non-GCB Subtype Research Articles

Clinical Features and Prognosis Analysis of 168 Cases of MYC/BCL2 Double Expression Diffuse Large B Lymphoma

Objective: A retrospective study of the clinical features and prognosis of MYC/BCL2 double-expressor diffuse large B-cell lymphoma (DEL). Methods: A retrospective analysis was conducted on patients diagnosed with MYC/BCL2 double-expressor diffuse large B-cell lymphoma (DEL) for the first time by pathological and fluorescence in situ hybridization (FISH) techniques at Fujian Medical University Affiliated Union Hospital from January 2017 to December 2021. Clinical features, immunohistochemistry, laboratory tests, treatment plans, and follow-up information of all patients were collected. Kaplan-Meier analysis was used for univariate analysis, and Cox proportional hazards model was used for multivariate analysis. Results: Clinical characteristics:Among the 168 patients with diffuse large B-cell lymphoma (DLBCL), 13 cases (7.7%) involved the primary central nervous system. Of the patients, 95 (56.55%) were male and 73 (43.45%) were female, resulting in a male-to-female ratio of 1.3:1. The age of onset was at least 60 years for 93 cases (55.36%), with the youngest being 22 years old and the oldest being 88 years old. The median age was 62 years old. A total of 113 cases (72.90%) were classified as stage III-IV by the Ann Arbor staging system, 40 cases (23.81%) had B symptoms, 82 cases (52.90%) had an International Prognostic Index (IPI) score of 3-4, 84 cases (54.19%) had involvement of two or more extranodal sites, 87 cases (51.79%) had a β2-microglobulin (β2-MG) level of ≥2.53mg/L, 122 cases (72.62%) had a serum albumin (SA) level of <35g/L, and 103 cases (61.31%) had elevated levels of lactate dehydrogenase (LDH).Survival status: Complete follow-up data were available for 162 DEL patients, with a median follow-up time of 20.38 (1-73) months. The median overall survival (OS) was not reached, while the median progression-free survival (PFS) was 37.1 months. Among them, and 58 (34.5%) died. Survival analysis was performed on 168 DEL patients, with 1-year, 3-year, and 5-year OS rates of 82.1%, 61.1%, and 58.9%, respectively. The 1-year, 3-year, and 5-year PFS rates were 69.5%, 51.4%, and 45.6%, respectively. Among the 93 elderly (≥60 years old) DEL patients, the 1-year, 3-year, and 5-year OS rates were 77.2%, 52.7%, and 48.6%, respectively, and the 1-year, 3-year, and 5-year PFS rates were 67.4%, 47.4%, and 43.7%, respectively. The 1-year and 3-year OS rates of the 13 patients with primary central nervous system DLBCL were 25.50%, and the 1-year and 3-year PFS rates were 16.70%.Clinical efficacy analysis: Among 168 patients with DEL, the complete remission (CR) rate was 62.5% (105/168), the partial remission (PR) rate was 3.5% (6/168), the overall effective rate (ORR) was 66.1%, and the relapse/progression rate was 25.6% (43/168). Comparing the efficacy of 88 patients treated with the R-CHOP regimen with 32 patients treated with sildalafil combined with the R-CHOP regimen, the results showed a higher CR rate (81.25% vs. 67.05%) and a lower relapse rate (3.13% vs. 12.5%) in patients treated with sildalafil combined with the R-CHOP regimen.4. Univariate analysis: Age over 60 years ( P=0.031), B symptoms ( P=0.031), advanced Ann Arbor stage (P=0.003), high IPI score ( P<0.001), elevated LDH ( P=0.031), and elevated β2-MG( P<0.001) were significantly correlated with poor prognosis in DEL patients, and were identified as adverse prognostic factors. Non-GCB subtype ( P=0.029), B symptoms ( P<0.001), advanced Ann Arbor stage ( P<0.001), high IPI score ( P<0.001), decreased serum albumin ( P<0.001), elevated LDH (P=0.085), and elevated β2-MG(P<0.001) were significantly associated with poorer progression-free survival (PFS) among DEL patients.5.Multivariate analysis revealed that elevated β2-MG levels and high IPI scores were independent risk factors for poor overall survival (OS) in DEL patients, with HR values of 2.363 and 7.090, respectively. B symptoms, elevated β2-MG levels, and reduced serum albumin (SA) levels were independent risk factors for poor progression-free survival (PFS) in DEL patients, with HR values of 1.819, 2.879, and 1.987, respectively. Conclusion: Elevated β2-microglobulin and high IPI scores in DEL patients are risk factors for OS, while B symptoms, elevated β2-microglobulin, and decreased serum albumin are risk factors for PFS. Keywords: Lymphoma, diffuse large B-cell, clinical features, prognosis, double- expression lymphoma

Ratio of Circulating CD8 + T Lymphocytes to M-MDSCs (CD8MMR): A Novel Prognostic Predictor for Treatment-Naïve DLBCL Patients

Introduction: CD8 + T lymphocytes are crucial in contemporary cell-and-immunotherapies as they can be functionally enhanced or manipulated. Monocytic myeloid-derived suppressor cells (M-MDSCs) are known for their intense immunosuppressive ability, and higher percentages of circulating M-MDSCs among peripheral-blood monocytes or white cells are associated with a worse prognosis. Recently, a low lymphocyte-to-monocyte ratio (LMR) has been linked to a poor prognosis in lymphoma patients. Consequently, we hypothesized that the ratio of circulating CD8 + T lymphocytes to M-MDSCs (CD8MMR) could be a novel and more precise predictor for the clinical outcomes of DLBCL patients before initiating treatment. Methods: This prospective, observational study enrolled 136 adults with treatment-naïve DLBCL from May 2019 to March 2023 in a tertiary medical center in Taiwan. Patients with primary CNS lymphoma or primary mediastinal B-cell lymphoma were excluded. Fresh peripheral blood samples were obtained to calculate the absolute counts of CD8 + T lymphocytes and M-MDSCs, defined as alive singlets with CD45 + CD3 + CD8 + and CD45 + CD15 - CD14 + CD11b + CD33 + HLA-DR low/- by flow cytometry, respectively. ROC analysis and AUC determined the cut-off values for LMR and CD8MMR. Results: For 136 treatment-naïve DLBCL patients, the median age was 70, with 61.8% being male. Among them, 37.5% were in stage IV, and 27.9% were classified as the germinal-center B-cell (GCB) subtype by IHC staining according to the Hans algorithm. Additionally, 43.4% were diagnosed with double expressor lymphoma (DEL). The IPI risk score distribution was as follows: 7.4%, 18.4%, 17.6%, 24.3%, 19.9%, and 12.5% were in scores 0, 1, 2, 3, 4, and 5, respectively. Of note, the median level of LMR was 2.17, the median level of absolute counts of circulating CD8 + T lymphocytes was 385/µL, the median level of absolute counts of circulating M-MDSCs was 102/µL, and the median level of CD8MMR was 3.56. DLBCL patients with high-risk IPI had a significantly lower CD8MMR than patients with intermediate or low-risk IPI (1.34 vs. 4.51 vs. 7.82, PH vs. L = 0.001, PH vs. I = 0.060, PI vs. L = 0.082). DLBCL patients with the non-GCB subtype (2.75 vs. 5.63, P = 0.031) also demonstrated a significantly lower CD8MMR. Additionally, there were trends of lower CD8MMR in elderly patients > 70 (2.74 vs. 3.91, P = 0.209) and patients with DEL (3.33 vs. 4.65, P = 0.101). After a median follow-up of 22.6 months for the 136 DLBCL patients, LMR was at first tested, and patients with LMR < 1.19 had significantly worse PFS (13.8 months vs. non-reach, Log Rank P = 0.004) and OS (non-reach vs. non-reach, Log Rank P = 0.019) than those with LMR ≥ 1.19. Next, we examined the CD8MMR, and patients with CD8MMR < 4.40 experienced significantly worse PFS (21.3 months vs. non-reach, Log Rank P < 0.001) and OS (non-reach vs. non-reach, Log Rank P < 0.001) compared to those with CD8MMR ≥ 4.40 [Figure]. We subsequently conducted a multivariate Cox regression analysis to validate whether CD8MMR < 4.40 could serve as an independent predictor of poor prognosis [Table]. The results showed that “CD8MMR < 4.40” was confirmed as an independent prognostic factor for both PFS (HR = 2.747; 95% CI = 1.275 to 5.918; P = 0.010) and OS (HR = 3.669; 95% CI = 1.462 to 9.211; P = 0.006) after adjusting for confounding factors, including age, sex, IPI risk scores, bulky mass > 10 cm, non-GCB type, DEL, and LMR < 1.19. In this study cohort, 106 (77.9%) patients received R-CHOP-like regimens as induction therapies, 25 (18.4%) patients received an R-EPOCH regimen, two (1.5%) patients received an R-HyperCVAD regimen, and three (2.2%) patients died before completing the first cycle of chemoimmunotherapy. Among the 106 DLBCL patients receiving R-CHOP-like regimens, 57 patients showed CD8MMR < 4.40 and reported a significantly lower rate of complete remission (73.7% vs. 95.9%, P = 0.006) at the end of induction therapy compared to the 49 patients with CD8MMR ≥ 4.40. Furthermore, patients with CD8MMR < 4.40 had significantly worse PFS (17.4 months vs. non-reach, P < 0.001) and OS (non-reach vs. non-reach, P < 0.001) compared to those with CD8MMR ≥ 4.40. Conclusion: A low ratio of circulating CD8 + T lymphocytes to M-MDSCs (CD8MMR) serves as a poor prognostic factor for both PFS and OS in treatment-naïve DLBCL patients. This finding warrants further investigation and highlights the possibility of risk-adapted strategies when treating DLBCL patients.

