. Methods: The primary endpoints are safety, tolerability, and determining the recommended phase 2 dose of UCART20x22. Additional endpoints include anti-lymphoma activity and cellular kinetics of UCART20x22. Key eligibility criteria are age 18-80y, lymphoma expression of CD20 and/or CD22 by local assessment, and ≥ 2 prior treatment regimens including autologous CD19 CAR T-cell therapy (CAR19), if eligible. After LD with FCA (fludarabine 30 mg/m 2 × 3d, cyclophosphamide 0.5g/m 2 × 3d, CLLS52 12 mg on D1, 24 mg on D2, D3), a single infusion of UCART20x22 is administered at a flat dose level ([DL1] 50 x 10 6 cells; [DL2] 150 x 10 6 cells; and [DL3] 450 x 10 6 cells). Results: As of 01 July 2023, 3 pts were enrolled and treated at DL1. Pt 1 is a 76yo female with double-expressor diffuse large B-cell lymphoma (DLBCL) relapsed after R-CHOP, radiation therapy, and polatuzumab vedotin with bendamustine/rituximab (BR) and was considered ineligible for CAR19 due to recent high dose bendamustine. Pt 2 is a 65yo female with R/R triple-hit lymphoma transformed from follicular lymphoma who was previously treated with radiation therapy, BR, dose-adjusted R-EPOCH, and two separate CAR19 treatments. Pt 3 is an 18yo female with R/R DLBCL transformed from marginal zone lymphoma who previously received chemoimmunotherapy, venetoclax, ibrutinib, BR, CAR19, obinutuzumab, glofitamab, tafasitamab, lenalidomide, and an experimental epigenetic modifier. Cytokine release syndrome (CRS) occurred in all patients. Pt 1 had G1 CRS and received tocilizumab (toci) x3 and dexamethasone (dex) x1. Pt 2 had G2 CRS and received toci and dex x1. Pt 3 had G1 CRS and received toci x1. All pts' CRS resolved with treatment. No immune effector cell associated neurotoxicity (ICANS) or GvHD was observed. There were no UCART20x22 dose limiting toxicities (DLTs), and there was 1 CLLS52 SAE and 1 DLT. The SAE was G3 CMV reactivation and was managed with IV antivirals. The DLT was in Pt 2, who entered the study with G1/2 cytopenias and a hypocellular bone marrow and had prolonged bone marrow aplasia after treatment thought to be due to cumulative chemotherapy exposure. This pt proceeded to allogeneic hematopoietic stem cell transplant (HSCT) from a matched donor while in complete remission from UCART20x22 treatment. Responses were assessed per Lugano criteria beginning on D28. At D28, all 3 pts responded with 2 CRs and 1 PR. Host lymphocytes remained suppressed through D28 for all pts. UCART20x22 expansion was observed by flow cytometry in 3/3 pts and was initially detected on D7 followed by peaks between D9-14. Peak expansion ranged from 16-682 cells/μL and was predominantly CD8 T-cells. Ferritin levels and increases in serum cytokines, including IFNγ, TNFα, IL-2, and IL-6, correlated closely with UCART20x22 expansion. Summary: Treatment with UCART20x22 at DL1 has been safe and well-tolerated, with no UCART20x22 DLTs or ICANS, and CRS was <G3 in all pts. There was 1 DLT of prolonged bone marrow aplasia in a pt with impaired hematopoietic reserve at study entry. UCART20x22 was active at DL1, with all 3 pts responding, including 2 CRs in pts who had previously received CAR19. UCART20x22 expansion was observed in all pts. These initial data support the continued study of UCART20x22 in R/R NHL, and updated results will be presented.

Outcomes of Patients with Transformed Diffuse Large B-Cell Lymphoma (DLBCL).

There is limited data regarding the results of treating transformed Diffuse Large B-Cell Lymphoma, including the influence of radiation therapy on their overall prognosis. Thus, we aimed to investigate the clinical outcomes of transformed DLBCL patients treated at a single institution. We reviewed clinical features and outcomes of 27 patients with biopsy confirmed transformed Diffuse Large B-cell Lymphoma (DLBCL) treated at a single institution from 2005 and 2014. Response to therapy was based on the Deauville criteria. Kaplan-Meier method was used to estimate time to event outcomes and compared using log-rank tests. Associations between local recurrence and relevant clinical variables were analyzed using logistic regression. The median age of patients was 63 (43-84). Eleven patients (40.7%) presented with bulky disease. The majority of patients (n = 16, 59.3%) had the activated B-cell (ABC) subtype, while 11 (40.7%) patients had germinal center B-cell (GCB) subtype. Most of the patients had early-stage disease 19 (70.4%). Double- and triple-hit expression was noted in 6 (22.2%) and 3 (11.1%) patients, respectively. The median international prognostic index (IPI) was 2. Median follow up was 37 months (range 2-168). Twelve (44.4%) patients received consolidative RT after systemic therapy with a median dose of 36 Gy (30-45). At 72 months, our RT cohort had an overall survival (OS), relapse-free survival (RFS) and local control (LC) rates of 63%, 64% and 81%, respectively. The OS did not differ between patients that received RT compared to those who did not (p = 0.124), although RT conferred statistically significant improvement in OS on MVA (HR 0.07; 95% CI, 0.007 to 0.718; p = 0.025). In the group that received RT, local control was worse among patients with double or triple-hit disease (p = 0.034). Patients with transformed Diffuse Large B-cell Lymphoma treated with RT may have better outcomes than patients who did not receive RT. Presence of translocation (double-hit and/or triple-hit lymphomas) was significantly associated with worse local control in our RT cohort.