Zanubrutinib Combined Regimen As Bridging Treatment on Promising Clinical Outcome of CD19 Chimeric Antigen Receptor T-Cell Therapies in Relapsed or Refractory Large B-Cell Lymphoma

Background Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) shows poor clinical outcomes. Anti-CD19 chimeric antigen receptor-T cells (CAR-T) therapy is emerging as the second-line therapy of choice, but in real-world studies its efficacy and long term follow is not promising. Effective bridging therapy can reduce tumor burden and improve CAR-T efficacy. Zanubrutinib, a selective Bruton tyrosine kinase (BTK) inhibitors, combined regimens have established therapeutic activity by targeting B-cell receptor signaling and have individually shown efficacy in patients with R/R DLBCL. Thus, the present study evaluated the efficacy and safety of zanubrutinib-combined regimen as bridging treatment in CD19 CAR-T therapy. Methods This retrospective study includes all patients participant in the clinical trail (NCT 02537977) between January 2020 and December 2022. All 21 heavily treated patients with r/r DLBCL who received zanubrutinib-combined regimens as bridging therapy are included. Patient clinical characteristics were collected upon patient enrollment. Efficacy outcomes included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS), whereas safety outcomes included incidence of adverse events. The zanubrutinib combined regimens are zanubrutinib with IMIDs, chemotherapy and/or rituximab. Results: Of the 21 evaluable patients, median age of patients is 57 year old and median treatment line is 3. 19 patients (90%) are non-GCB subtype and 18 patients(86%) are IPI score >3. 14 patients(67%) are double expression and 7 patients (33%) have TP53 mutation. Objective response rate(ORR) is 81%; of whom, 10 patients(48%) got complete remission(CR) and 7 patients(33%) got partial remission(PR). With a median follow-up of 25 months, the median PFS was 12.8 months and median OS was not yet reached. 8 CR patients and 3 PR patients maintain long-term remission and are still under followup. 18 patients (86%) has grade 1-2 cytokines release syndrome(CRS) and 3 patients(14%) has grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS). No severe CRS and >1 grade ICANS were observed. The most common hematologic toxicities included grade ≥3 neutropenia (90%) and grade 3/4 thrombocytopenia (71%). No patients had grade ≥3 non hematologic toxicities. Conclusion: Zanubrutinib-combined regimens are effective and safe as bridging therapy in CD19 CAR-T therapy. More prospective studies are further command.

Phase II Clinical Trial of R-CHOP Combined with Lenalidomide in the First-Line Treatment for Patients with High Risk Diffuse Large B Cell Lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma (NHL). Currently, R-CHOP (Rituximab-Cyclophosphamide, Epirubicin, Vincristine and Prednisone) is world-widely used in the first-line treatment for DLBCL, producing a long-term survival rate of approximately 60%. The unmet need is focused on high-risk DLBCL patients, having high International Prognostic Index (IPI) scores, non-germinal center-like B cell (non-GCB) origin, double expression of MYC and BCL2 protein, or TP53 aberrations. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of DLBCL by increasing interferon-stimulated gene transcription and activation of immunomodulatory mechanisms. At present, it has been approved for the treatment of multiple myeloma with good efficacy and safety. This is a prospective single arm, multi-center, phase II clinical trial and the goal of our trial is to assess the efficacy and safety of R-CHOP combined with lenalidomide in the first-line treatment for patients with high risk diffuse large B cell lymphoma. Methods: We did an investigator-initiated, open-label, phase 2 trial at the Affiliated Cancer Hospital of Zhengzhou University in China. Patients were eligible if they were aged over 18 years, had histologically confirmed DLBCL. The key inclusion criteria included untreated and medium to high /high risk DLBCL (International Prognostic Index (IPI) score 3-5, aaIPI score 2-3 or NCCN-IPI score≥ 4/ Immunohistochemical staining of double expression (BCL2 ≥ 50% and C-MYC ≥ 40%) or P53 protein mutation positive ≥ 50%). Participants in RL-CHOP cohort were given Lenalidomide orally 25 mg per day on days 1 through 10 of each cycle and delivered concomitantly with standard dose R-CHOP-21 chemotherapy. We also set another group treated with conventional R-CHOP-21 used as a matched contemporary cohort. The primary clinical endpoint was 2-year progression-free survival (PFS). The secondary efficacy endpoints were objective response rate (ORR), 2-year overall survival (OS) and safety. This trial is registered with ClinicalTrials.gov, number NCT04214626. Results: Between December 2019 and January 2023, 63 patients with untreated DLBCL enrolled and were evaluable in the investigator-initiated phase II study. The median age was 59 years (range: 25-76 years); 49.2% of patients were older than age 60 years. 46 of 63 patients (73.02%) had a ECOG score of 0-1; 43 of 63 patients (68.25%) had stage III-IV disease, and 33 of 63 patients (52.38%) had ≥2 extranodal localizations. The IPI score was high-intermediate and high in 73.02% (46/63) of patients. 51 of 63 patients (80.95%) were non-GCB subtype; 25 of 63 patients (39.68%) were double expressor. The TP53 protein was positive in 34.55% (19/55) of patients. In RL-CHOP cohort, the complete response rate was 80.95%, with overall response rate achieving 100%. The median follow-up time was 18.25 (3.73-42) months, median PFS and OS were not reached, and 2-year PFS and OS was 71.1% (+-6.7) and 87.4% (+-4.5), respectively. Comparing with historical matched contemporary cohort, the 2-year PFS and OS rates were improved for medium to high /high risk DLBCL. The most common adverse events of treatment were hematologic toxicities. Grade 3/4 AEs occurred in ≥20% patients were neutropenia, leukopenia, anemia, thrombocytopenia and decreased serum potassium, without bleeding complications. No grade 4 non-hematological adverse event was reported. Conclusion: RL-CHOP is effective and safe in patients with newly diagnosed DLBCL with high risk. Relevance of molecular biomarkers on RL-CHOP response warrants further investigation in DLBCL.

Genetic Subtyping Using Whole Exome Sequencing across Large B Cell Lymphoma Entities in a Series of 109 DLBCL and Plasmablastic Lymphoma Cases

Background: Diffuse Large B Cell Lymphomas are still a significantly heterogeneous disease with few molecularly defined entities. Recently genomic based algorithms have been proposed for DBCL NOS disease classification, including the Lymphgen tool 1. Other large B cell lymphoma entities, including High Grade BCL Dual Hit or plasmablastic lymphoma remain poorly characterized according to this molecular based clustering methods. Here we sought to apply this tool for disease classification in a series of 109 large B cell lymphoma cases derived from GELTAMO clinical trials (NCT01848132 and NCT2015-005390-21) and retrospective multicentric series to confirm its application across different large B cell lymphoma entities. Methods: We retrieved FFPE and liquid biopsy diagnostic samples from 109 cases and performed histopathological confirmation, FISH testing and whole exome sequencing (Agilent Sureselect v6) by NGS. Sequencing data and available fusion data for BCL2 and BCL6 were used to classify the cases according to Lymphgen (https://llmpp.nih.gov/lymphgen). Results: 109 cases were classified as different large B cell lymphoma entities, including 31 DLBCL NOS GCB type, 8 HGBCL/DLBCL DH/NOS, 41 DLBCL NOS non-GCB type, 22 plasmablastic lymphoma, 3 THRLBCL, 1 PMBL, 2 DLBCL NOS and 1 3B Follicular Lymphoma. After WES median Tumor Mutation Burden was 972. Overall genetic classification by Lymphgen was obtained in 53 cases (49%) with the remaining 56 cases considered unclassified (molecular subtype other/NOS). Of the genetically identified cases, 18 were classified as ST2 (33%), 15 were considered EZB (28%), 5 BN2 (10%) and 3 MCD (5%). Interestingly 12 (22%, overall, 11%) were genetically composite, including 8, ST2/EZB; 1, ST2/BN2; 1, ST2/MCD and 2 ST2/EZB/MCD. According to histopathological subtype 17 GCB type DLBCL were classified as EZB (8, 47%), ST2 (5, 30%) and BN2 (1, 6%). 3 cases (17%) were genetically composite. 6 out of 8 cases of HGBCL/DLBCL DH/NOS were classified as either EZB (2), ST2 (2) or composite EZB/ST2 (2). 19 non-GCB type DLBCL were classified as ST2 (5, 26%), BN2 (4, 21 %), EZB (3, 16 %) and MCD (3, 16%). 4 cases were genetically composite (21%). Interestingly, plasmablastic lymphomas showed ST2 genetic features, found in 5 out of 22 (23%) and composite ST2/EZB in 2 cases. Genes overrepresented in the ST2 cluster include TET2, PRRC2C, STAT3,DOCK8 and CLTC. Plasmablastic Lymphomas classified as other showed recurrent mutations in PRRC2C, PABPC1, TRRAP, PRKCB, EP300 and TP53. Conclusions: ST2 and EZB genetic groups stand as the most frequent genetic subtypes of DLBCL NOS. While ST2 is found in either GCB and non-GCB subtypes, EZB is found preferentially in cases with the GCB phenotype, including GCB type DLBCL and HGBCL/DLBCL DH. In comparison with already published series we found a relatively higher prevalence of the ST2 genetic subtype among DLBCL NOS. MCD and BN2 genetic subtypes are restricted to the non-GCB subtype, but its prevalence is relatively low. Importantly, a significant fraction of cases (~50%) is still genetically unclassified (molecular subtype NOS) using WES and FISH-derived fusion data. Proper identification of the A53 cluster and relevant dysregulated pathways in the molecular NOS group require more research. Specific large B cell lymphoma entities such as plasmablastic lymphoma show features of ST2 genetic subtype in a significant fraction of tumors. These features include JAK/STAT3 pathway activation related genes and fit well with already published data 2,3. In addition, recurrent mutations in TET2, involved in epigenetic regulation of gene expression and TRRAP, target of C-MYC transcriptional activation, were enriched in this molecular subtype of plasmablastic lymphoma. Importantly ~70% remained unclassified. Mutations in this group are recurrently found in PRRC2C as well as PABPC1, both involved in VEGFA-VEGFR2 signaling. In summary, DLBCL NOS molecular subtyping supplies exact molecular classification in ~50% of our cohort. ST2 and EZB subtypes constitute ~60% of the cases with a fraction of ~15% of composite ST2/EZB. Application of the molecular classifier to plasmablastic lymphomas shows intraspecific genetic diversity and points toward dysregulation of JAK/STAT, MAPK/Erk and VEGFA-VEGFR2 signaling as well as MYC transcriptional activation. REFERENCES 1. PMID: 32289277 2. PMID: 33225311 3. PMID: 32273478

Molecular Monitoring with Tumor DNA and Nivolumab Maintenance: A Pilot Study in Diffuse Large B-Cell Lymphoma

Introduction Molecular minimal residual disease (MRD) has been shown to correlate with disease relapse in patients (pts) with Diffuse Large B cell Lymphoma (DLBCL)[1-4]. However, the clinical impact of detectable MRD (dMRD) prior to clinical relapse is unknown. Checkpoint inhibitor (CPI) therapy has some activity in certain pts with DLBCL, particularly after CAR-T therapy attributed to effects on T cell activity [5-8]. We hypothesized that early intervention with CPI may lower relapse rate in pts with molecular recurrence only. Patients were monitored for detectable MRD (dMRD) by circulating tumor DNA (ctDNA) using the clonoSEQ assay monthly. We conducted a pilot study in high risk pts in remission after most recent line of therapy (LOT) in which maintenance nivolumab (MN) was offered at time of dMRD to assess effect on MRD clearance and relapse rate. Methods Pts >18 years with high-risk DLBCL (ABC-subtype, high grade B cell lymphoma (HGBCL), MYC translocation, relapsed/refractory disease, or Ki67 >90%) who had achieved CR on most recent LOT. Pts had monthly MRD and every 4 month CT or PET/CT and exam for 2 years. If conversion to dMRD without clinical relapse, pts received MN (240 mg IV q2 weeks) until clinical relapse or toxicity for up to 2 years (26 cycles). Pts with overt relapse at time of dMRD were ineligible for MN and taken off trial. The primary endpoint was rate of conversion from +MRD to undetectable MRD (uMRD) in pts receiving . Results 20 pts were screened and 15 pts were enrolled between June 2018 and March 2022. 5 pts had non-GCB subtype, 2 had GCB subtype, and two had unknown cell of origin. 4 had transformed DLBCL (2 from FL, 1 from MZL, and 1 Richter's), 2 pts had HGBCL (one of which was also transformed). Of the 8 pts who enrolled after 1 prior LOT, none had molecular or clinical relapse (Figure). In 7 pts who enrolled after 2+ prior LOT, 3 had dMRD. 2/3 had clinical relapse at same time as dMRD and were taken off study– one at initial enrollment with dMRD at first ctDNA screening and one converting from uMRD to dMRD after 135 days post enrollment. In the single patient who was eligible for MN, dMRD preceded radiographic relapse by 105 days, 410 days after enrollment. The pt went on to receive 2 cycles of MN before clinical progression and was taken off study. This pt failed to achieve conversion of dMRD to uMRD. Conclusions Due to small sample size and lower than expected relapse rates, we were unable to demonstrate conversion of dMRD to uMRD using MN. Of the 3 pts with dMRD, 2 were not eligible for MN due to clinical relapse at same time as dMRD and both died due to lymphoma progression. In the single pt who received MN, this intervention did not achieve uMRD or prevent relapse. However, this pt went on to achieve CR to subsequent CAR-T and remains in prolonged remission despite multiple relapsed/refractory disease. It is unknown if this patient's positive outcome was impacted by the effect of nivolumab on T cells and/or related to slower disease kinetics as this pt had a 3 month period between dMRD and radiographic detection and long duration of response to prior lines. A larger study is needed to adequately assess impact of early intervention MN using MRD kinetics, particularly in patients receiving CAR-T. All pts with dMRD had radiographic relapse within 4 months of molecular relapse. No pts with uMRD by ctDNA had clinical relapse on study during 2 year follow up, confirming uMRD correlates with disease free survival. If a strong negative predictive value is confirmed in a larger study, ctDNA MRD testing may be used to spare pts from imaging or to guide additional work up in pts with inconclusive findings on scans.