Diagnostic and prognostic values of flow cytometry in diffuse large B-cell lymphoma with bone marrow involvement

Objective: To analyze the diagnostic and prognostic values of flow cytometry (FC) in diffuse large B cell lymphoma (DLBCL) with bone marrow involvement (BMI). Methods: The clinical data of 412 patients with newly diagnosed DLBCL, including 243 males and 169 females, aged 64 (28-92) years old, in the Department of Hematology at Peking University Third Hospital from December 2012 to June 2022 were retrospectively analyzed. All patients underwent bone marrow biopsy (BMB) and bone marrow FC. The patients with BMI by FC were further detected by fluorescence in situ hybridization (FISH) for gene analysis. The positive rates and consistency of BMI detected by BMB and FC were evaluated. According to the results of BMB and FC examinations, all patients were divided into four groups: the BMB+FC+group (115 cases), the BMB-FC+group (50 cases), the BMB+FC-group (8 cases, the results did not include in statistical analysis because of small sample size), and the BMB-FC-group (239 cases). The clinical features, treatment response rates, 5-year survival rates, and immunophenotype characteristics by FC in different groups were analyzed. Results: Among the 412 patients with DLBCL, the positivity rates of BMB and FC for BMI detection were 29.9% (123/412) and 40.0% (165/412), respectively. Good consistency between BMB and FC was found (Kappa=0.841, P=0.001). The numbers of extranodal involvement≥2, splenomegaly, huge mass, higher Ki-67 score, higher international prognostic index (IPI) score, thrombocytopenia, and elevated lactate dehydrogenase level were more prevalent in the BMB+FC+group than those in the BMB-FC+group and the BMB-FC-group (all P<0.05). The treatment response rate in BMB+FC+group was 63.5% (73/115), which was lower than those in BMB-FC+group (88.0%, 44/50, P=0.048) and BMB-FC-group (90.0%, 215/239, P=0.032), respectively. The 5-year overall survival rates in three groups were (53.6±9.7) %, (72.5±8.6) %, and (75.2±7.6) %, respectively, with a statistically significant difference (P=0.037). According to the FISH results of bone marrow, 102 cases were diagnosed as not otherwise specified (NOS), 48 cases were diagnosed as double hit lymphoma (DHL), and 15 cases were diagnosed as triple hit lymphoma (THL). Compared with NOS subtypes, the tumor cells in DHL or THL subtypes had higher proportion of increased side scatter (SSC), higher positive rates of CD10 expression, CD38 strong expression and CD56 expression, and lower proportion of surface immunoglobulin light chain restriction (all P<0.05). Conclusions: FC is well consistent with BMB in diagnosing DLBCL with BMI. Combined with FISH detection, FC can contribute to the auxiliary diagnosis and risk stratification for DHL and THL, and provide reference for the prognostic evaluation in DLBCL with BMI.

Chromosomal Aberration t(14;17)(q32;q21) Simultaneously Activates HOXB5 and miR10a in Triple-Hit B-Cell Lymphoma.

BCL2, BCL6 and MYC are major oncogenes in B-cell lymphoma. Their aberrant activation frequently occurs via chromosomal translocations which juxtapose light or heavy chain immunoglobulin (IG) genes to BCL2 and MYC or fuse diverse partner genes with BCL6. So-called double-hit lymphomas usually carry BCL2 and MYC rearrangements, while triple-hit lymphomas additionally bear BCL6-fusions. All these translocations are of diagnostic relevance and usually denote poor prognosis. Here, we genomically characterized classic follicular lymphoma (FL) cell line SC-1, thereby identifying t(14;18)(q32;q21) juxtaposing IGH and BCL2, t(8;14)(q24;q32) juxtaposing IGH and MYC, and t(3;3)(q25;q27) fusing MBNL1 to BCL6. In addition, we found that SC-1 carries a novel chromosomal rearrangement, t(14;17)(q32;q21), which, though present at establishment, has remained unreported until now. We further show that t(14;17)(q32;q21) juxtaposes IGH with the HOXB gene cluster at 17q21 and affect the oncogenic activation of both homeobox gene HOXB5 and neighboring micro-RNA gene miR10a. Moreover, we detected aberrant overexpression of HOXB5 in subsets of Burkitt lymphoma, FL, and multiple myeloma patients, confirming the clinical relevance of its deregulation. In SC-1, HOXB5 activation was additionally supported by co-expression of hematopoietic stem cell factor ZNF521, indicating an aberrant impact in cell differentiation. Functional investigations showed that HOXB5 represses the apoptotic driver BCL2L11 and promotes survival in the presence of etoposide, and that miR10a inhibits BCL6 and may thus play an oncogenic role in later stages of lymphomagenesis. Collectively, we characterize triple-hit B-cell line SC-1 and identify the aberrant expression of HOXB5 and miR10a, both novel oncogenes in B-cell lymphoma.