High Prevalence of MYD88 and CD79B Mutations and PD-L1 Alterations in Bone Marrow Large B Cell Lymphoma Associated with Hemophagocytic Lymphohistiocytosis

Introduction: Bone marrow large B cell lymphoma associated with hemophagocytic lymphohistiocytosis (HLH) is a rare disease with a highly aggressive clinical course. Until now, the molecular genomics of bone marrow large B cell lymphoma associated with HLH remains undetermined. Methods: Cases of bone marrow large B cell lymphoma with HLH were included. The inclusion criteria were as follows: (1) diagnosis of large B cell lymphoma presenting initially in the bone marrow; (2) no overt lymph node involvement or tumor formation; (3) fulfillment of five or more of the eight HLH-2004 diagnostic criteria. (4) cases with available samples. Genomic DNA was extracted from bone marrow cells or the plasma. A panel comprising 475 hematological malignancy-related genes were used for targeted gene sequencing. The prepared library was sequenced using Illumina HiSeq 4000. Molecular classification was predicted by using the LymphGen algorithm. Results: A total of 23 cases were included. The median age was 63. 71.4% of patients were the non-GCB subtype (10/14) based on the Hans algorithm and 50% (9/18) of cases showed CD5 expression. Somatic mutations were identified in all the cases. The most common disrupted genes were PIM1 (12/23, 52.2%), PD-L1 (9/23, 39.1%), MYD88 (8/23, 34.8%), BCL6 (6/23, 26.1%), CD79B (6/23, 26.1%), IRF4 (6/23, 26.1%) and TET2 (6/23, 26.1%) (Figure 1). For nine cases with PD-L1 aberrations, seven cases had PD-L1 structural variants (SVs) and one case showed PD-L1 amplification (Figure 2). All the six cases with BCL6 aberrations showed BCL6 SVs (Figure 2). For six cases with BCL6 SVs, four cases showed immunoglobulin heavy chain ( IGH)- BCL6 translocations and two cases harbored IKZF1- BCL6 translocations. Regarding molecular classification, eight cases (34.8%) were classified as the MCD subtype, two cases the BN2 subtype, two cases the ST2 subtype, and two cases the composite subtype. Conclusion: To the best of our knowledge, this is the first study exploring the molecular genetics of bone marrow large B cell lymphoma with HLH. Our study suggesting bone marrow large B cell lymphoma with HLH showed frequent MYD88/CD79B and PD-L1 alterations. Our study provided clues for the pathogenesis of bone marrow large B cell lymphoma with HLH and identified potential druggable targets for future clinical trials.

The significance of concurrent MYC and BCL2 expression in Egyptian patients with diffuse large B-cell NHL

This study aimed to investigate the frequency of MYC/BCL2 co-expression using immunohistochemistry (IHC) in Egyptian patients with diffuse large B-cell lymphoma (DLBCL) and its impact on treatment outcomes, overall survival (OS), and disease-free survival (DFS). A total of 100 adult patients with newly diagnosed DLBCL were included in the study and treated with the R-CHOP regimen. The cases were classified into germinal center B-cell (GCB) and non-germinal center B-cell (non-GCB) subtypes based on IHC. Our results showed that a subset of the studied cases didn’t achieve complete remission (CR) after chemotherapy (22%). Co-expression of MYC and BCL2 was observed in 20% of the cases, with most of them belonging to the non-GCB subtype. The CR rate was significantly lower in the double-expressor (DE) group (45%), while no significant difference was observed in CR between the GCB and non-GCB cases. The DE group was associated with advanced stage, extra-nodal involvement, and high-risk R-International Prognostic Index (R-IPI). Both the non-DE and GCB groups demonstrated better DFS, while the non-DE group only showed better OS. Both non-DE status and GCB subtype were identified as independent factors affecting DFS. In conclusion, the concurrent expression of MYC and BCL2 in DLBCL indicates an inferior outcome and lower chemotherapy response. The current chemotherapeutic strategy has not effectively improved the outcomes of this aggressive lymphoma subtype. Therefore, the identification of these patients is crucial for selecting appropriate treatment options.

The ZR2-Minichop Study：Zanubrutinib, Lenalidomide, Rituximab, Cyclophosphamide, Vincristine, Epirubicin and Prednisone in Elderly Patients with Previously Untreated Diffuse Large B-Cell Lymphoma

Background: Diffuse large B cell lymphoma (DLBCL) is common in the elderly group. New schedules are required due to the toxicity in classic chemotherapy regimens. Previous studies reported that non-germinal center B cell (non-GCB) subtype aggregates in elderly patients with abnormality in the activated B cell receptor signal and nuclear factor kappa-B pathway. We conducted a prospective trial to investigate the efficacy and safety of the ZR2-miniCHOP schedule (zanubrutinib, lenalidomide, rituximab, cyclophosphamide, vincristine, epirubicin and prednisone) in elder patients newly diagnosed with non-GCB DLBCL. Methods: This multicenter, single-arm, phase II clinical study started in June 2021 at 6 centers in China. Eligible patients were newly diagnosed non-GCB DLBCL, aged 60 to 80 with Eastern Cooperation Oncology Group (ECOG) performance status over 2, or patients aged over 80 with ECOG status of 0-4. The purpose of this study is to discuss the response, safety and long-term survival with the primary endpoint of objective response rate (ORR) and the secondary endpoint of overall survival (OS) and progression free survival (PFS). Patients would receive 2 cycles induction period with zanubrutinib 160mg twice per day on d1-28, lenalidomide 25mg d1-10 and rituximab 375mg/m 2 d1, after then combine 4-6 cycles miniCHOP schedule as: cyclophosphamide 400mg/m 2 d2, vincristine 1.4mg/m 2 d2, epirubicin 35mg/m 2 d2 and prednisone 45mg/m 2 d2-6. Evaluation is conducted after 6 cycles of treatment including the ORR and CR rate by positron emission tomography-computed tomography (PET-CT) and 2-years OS and PFS. Results: At the data collection duration from June 2021 to June 2023, 45 patients were recruited in this trial. 34 patients were considered for result analysis as 2 withdrawals and 1 drop-out. 6 patients are still in the process of treatment. The median age of the 45 patients enrolled was 79 (range 65-90) years, with 34 (75.6%) patients over 75 years old. The ORR was 91.2% (31/34). Among the 34 assessable patients, 61.8% (21/34) of them reached CR after 6 cycles routine therapy. Besides, 29.4% (10/34) patients just achieved PR, and for these patients we then continued administered more 2 cycles with ZR2+miniCHOP. Later 5 of these 10 patients achieved CR. One patient stayed SD and 3 patients appeared disease progressing. The most common hematology-relevant adverse events (AEs) were neutropenia (13/34) and thrombocytopenia (10/34). Eleven patients appeared AEs over grade 3 occurred during therapy with neutropenia, thrombocytopenia, infectious pneumonia events. All these patients have been administrated the normal dose as the treatment resistance and the toxicity did not affect therapy. Two patients died of progressive disease. No treatment-associated death was observed. Generally analyzing, this trial achieved acceptable response and safety. Conclusion: The ZR2-miniCHOP schedule represented satisfactory efficacy and safety in elderly non-GCB DLBCL.

Clinical and Molecular Features of Patients with Double/Triple Hit Large B-Cell Lymphoma

Background: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) is a recently defined category in the latest revision of the World Health Organization (WHO) classification, which comprises approximately 5-15% of newly diagnosed DLBCL. This subtype of lymphoma poses numerous challenges from diagnosis to treatment of patients. On the one hand, diagnosis of HGBL-DH/TH requires the accomplishment of fluorescent in-situ hybridization (FISH) to identify the translocations of MYC(8q24) and BCL2(18q21) or/and BCL6(3q27), with no consensus being reached regarding the most applicable population for FISH tests due to high cost and low prevalence. On the other hand, HGBL-DH/TH displays aggressive features, including a high incidence of advanced disease at diagnosis, and inferior efficacy to standard-induced chemical immunotherapy with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Despite several treatment strategies including intensified protocols and hematopoietic cell transplantation (HCT) have been explored, there is no standard regimen to improve outcomes for HGBL patients, and participation in clinical trials is encouraged. Aims: This study aims to investigate the clinical characteristics and prognostic outcomes of HGBL-DH/TH patients, and performed genomic and transcriptomic analyses to link the oncogenic mutations and tumor microenvironment alterations to different DLBCL subgroups. Method:In this study, 107 patients with newly diagnosed HGBL-DH/TH were analyzed. Histological diagnoses were established according to the WHO classification. These HGBL-DH/THs were termed DHL-BCL2 (MYC and BCL2 translocation), DHL-BCL6 (MYC and BCL6 translocation), and THL in our study. DNA sequencing was carried out on 95 patients, for detection of genetic aberrations. Results: Patients with DHL-BCL2 (n=44) and THL (n=15) have similar clinical features, including elevated serum lactate dehydrogenase (LDH), increased proportion of double expression (DE) as well as increased prevalence of non-GCB subtype compared to those with DHL-BCL6 (n=48). With the median follow-up time of 19.3 months (2.5-114.2 months), patients in the DHL-BCL2 and THL group had dismal survival (2-year PFS: 49.3% and 34.2%; 2-year OS: 64.0% and 55.6%) when compared with those in the DHL-BCL6 group (2-year PFS: 63.5%; 2-year OS: 89.3%) (Figure A). Moreover, intensive chemoimmunotherapy or combination with novel targeted agents seemed to improve outcomes in patients with DHL/THL compared with those who received a standard R-CHOP regime. The use of autologous stem cell transplant (ASCT) did not yield a significant improvement in survival for patients who achieved remission after receiving first-line R-DA-EDOCH therapy. However, it is worth noting that there was a trend towards prolonging progression-free survival (PFS) and overall survival (OS) in patients who achieve complete remission (CR) through RCHOP-based treatment and undergo ASCT (Figure B). Compared to DHL-BCL6, the primary genetic lesions in DHL-BCL2 and THL were alterations associating epigenetic regulators like EZH2 and CREBBP, while the primary genetic lesions in DHL-BCL6 were alterations associating cellular differentiation and transcription factors like BTG2, PTPN6, CD70, and BCL6. Correlatively, the EZB genotype was more frequently observed in patients with DHL-BCL2 and THL, while the BN2 genotype was more frequently observed in patients with DHL-BCL6. Conclusion: DHL-BCL2/THL exhibits similar clinical characteristics, prognostic outcomes, and molecular features that set it apart from DHL-BCL6. This implies the need for testing novel agents or therapeutic strategies to expedite treatment development for patients with DHL-BCL2/THL.

The Activation Status of RSK2, but Not PDPK1 and AKT, Associates with the Prognosis of Diffuse Large B Cell Lymphoma

B-cell lymphoma (BCL) is the most common hematologic malignancy with various genetically and molecularly distinct subtypes. Although the prognosis of most BCL subtypes has been dramatically improved by the advances of disease subtype-directed, molecularly targeted therapies, some patients remain incurable with currently available therapeutics. We previously identified that a serine/threonine kinase 3-phosphoinositide-dependent kinase-1 (PDPK1) and its major downstream substrate serine/threonine kinases, AKT and RSK2, play pivotal roles in the pathophysiology of multiple myeloma and mantle cell lymphoma (MCL) (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012; Matsumura-Kimoto Y, Cancer Med 2020). In particular, we found the crucial roles of the constitutive activation of the N-terminal kinase domain (NTKD) of RSK2 in cell survival, resistance to apoptosis, and cell proliferation of myeloma cells and MCL cells. However, the activation status of PDPK1, AKT, and RSK2-NTKD and their impacts on prognosis have yet to be comprehensively investigated in various subtypes of BCLs. To answer this, we here investigated the activation status of PDPK1, AKT, and RSK2-NTKD and their prognostic impacts in two major subtypes of BCLs, diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). To evaluate the activation status of the three kinases in DLBCL and FL, we performed the immunohistochemical analyses to characterize the expression profiles of phosphorylated (p)-PDPK1, p-AKT, and p-RSK2-NTKD in tumor-biopsied samples from 277 pts with DLBCL and 121 patients (pts) with FL diagnosed between 2010 and 2022 at our institution, and the expression levels of three target kinases were classified into four categories; negative (0), weak (1), moderate (2), and strong (3). As a result, in DLBCL, p-PDPK1, p-AKT, and p-RSK2 were positive in 98%, 68%, and 100%, respectively. More precisely, p-PDPK1 was 0-1 in 160 (59%) and 2-3 in 111 (41%). p-AKT was 0-1 in 225 (82%), and 2-3 in 48 (18%). p-RSK2 was 1 in 177 (65%), and 2-3 in 96 (35%). OS was significantly shorter in patients with moderate/strong expression (2-3) of p-RSK2 than those with weak expression (1) of p-RSK2 (5-year OS rate 52% vs. 73%, P<0.01), and, importantly, p-RSK2 status was identified as the independent prognostic factor by the multivariate analysis evaluating p-RSK2 expression and elements included in the International Prognostic Index as variables (HR 1.6, P=0.03). In addition, the prognostic relevance of RSK2 activity was evident in non-GCB type DLBCL but not GCB type DLBCL. In FL, p-PDPK1, p-AKT, and p-RSK2 were positive in 76%, 53%, and 69%, respectively. More precisely, p-PDPK1 status was 0-1 in 94 (89%) and 2-3 in 11 (10%), p-AKT status was 0-1 in 81 (77%) and 2-3 in 24 (23%)), and p-RSK2 was 0-1 in 67 (64%), and 2-3 in 38 (36%). In contrast to the case with DLBCL, the OS of FL was not associated with the activation status of p-PDPK1, p-AKT, and p-RSK2. In conclusion, the present analysis suggests the functional involvement of p-RSK2 activity and the role of future therapeutic targeting, especially in DLBCL, especially in high-risk non-GCB subtype.

Development and Validation of a Clinical Prediction Model for the Efficacy of CD19 CAR T-Cell Therapy in DLBCL

Currently, Autologous CD19 chimeric antigen receptor T-cell therapy (CART) has become the most important treatment modality for relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) beyond conventional multi-line treatments. However, it is worth noting that over 60% of CD19 CAR-T recipients will eventually experience relapse. A substantial amount of clinical research has been conducted to explore the reasons for the ineffectiveness or relapse of CAR-T therapy. Based on a comprehensive analysis of clinical data, we selected various combinations of predictive factors and used the TRIPOD guidelines to construct a logistic regression model for predicting the efficacy of CD19 CAR-T therapy. This study was conducted as a retrospective cohort study, including patients with confirmed diagnosis of DLBCL who underwent CD19 CAR-T therapy and met the following criteria: (1) aged 18 years or older but not exceeding 80 years; (2) voluntarily participated in the study and signed an informed consent form. The exclusion criteria for this study were: (1) addition of other chemotherapy drugs or targeted drugs within 90 days before assessing the best response after infusion, except for those who achieved complete response (CR) or progressive disease (PD); (2) death caused by non-tumor progression factors within 90 days after infusion. After the inclusion and exclusion criteria, a total of 54 patients with DLBCL undergoing CD19 CART at Shanghai Tongji Hospital of Tongji University ultimately participated in this study from January 1, 2019, to April 31, 2023. The derivation cohort consisted of 45 patients enrolled in clinical trials (NCT02537977, NCT03154775, CTR20201986, CTR20200561). The median age of the patients in the derivation cohort was 60 years (IQR: 50-67 years). Out of the 45 participants, 25 (55.6%) were male, and 29 (64.4%) had the non-germinal center B-cell (non-GCB) subtype, 1 had grade 3-4 Cytokine Release Syndrome (CRS). The validation cohort consisted of 9 patients involved in real-world studies. The median age of the patients in the validation cohort was 55 years (IQR: 52.5-69 years). Out of the 9 participants, 5 (55.6%) were male, and 6 (66.7%) had the non-GCB subtype, 2 had grade 3-4 CRS. The demographic and clinical characteristics of the derivation cohort were similar to those of the validation cohort. Based on the best CD19 CART response at 90 days, we divided the derivation cohort into two groups, CR and non-CR. Then, we selected a series of candidate predictive factors from baseline data such as clinical features, genetic features, and laboratory features before CART (Table 1). The results of univariate and multivariate logistic regression analysis were used to select specific factors (Gender, ECOG, TP53) as model prediction indicators based on p-values (p<0.05). There was no significant correlation between these factors (p>0.1). A score nomogram (Model1) was plotted using the regression coefficients of each predictive factor as weights (Fig.1 A), C-index (Model1) =0.872 (Fig.1 C). To facilitate better clinical use, we also constructed a risk nomogram (Model2) to assess the degree of risk (Fig.1 D). Model2 considers three factors: Male, ECOG≥2, TP53 altered. If a person has not any of these risk factors, they are categorized as low risk. If there is one risk factor present, they are deemed to be at moderate risk, whereas having two or more risk factors classifies them as high risk. C-index (Model2) =0.845 (Fig.1 F). Finally, the model's clinical efficacy was validated using real-world CART-treated patients, and the C-index was calculated as Model1=0.875 (Fig.1 G), Model2=0.7 (Fig.1 H), confirming the clinical impact of the model. In conclusion, this study has successfully developed an easily implementable clinical prediction model. For the first time, we have used CART clinical studies as the derivation cohort and real-world results as the validation cohort. Our scoring system provides clinicians with accurate and objective efficacy information, helping them identify the appropriate population for CAR-T therapy in DLBCL.

Loncastuximab in High-Risk and Heavily-Pretreated Relapsed/Refractory Diffuse Large B-Cell Lymphoma: A Real World Analysis from 21 US Centers

Background: In the LOTIS-2 study, patients (pts) with relapsed/refractory (R/R) DLBCL treated with loncastuximab-tesirine (lonca), a CD19 directed antibody-drug conjugate, demonstrated an overall response rate (ORR) and complete response rate (CRR) of 48.3 % and 24.1%. However, there is a paucity of data evaluating outcomes with lonca in the real-world setting (RWS). Hence, we performed a multicenter retrospective study to describe pt characteristics and clinical outcomes in R/R DLBCL pts receiving lonca in this setting. Methods: This retrospective study included pts with R/R DLBCL treated with commercial lonca at 21 academic centers in USA. Clinicopathologic data, treatment outcomes and adverse event (AE) data were collected. Progression-free survival (PFS) and overall survival (OS) were estimated using Kaplan-Meier method and characteristics associated with survival and CR calculated using Cox proportional hazards model and logistic regression. Response was assessed per institutional standards. Results: 187 pts were analyzed with a median follow-up of 12.5 months (mo). Median age was 68 years (range 22-95), 64% male, and 85% white ( Table 1). Most common histology was DLBCL (55% de novo, 22% transformed from low-grade) and 19% were double hit (DH). Thirty-two percent (n=59) had primary refractory disease, 17% (n=31) prior autologous transplant, and 60% (n=112) prior CAR T-cell therapy (CART). Median number of treatment lines before lonca was 4 (1-11) with 81% (n=151) receiving lonca in 4 th line (4L) or later and 8 pts treated off-label in 2 nd line. More pts receiving lonca in ≥4L had prior CART (72% vs 8%, p <0.001). CD19 status was confirmed prior to lonca in 128 pts (68%) with 109 (58%) confirmed CD19+. The ORR/CRR were 33% and 14%, respectively. Median PFS was 2.1 mo (95%CI=1.8-2.6) and median OS 4.6 mo (95%CI=3.7-5.8). 12-month PFS and OS were 12% and 20%. The CRR in 2L/3L, 4L, and >4L were 15%, 13%, and 15% with ORR of 44%, 26%, and 33%, respectively. Figure 1 shows PFS stratified by response with median PFS not reached in those achieving CR. Patients with a CR to last therapy prior to lonca had superior median PFS (8.8 vs 2.0 mo, p<0.01) and OS (10.8 vs 4.5 mo, p =0.01). Factors associated with achieving CR to lonca included CR to last prior therapy (OR 8.3, p<0.01) and nonGCB subtype (OR 3.9, p=0.02). No pts with bulky disease (>10 cm) had objective response. In multivariable analysis (MVA) including factors in Table 1, elevated LDH (HR 1.8, p=0.02; 95%CI=1.1-2.9) and bulky disease (HR 1.7, p=0.03; 95%CI=1.1-2.6) were associated with inferior PFS while CR to last therapy was associated with superior PFS (HR 0.2, p=0.02; 95%CI=0.05-0.76). Bulky disease (HR 2.3, p<0.01; 95%CI=1.4-3.5), HGBL histology (HR 6.1, p<0.01; 95%CI=2.6-14.5), and elevated LDH (HR 2.0, p<0.01; 95%CI=1.2-3.3) were associated with inferior OS, while CR to last therapy (HR 0.4, p=0.05; 95%CI=0.13-0.98) was associated with superior OS in MVA. Neither CD19 status nor prior CART were associated with PFS, OS, or CR. In pts receiving lonca after CART, ORR/CRR were 31% and 15%, respectively, and median PFS/OS were 2.0mo (95%CI=1.6-2.7) and 4.6mo (95%CI=3.2-6.1). In pts who received tafasitamab (n=15) and CART (n=7) after lonca, CR was 13% and 29%, respectively. The most commonly documented AE was cytopenias (45%), followed by peripheral edema, and rash (27% and 27% respectively). Infection was seen in 5% and AEs led to lonca discontinuation in 14%, including peripheral edema (24%) and rash (28%). Conclusions: In this heavily pre-treated population enriched with prior CART exposure, HGBL, and DHL, ORR and CRR to lonca were lower than previously reported. Nonetheless, we found that pts who achieve a CR to lonca have favorable outcomes and factors associated with achieving a CR include lack of bulky disease, nonGCB subtype, and achieving a CR to most recent therapy prior to lonca. Receipt of prior CART did not negatively impact outcomes to lonca. Furthermore, responses were seen with CD19-directed therapies following lonca failure, suggesting sequencing of CD19 therapy pre and post lonca was successful.

Nivolumab in Relapsed or Refractory Primary CNS Lymphoma: Multicenter, Retrospective Study

BACKGROUND Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin's lymphoma confined in the brain and/or spinal cord. While high-dose methotrexate (HDMTX) has been considered the standard of care for newly-diagnosed patients, the optimal treatment strategies for relapsed disease remain challenging, and the outcomes are dismal. Given that 40~50% of PCNSL tissues exhibit aberrancy on 9p24.1 ( PD-L1/PD-L2), immune-checkpoint inhibitors (ICI) may work for the disease. However, there still have been limited data due to the rarity of the disease and the limited accessibility of the drug. To further elucidate the role of ICI in PCNSL, we carried out a nationwide retrospective analysis. METHOD Patients treated with nivolumab for relapsed PCNSL were included in the analysis. Medical records were retrospectively reviewed, and characteristics, including age, sex, prior treatment, risk factors, response, and survival outcomes, were collected. RESULTS From November 2018 to September 2022, 22 consecutive patients were included in the analysis from 4 tertiary institutes in Korea. The median age at diagnosis was 66, and male: female ratio was 1:1. The histologic subtype was diffuse large B-cell lymphoma in all cases, and in 15 cases of which cell-of-origin was identified, 12 cases (80%) were non-GCB subtype. In terms of frontline-treatment, 21 patients (95%) had received HDMTX + procarbazine + vincristine, and rituximab was administered in 73% (16/22) of patients. Nivolumab (at a dose of 3mg/kg every 2 weeks) was treated after median 3 lines (range, 2 - 6) of therapy and at the median age of 67 (range, 37 - 82). Four (18%), twelve (55%), and six (27%) patients were classified as low-, intermediate-, and high-risk groups by IELSG criteria, respectively. Eleven patients (50%) were refractory to the last treatment prior to nivolumab. With a median follow-up duration of 22.3 months (95% CI, 13.1 - 31.5), nine patients (41%) had an objective response (6 complete responses, 3 partial responses). The median time to response was 3.0 months (95% CI, 0.7 - 5.3 months), and the median duration of response was 20.9 months (95% CI, 1.7 - 40.0). The median progression-free survival (PFS) was 2.1 months (95% CI, 0.2 - 4.0), and the 1-yr PFS rate was 36.9% (± 10.9%). The median overall survival (OS) was 18.9 months (95% CI, 5.0 - 32.8), and the 1-yr OS rate was 53.1% (± 13.9%). As expected, responders showed a strong tendency for prolonged OS (36.3 mo. vs. 8.6 mo., p = 0.058). While earlier use (3L vs. 4L or later) of nivolumab was not associated with a higher response rate, it resulted in statistically non-significantly prolonged OS (36.3 months vs. 8.6 months, p = 0.164). Nivolumab was very well-tolerated as no dose reduction was required, and treatment delay was required in two cases (one infection and one seizure). CONCLUSION Given that only restricted treatment strategies are available for relapse PCNSL patients, our study suggests that nivolumab can be a suitable option for the disease. Further studies are warranted to determine predictive biomarkers for nivolumab treatment given the excellent survival duration in responders.

Smart Stop: Lenalidomide, Tafasitamab, Rituximab, and Acalabrutinib Alone and with Combination Chemotherapy for the Treatment of Newly Diagnosed Diffuse Large B-Cell Lymphoma

Background: Despite recent advances, first-line (1L) chemoimmunotherapy (CIT) for patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) is largely unchanged for decades.In the Smart Start trial, we established the feasibility of targeted chemotherapy-free 1L therapy with rituximab (R), lenalidomide (L), and ibrutinib, prior to chemotherapy in patients with newly diagnosed DLBCL with an overall response rate (ORR) and complete response rate (CRR) of 86% and 36%, respectively, prior to chemotherapy, and a 2 year progression free survival rate of 91.3% following 6 cycles of standard CIT (Westin et al, JCO 2023, PMID: 35952327). One patient withdrew from the study after achieving CR and prior to receiving CIT and remains in remission without further therapy at >4 years. BTK inhibitors like acalabrutinib (A) and the immunomodulatory agent L result in synthetic lethality in non-GCB DLBCL models. Both the CD20 antibody R and CD19 antibody tafasitamab (T) demonstrate clinical activity when combined with L in patients with DLBCL. L, T, R, and A are immunomodulatory, driving an anti-tumor immune response. Based upon these data, we are conducting the Smart Stop trial (NCT04978584) to evaluate if the number of CIT cycles can be reduced or omitted after response to targeted therapy, and now report the preplanned interim results from cohort 1. Methods: Eligibility criteria include patients who are 18y+ with previously untreated DLBCL (initially the Hans algorithm was used to select non-GCB DLBCL, but this criteria was removed this criteria) with adequate organ and bone marrow function. Patients with known CNS involvement of lymphoma, recent thrombosis, or inability to tolerate prophylactic anticoagulation are ineligible. Patients receive lenalidomide 25mg on days (d) 1-10, tafasitamab 12mg/kg IV d1, 8, and 15, rituximab 375mg/m2 IV d1, and acalabrutinib 100mg PO twice/d in a 21 day cycle. All patients receive LTRA for 4 cycles, followed by PET/CT scan (CR defined as a 5PS of 1, 2, or 3). In cohort 1, patients receive an additional 6 cycles of LTRA combined with a response adapted number of cycles of CHOP therapy. After 4 cycles of LTRA, patients with CR receive 2 cycles of CHOP, and all other patients receive 6 cycles of CHOP. In the upcoming cohort 2, patients with CR after 4 cycles of LTRA are planned to receive no cycles of CHOP. The primary objectives are to determine the 1A: ORR after 4 cycles of uLTRA and 1B: CRR at of LTRA +/-CHOP at the end of therapy. Results: Cohort 1 enrolled 30 patients from May 2022 - July 2023, with all patients expected to be evaluable for 1A endpoint and 22 patients (73%) evaluable for endpoint 1B by the ASH meeting. The median age was 61 years (range: 32-85), 30% were >= 70 years, and 50% were female. 67% of patients have poor risk R-IPI, 77% had advanced stage, and 83% had an elevated LDH. 83% had the non-GCB subtype and 17% had the GCB subtype of DLBCL. After 4 cycles of LTRA, the ORR is 100% and the CRR is 64% (endpoint 1A, n = 14/22 evaluable at abstract submission, figure 1 schema and swimmer plot). After an additional 2 cycles of LTRA-CHOP, the ORR is 100% and the CRR is 95% (n = 18/19 evaluable at abstract submission). At end of all therapy, the CRR is 100% (endpoint 1B, n = 12 evaluable at abstract submission), including 7 patients who received LTRA for 10 cycles and CHOP for 2 cycles (early CR) and 5 patients who received LTRA for 10 cycles and CHOP for 6 cycles. To date, no patient has progressive lymphoma, including the first 6 patients who have 3 and 6 month follow up scans. 47% of patients experienced rash (13% grade 3), and 40% of patients required a dose reduction of lenalidomide. Conclusions: The Smart Stop trial demonstrates that combination of lenalidomide, tafasitamab, rituximab, and acalabrutinib is highly effective as an initial chemotherapy-free combination in patients with newly diagnosed DLBCL, and may allow for a response adapted reduction in chemotherapy. Response and time to event data will be updated for presentation at the meeting. We will soon open cohort 2 which will enroll an additional 30 patients where those with early CR after 4 cycles of LTRA will receive no cycles of CHOP.

Impact of “Cell-of-Origin” on Outcome after Axicabtagene-Ciloleucel CAR-T Cell Therapy in Relapsed/Refractory Aggressive Non-Hodgkin B-Cell Lymphomas

Introduction: Despite significant advances in characterizing aggressive B-cell non-Hodgkin lymphomas (aNHL) based on molecular and genetic signatures, the Hans algorithm, determined by immunohistochemistry, remains the most commonly used approach to differentiate aNHLs based on the cell-of-origin (COO) into germinal center B-cell (GCB) and non-GCB types. Although the Hans algorithm has demonstrated reliability as a predictor of better outcomes in newly diagnosed GCB cases, conflicting evidence has emerged, particularly in the relapsed/refractory (R/R) setting, and there are no definitive data about the impact of COO in patients (pts) receiving CAR-T cells. Thus, we conducted a retrospective analysis to evaluate the impact of COO extended to R/R aNHLs who underwent treatment with axicabtagene-ciloleucel (axi-cel). Methods: We retrospectively collected data from pts with R/R aNHLs treated with axi-cel at Fred Hutchinson Cancer Center between February 2018 and August 2022. The Hans algorithm, using CD10, BCL6 and MUM1 markers, was employed to stratify the pts into two cohorts: GCB and non-GCB. Paired biopsies of diagnosis and relapse before CAR-T eligibility were reviewed at our institution. Response to CAR-T was assessed at one month per Lugano PET/CT criteria. Univariate cox proportional hazard regression was used to estimate associations between baseline factors and outcomes. Statistically significant factors ( p<.05) in univariate analysis were further evaluated by multivariate analysis. Continuous variables were compared by Mann-Whitney U test and categorical variables by Fischer's exact test. Progression-free survival (PFS) was calculated from the date of CAR-T cell infusion to the date of death from any cause, disease progression or relapse. Overall survival (OS) was calculated from the date of CAR-T cell infusion to the date of death from any cause. Results: Sixty-four pts were included in this analysis, with an equal distribution of pts in the two cohorts: 32 with GCB subtype and 32 with non-GCB subtype. Baseline pts' characteristics and histology subtypes are listed in Table 1. Transformed follicular lymphoma and primary mediastinal B-cell lymphoma were not included in this analysis. The presence of bulky disease (defined as >5cm diameter) was higher in the GCB subtype (56% vs 22%, p<.001). Although primary refractory cases were more frequent in the GCB cohort (87.5 vs 66%), statistical significance was not reached (NR)( p=.07). The same proportion of pts received bridging therapy in the two groups (53% vs 53%, p=.99). Pts evaluable for response assessment were 62. Median time to best response was 29 days (range, 21-186) for GCB pts and 31 days (range, 22-182) for non-GCB pts ( p=.91). The overall response rate was 70% for GCBs (34.5% complete response [CR], 34.5% partial response [PR]) and 75% for non-GCBs (44% CR, 31% PR). After a median follow-up of 24.5 months (range, 9-121), comparable outcomes were observed for both the COO groups: despite the estimated median OS of 14 months (95% CI, 4.73-NR) for GCB vs 40 months (95% CI, 12.55-67.46) for non-GCB, we did not find any significant differences ( p=.73) (Figure 1A). Estimated median PFS was 10 months (95% confidence interval [CI], 3.5-16) for GCB and 11 months (95% CI, 0-24) for non-GCB ( p=.57) (Figure 1B). The 2-year OS and PFS were: GCB, 45% (95% CI, 30-69) and 44% (95% CI, 29-67) respectively; non-GCB, 55% (95% CI, 38-79) and 29% (95% CI, 16-55), respectively. Treatment-related toxicities did not differ between GCB and non-GCB, with cytokine-release syndrome rates of 97% vs 94% ( p=.99) and neurotoxicity rates of 53% vs 62.5% ( p=.61), respectively. The incidence of infectious complications was 25% vs 19% ( p=.76). On univariate analysis, the impact of COO on OS (HR=0.82, 95% CI 0.39-1.73, p=.60) and PFS (HR=1.18, 95% CI 0.61-2.28, p=.62) was undetermined. After adjusting for bulky disease, elevated LDH, absolute lymphocyte count≤1000/uL, performance status≥2, >2 lines of therapy and response status before axi-cel infusion, the impact of COO on OS (adjusted HR [aHR]=1.73, 95% CI 0.71-4.22, p=.23) and PFS (aHR=2.01, 95% CI 0.93-4.35, p=.08), remained undetermined. Conclusion: We could not confirm an association between COO, as determined by Hans algorithm, and outcomes in R/R aNHLs who received axi-cel at our center. Prospective analyses on larger datasets and other types of CAR-T products are needed.

Phase Ib/II Study of Multi-Targeted Therapy with Venetoclax, Ibrutinib, Prednisone, Obinutuzumab, and Lenalidomide (ViPOR) in Relapsed/Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Foundational studies have revealed essential oncogenic pathways in DLBCL, triggering the development of drugs targeting distinct survival pathways in this malignancy. While many of these agents are active in DLBCL as monotherapy, they rarely induce deep responses or cure. Based on our identification of drug synergy in DLBCL models, we hypothesized that targeting multiple survival pathways concurrently could be curative in DLBCL. We developed a 5-drug combination regimen (ViPOR) that targets DLBCL survival sustained by constitutive B-cell receptor (BCR) signaling (ibrutinib, lenalidomide, prednisone) and by BCL2 (venetoclax), and also enlists the innate immune system using obinutuzumab. To maximize drug exposure and minimize toxicity, we administered all agents in non-continuous cycles for fixed duration in R/R DLBCL. Methods: R/R DLBCL pts with adequate organ function were eligible. In Ph I, pts were treated at 4 doses of venetoclax (200-800 mg) PO D2-14 to identify the MTD. An initial 12d venetoclax ramp-up was given in combination with fixed-dose ibrutinib 560 mg PO D1-14, prednisone 100 mg PO D1-7, obinutuzumab 1000 mg IV D1-2, and lenalidomide 15 mg PO D1-14. Ph II expansion cohorts of R/R GCB and non-GCB DLBCL were included at the MTD. Max 6C of ViPOR q21d were given without maintenance. TLS, G-CSF, and PCP prophylaxis were given to all pts. Baseline CT, PET, BM, and tumor biopsies were performed with CT after C1, 2, 4, and 6 and PET after C6. CT was then performed q3m x 1y, q4m x 1y, q6m x 1y, then q12m x 2y. Tumor genomics and ctDNA (clonoSEQ) were studied. Results: 50 DLBCL pts were enrolled (25 DLBCL NOS, 17 HGBCL-DH-BCL2, 3 HGBCL-DH-BCL6, and 5 THRLBCL). 52% and 48% were GCB and non-GCB subtype by IHC, respectively, with transformed lymphoma in 34%. Median age was 61y (range 29-77), with stage 3-4 disease in 92%, elevated LDH in 86%, >2 extranodal sites in 56%, and IPI >3 in 68% of pts. Median prior txs were 3 (range 1-9), with 40% post-CAR-T pts and 58% refractory. A single DLT of G3 intracranial hemorrhage occurred, and venetoclax 800 mg was identified as the MTD. Heme AEs were most common, with G3-4 neutropenia in 24%, thrombocytopenia in 23%, and anemia in 7% of cycles. Febrile neutropenia occurred in 3 (1%) cycles. The only G3-4 non-heme AE in >10% pts was hypokalemia (28%). G3 A.fib occurred in 3 pts, and G4 TLS occurred in 1 pt, which resolved. Other common any grade non-heme AEs (% pts) included diarrhea (68%), hypokalemia (67%), nausea (45%), rash (35%), and fatigue (33%). Dose reductions occurred in 17% of pts, and 8% discontinued tx due to AE. Of 48 evaluable pts (2 came off tx prior to restaging), ORR was 54% (26/48), and CR was 38% (18/48). Responses were observed across all molecular DLBCL subtypes, including a CR rate of 62% (8/13) in non-GCB DLBCL, 53% (8/15) in HGBCL-DH-BCL2, 25% (2/8) in other DLBCL, and a PR rate of 33% (4/12) in GCB DLBCL (non-DH) (Fig. 1A). CR rate was 20% (4/20) and 19% (5/27) in post-CAR-T and refractory pts, respectively. With a median FU of 40m, 72% of CRs are ongoing, with a 2-year PFS and OS of 34% and 36%, respectively. By histology, 2-year PFS was 47%, 38%, 38%, and 8% in HGBCL-DH-BCL2, non-GCB DLBCL, other DLBCL, and GCB DLBCL (non-DH), respectively (Fig. 1B). 2-year PFS was 30% and 21% in post-CAR-T and refractory pts, respectively. MRD was undetectable in 38% (16/42) of pts at end of therapy (EoT), and in 93% (14/15) of pts in PET CR at EoT. Elevated baseline ctDNA or detectable ctDNA during or at EoT were associated with significantly inferior PFS and OS, as were quantitative PET parameters (elevated baseline TMTV and TLG). Two genetic subtypes known to rely on BCR-dependent NF-kB signaling - MCD and N1 - had a significantly higher CR rate (5/6, 83%) than all other genetic subtypes (4/22, 18%; p=0.0066). Conclusions: This is the first study to show the feasibility and curative potential of multi-targeted therapy in R/R DLBCL. ViPOR was well tolerated across all ages in R/R DLBCL with rare febrile neutropenia. ViPOR was most effective in non-GCB DLBCL, as expected from its reliance on BCR signaling and BCL2. ViPOR was also highly active in HGBCL-DH-BCL2, possibly due to inhibition of MYC-driven apoptosis by BCL2 in this subtype. Durable remissions and likely cure were observed in non-GCB DLBCL (38%) and HGBCL-DH-BCL2 (47%), including pts relapsed after or refractory to CAR-T (30%). Multicenter Ph II testing is in development to confirm the activity of ViPOR in R/R non-GCB DLBCL and HGBCL-DH-BCL2.

CDC6, a key replication licensing factor, is overexpressed and confers poor prognosis in diffuse large B-cell lymphoma

BackgroundCell division cycle 6 (CDC6) is a key licensing factor in the assembly of pre-replicative complexes at origins of replication. The role of CDC6 in the pathogenesis of in diffuse larger B-cell lymphoma (DLBCL) remains unknown. We aim to investigate the effects of CDC6 on the proliferation, apoptosis and cell cycle regulation in DLBCL cells, delineate its underlying mechanism, and to correlate CDC6 expression with clinical characteristics and prognosis of patients with DLBCL.MethodsInitial bioinformatic analysis was performed to screen the potential role of CDC6 in DLBCL. Lentiviral constructs harboring CDC6 or shCDC6 was transfected to overexpress or knockdown CDC6 in SUDHL4 and OCI-LY7 cells. The cell proliferation was evaluated by CCK-8 assay, cell apoptosis was detected by Annexin-V APC/7-AAD double staining, and cell cycle was measured by flow cytometry. Real time quantitative PCR and western blot was used to characterize CDC6 expression and its downstream signaling pathways. The clinical data of DLBCL patients were retrospectively reviewed, the CDC6 expression in DLBCL or lymph node reactive hyperplasia tissues was evaluated by immunohistochemistry.ResultsIn silico data suggest that CDC6 overexpression is associated with inferior prognosis of DLBCL. We found that CDC6 overexpression increased SUDHL4 or OCI-LY7 cell proliferation, while knockdown of CDC6 inhibited cell proliferation in a time-dependent manner. Upon overexpression, CDC6 reduced cells in G1 phase and did not affect cell apoptosis; CDC6 knockdown led to significant cell cycle arrest in G1 phase and increase in cell apoptosis. Western blot showed that CDC6 inhibited the expression of INK4, E-Cadherin and ATR, accompanied by increased Bcl-2 and deceased Bax expression. The CDC6 protein was overexpressed DLBCL compared with lymph node reactive hyperplasia, and CDC6 overexpression was associated with non-GCB subtype, and conferred poor PFS and OS in patients with DLBCL.ConclusionCDC6 promotes cell proliferation and survival of DLBCL cells through regulation of G1/S cell cycle checkpoint and apoptosis. CDC6 is overexpressed and serves as a novel prognostic marker in DLBCL.

Prediction of Survival in Diffuse Large B Cell Lymphoma According to Immunohistochemistry

BACKGROUND: The introduction of chemoimmunotherapy in the treatment of diffuse large B cell lymphoma (DLBCL) dramatically improved the outcomes compared to chemotherapy alone. However, a significant part of the patients become refractory and eventually relapse. AIM: The purpose of this study is to analyze the survival differences between different groups of DLBCL according to Han’s algorithm. MATERIALS AND METHODS: We will study the medical records of 244 patients treated with RCHOP as first-line therapy who are diagnosed with de novo DLBCL in a cohort of 10 years in the University Clinical Center of Kosovo. According to immunohistochemical markers, the patients will be classified into two major groups, germinal center B cell-like (GCB) and non-germinal center B cell-like (non-GCB) subtypes, and five subgroups (Group 1, Group 2, Group 3 or double positive, Group 4, and Group 5 or triple negative [TN]). RESULTS: The patients in the GCB group have better progression-free survival (PFS) and overall survival (OS) than the non-GCB group. Despite this, double positive (DP) (CD10+MUM1+) and TN (CD10−Bcl6−MUM−) subgroups showed different PFS and OS to the same cell-of-origin group. The DP group showed similar OS and PFS with the non-GCB group, whereas the TN group showed similar OS and PFS with the GCB group. CONCLUSION: These factors will provide valuable knowledge for predicting the prognosis and redirecting the choice of treatment for different groups of DLBCL.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.